Research ArticleChinese Medicine Bu Xu Hua Yu Recipe for the Regulation ofTregTh17 Ratio Imbalance in Autoimmune Hepatitis

Lei Wang1 Huihui Du1 Yibo Liu1 Lingtai Wang12 Xiong Ma13 and Wei Zhang1

1Department of Hepatology Shanghai University of Traditional Chinese Medicine Longhua HospitalNo 725 South Wan Ping Road Shanghai China2Shanghai University of Traditional Chinese Medicine Shuguang Hospital Shanghai China3Department of Gastroenterology Renji Hospital Shanghai Institute of Digestive DiseaseShanghai Jiao Tong University School of Medicine China

Correspondence should be addressed to Wei Zhang 18918104444189cn

Received 12 October 2014 Accepted 26 February 2015

Academic Editor Vincenzo De Feo

Copyright copy 2015 Lei Wang et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ObjectivesThe aim of this study is researching the role of the Regulatory T cell (Treg)T helper cell-17 (Th17) cell ratio imbalance inthe pathogenesis of autoimmune hepatitis (AIH) and the use of the ldquoBu Xu Hua Yurdquo recipe in the treatment of AIHMaterials andMethods Sixty adult male C57BL6 mice were divided into six different groups 120572-Galcer was injected abdominally for productionof the animal models Liver function tests histological examinations liver tissue Regulatory T cell and T helper cell-17 levels testswere carried out TGF-1205731 IL-10 IL-17 and expression of mRNA and protein levels of Foxp3 and ROR-120574t were also assessed ResultsBu Xu Hua Yu method increased the levels of Regulatory T cell IL-10 and the expression of Foxp3 (119875 lt 005) in mice liver tissuesFurthermore there were decreases in the levels of T helper cell-17 IL-17 and expression of ROR120574t mRNA and protein (119875 lt 005)The ratio of TregTh17 was increased (119875 lt 005) Conclusion Mice with AIH have a TregTh17 ratio imbalance Bu Xu Hua Yumethod was able to restore the cellular balance of TregTh17 through the regulation of the expression of ROR120574t and Foxp3 and canplay an important role in the treatment of AIH

1 Introduction

Autoimmune hepatitis (AIH) is a type of inflammatory liverdisease whose etiology is not fully understood and withno standard treatment protocol As of now single doseprednisone or low dose prednisone plus azathioprine isthe main treatment protocol for AIH with alleviation ofsymptoms achieved in 60 to 80 of cases [1] Howeverit must be noted that 50 to 86 of patients witness arecurrence of the disease after stopping medications while13 of patients stop the treatment too early due to the severityof the side effects and 9of patientsrsquo situationwill deteriorateeven though they are administered medications accordingto guidelines [2] The pathophysiology of AIH is relativelycomplicated and the incidence of the disease is closelyrelated to genetics autoimmune antibodies environmentalfactors and imbalance of the immune system In these lastyears studies have shown that the activation of autoimmune

responsive T cells and B cells is an important step in thepathophysiology of the disease and the loss of balance in thelevels of these two cells is essential to the pathogenesis of AIHHowever these assumptions have as of now not been proven

The use of Chinese medicine for the treatment of autoim-mune hepatitis has proven its merits in mainland Chinaand is able to make up for the problems encountered withwestern medicine The need for better understanding themechanisms of these treatment protocols has put a spotlightonto those types of studies and is bound to become amajor converging point We made use of the Bu Xu HuaYu principle of immunological regulation which can standfor restoring deficiency and removing blood clots (exactcontent of this treatment is given in Table 2) combinedwith Ursodeoxycholic acid capsules (Losan pharma GmbHGermany specification 250mg times 25 pills) to prevent AIHand this treatment protocol showed results which weresimilar to those obtained with prednisone combined with

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 461294 14 pageshttpdxdoiorg1011552015461294

2 Evidence-Based Complementary and Alternative Medicine

azathioprine Furthermore the Chinese medicine protocolwas better apt at controlling the symptoms of the diseaseFollowing the new developments in the study of AIH animalmodel this study made use of 120572-Galcer to induce C57BL6mice AIH animal model 120572-Galcer is able to cause NKTinduced liver damage and infiltration of CD4+ lymphocytesin liver tissues as seen in classic presentations of AIH ashas been previously reported in other studies by Biburgerand Tiegs and by Matsumoto et al [3 4] At the sametime modeling is rendered simple and time for modeling isrelatively short 120572-Galcer hepatitis model has differences inindividual susceptibility gene C57BL6 mice showed higher120572-Galcer hepatitis susceptibility We therefore combinedwhat we already know about themechanism of AIH and earlystages of clinical studies to investigate the function of T cellsespecially the role of regulatory T cells (Treg)T helper cells-17 (Th17) abnormalities in the etiology of AIH with especialemphasis on the mechanism of the Bu Xu Hua Yu treatmentmethod

2 Materials and Methods

This study was approved by the Institutional Animal Careand Use Committee of Shanghai University of TraditionalChineseMedicineThe registration number for our approvedexperiment was 11007 and the following measures were takento ameliorate animal suffering

(1) The person taking care of the experimental animalsduring the study is very experienced in this type ofwork has very good animal handling skills and tookvery good care of all animals involved in this study ona daily basis

(2) All experiments of the animals were done after induc-ing anesthesia so as to decrease any pain that theanimals might have felt

(3) All animals were euthanized after the experiment wascarried out

(4) After they were introduced in our laboratory settingsall animals were given individual cages and the cageswere cleaned on a very regular basis

21 Choice of Experimental Animal and Grouping Sixty maleC57BL6 mice of SPF grade and aged between 4 and 6 weekswere chosen The mice were obtained from Shanghai Si LaiKe Experimental Animal Limited Liability Company (licensenumber SCXK 2007-0005 Shanghai China) The animalswere bred at Shanghai University of Traditional ChineseMedicineAffiliated LongHuaHospitalrsquos SPF grade laboratoryanimal room We made use of randomized block designto randomly separate the 60 mice into control group (K)model group (M) steroids group (X) Chinese medicine losedose group (D) Chinese medicine moderate dose group (Z)and Chinese medicine high dose group (G) with each grouphaving 10 mice A preexperiment was used by the samegroup of researchers to study the viability of this study beforeengaging in a larger scale study with more mice

22 Main Materials and Reagents FACS flow cytometry wasproducts of BD company (Becton Dickinson and CompanyEast Rutherford New Jersey United States) CD4+CD25+regulatory T cell mice staining kit mainly contains 3types of antibody namely FTTC-anti-mouse-CD4+ APC-anti-mouse-CD25 and PE-Cy5-anti-mouse-Foxp3TheTh17cell staining kit (PE Rat Anti-Mouse IL-17A APC IFN-120574Anti-Mouse FTTC-anti-mouse-CD3e and PerCP Rat Anti-Mouse CD4) was obtained from BD Company The TGF-1205731IL-10 and IL-17 Elisa reagent kit was obtained from ShanghaiAo Biological Company (Shanghai China) the antibodieswere obtained from RampD systems (Minneapolis USA) TheTrizol reagent was bought from Invitrogen Company (Cal-ifornia USA) Foxp3 ROR120574t and the reference GAPDHprimers were synthesized by Shanghai ShineGene Biotech-nology Companies (Shanghai China) cDNA synthesis kitand PCR amplification kit were provided by TaKaRa CoBiological Engineering (Dalian China) protein extractionkit was provided by the Bestbio Biotechnology Companies(Shanghai China) Mouse Anti-Human Foxp3 (2A11GG)polyclonal antibody and Rabbit Anti-Human ROR120574t (H-190)polyclonal antibody were purchased from Santa-Cruz Com-pany (Santa Cruz Biotechnology Inc) Reference antibodyHistoneH31 (Ab-10) was obtained fromSignalWayAntibody(Maryland USA)

23 Experiment Method

231 Drug Intervention and Preparation of AIH Mice Model

Bu Xu Hua Yu Drug Preparation A mouse (20 g) is more orless equivalent to a human adult (approximately 60Kg) Forthe normal daily dosage the following are required for theBu Xu Hua Yu recipe dried Rehmannia roots 005 g Angelica004 g Astragalus 005 g red peony root 005 g Chuanxiong003 g and Sedum sarmentosum 010 g

Dosage Dosage was calculated according to the ldquoEstimatingthe Safe Starting Dose in Clinical Trials for Therapeuticsin Adult Healthy Volunteersrdquo guidelines Low dose groupreceived a dose equivalent to 10 times the normal clinicaldose of the normal adult and one time the dose for micemediumdose group received a dose equivalent to 20 times thenormal clinical dose of a normal adult and 2 times the dosefor mice high dose group received a dose equivalent to 40times the normal clinical dose of a normal adult and 4 timesthe dose for mice steroid control group received prednisone01mg(micesdotday) Mice in the model control group and inthe blank control group were given isovolumetric normalsaline solution 02mLmice

Each group was first given the respective medications for7 days and on the 7th day except for the control group thefive other groups all received an intra-abdominal injection of2 120583g10 g 120572-Galcer (Enzo Life Sciences)

Histological Analysis of the Liver of Mice with AIH The liveris fixed with 10 neutral formalin and embedded in paraffinwax and slicedThe slides were stained with hematoxylin andeosin The degree of inflammation of the liver was graded as

Evidence-Based Complementary and Alternative Medicine 3

follows grade 0 = normal liver tissue grade 1 = mild infil-tration of inflammatory cells with rare hepatic cells necrosisgrade 2 = medium damage of hepatic cells accompanied byinfiltration of inflammatory cells and regional hepatic cellnecrosis grade 3 = wide range inflammatory cells infiltrationin portal area and hepatic lobules accompanied with widerange hepatic cells necrosis

Biochemical Indexes Ten to twelve hours after injection of themodeling agent blood was drawn from the micersquos eyeballwas kept at 4∘C for 2 hours and was centrifuged at 3000 rpmfor 15mins After centrifugation the serum sample was col-lected and was analyzed for alanine aminotransferase (ALT)aspartate aminotransferase (AST) and alkaline phosphatase(AKP) All experiments were done strictly following theinstructions of Nanjing Jian Cheng Ke Ji (Nanjing China)who provided us with the liver function test kit

24 Flow Cytometry Was Used to Detect the Number of TregCells and Th17 Cells in the Hepatic Tissue

241 Treg Cell We used monocytes extracted from lym-phocytes of the liver the cell concentration was set at 1 times107mL A 100 120583L hepatic monocytes suspension was addedto 025 120583L FITC anti-mice CD4 monoclonal antibody 03 120583LAPC-anti-mice CD25 +monoclonal antibody and 25 120583L PE-anti mice Foxp3monoclonal antibody incubation FACS flowcytometry was used to detect the number of Treg cells and thecontent of Foxp3 in liver

242 Th17 Cell We used monocytes extracted from lym-phocytes of the liver the cell concentration was set at 1 times107mLA 100120583Lhepaticmonocytes suspensionwas added toPerCP labeled anti-CD4 CD3e after incubation with brokenmembrane agent intracellular cytokine staining and FITClabeled IL-17A 05 120583g incubation FACS flow cytometry wasused to detect Th17 cytokines IL-17 and IFN-120574 which aresecreted by CD4+ cells

25 ELISA Method for the Detection of Liver Homogenate IL-10 IL-17 and TGF-1205731 Levels The experiment was carried outaccording to the strict instructions of the ELISA kit

26 RT-PCR Was Used to Analyze the Expression of Foxp3mRNA and ROR120574t mRNA (1) Primer synthesis the specificsequence is as follows

Foxp3 Forward Prime 51015840-ACCCAGGAAAGACAG-CAACC-31015840 Reverse Primer 51015840-CTCGAAGACCTT-CTCACAACCA-31015840 length of products 107 bpROR120574t Forward Primer 51015840-CCATTGACCGAA-CCAGCC-31015840 Reverse Primer 51015840-TCTGCTTCTTGG-ACATTCGG-31015840 length of products 111 bpGapdh Forward Primer 51015840-TGTGTCCGTCGT-GGATCTGA-31015840 Reverse Primer 51015840-TCTGCTTCT-TGGACATTCGG-31015840 length of products 77 bp

(2) Extraction of total RNA and RT-PCR procedure 50ndash100mg tissue was collected from each group Trizol kit was

used to extract total RNA after using the spectrophotometerat 260280 nm in the examination of the quality and purityof total RNA Using the cDNA synthesis kit we performedtranscription of mRNA to cDNA We then used cDNAcorresponding specific primers for PCR amplification withthe real-time PCR 20120583L reaction volume includes PremixEx TaqTM (2x) 10 120583L upstream and downstream primers04 120583L Taq Probe 08 120583L Rox Reference Dye (50x) 04120583LcDNA 2120583L and DEPC water 6120583L PCR procedure was doneas follows 95∘C for 30 s after the initial hot-start followed by45 PCR cycles at 95∘C for 5 s annealing temperature of 60∘Cfor 30 s

(3) Extraction of 10 120583L PCR reaction product 10 120583L PCRreaction product was extracted and placed on a 2 agarosegel electrophoresis for 50min and the image acquisition isdone by image analyzer Using glyceraldehyde-3-phosphatedehydrogenase (Gapdh) reference as the benchmark weperformed semiquantitative analysis Real-time PCR resultsare shown as follows 2-ΔΔCt (2-ΔΔCt = RQ) represents thetarget genes relative to GAPDH Higher values for 2-ΔΔCtcorrespond to higher levels of gene expression and smallervalues correspond to lesser levels of gene expression

27 Western Blot Technique Used to Measure the Expressionof Foxp3 and ROR120574t Protein in Hepatic Tissue After 50ndash100mg of hepatic tissue was homogenized tissue proteinswere extracted BCAmethodwas used formeasuring the con-centration of protein levels and for dilution Sodium dodecylsulfate polyacrylamide gel electrophoresis was carried outand the protein was transferred to the PVDF film PonceauS staining was used for the examination of the strip film 1 timesTBST buffer skim milk closed 1 h 1 100 Foxp3 ROR120574t and1 200 reference antibody were separately added respectivelyand were incubated overnight at 4∘C phosphate bufferwas added after washing the membrane with horseradishperoxidase-labeled IgG electrochemiluminescence color wasobtained through X-ray exposure developing and fixing wasdone and Gel-Pro analysis software was used to locate andanalyze the strip

28 Statistical Analysis All data obtained in this study wasanalyzed using the SPSS160 software Data for each groupis represented in the form (119883 plusmn 119878) multisamples groupswere compared with single factor analysis of variance (one-way ANOVA) Skewness distribution was analyzed usingnonparametric test119875 lt 005was considered to be statisticallysignificant 119875 lt 001 was considered to be statistically highlysignificant

3 Results

31 Tissue Histology Examination of AIHMouse Model and ofDifferent Groups after Drug Usage Hepatic tissue of mouseafter hematoxylin-eosin staining shows that after inductionwith 120572-Galcer the C57BL6 mice showed varying degrees ofpathological changes of grade 1 to grade 2 inflammation theportal area and its surroundings showed obvious infiltrationof inflammatory cells when compared to the model group

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Liver function indexes for each group

Group(119899 = 10) ALT (UL) AST (UL) AKP (UL)

K 3032 plusmn 954 309 plusmn 807 3546 plusmn 1216

M 28616 plusmn 5288 25357 plusmn 3146 29002 plusmn 5923X 18687 plusmn 4265 16619 plusmn 3388 1473 plusmn 3825

D 19222 plusmn 4432 17063 plusmn 26 17986 plusmn 3785

Z 18605 plusmn 4079 16560 plusmn 2241 16889 plusmn 2025

G 15699 plusmn 3459 14764 plusmn 2583 13786 plusmn 4592

When compared to the model group 119875 lt 005

steroids group and the Chinese medicine moderate dosegroup showed signs of mild inflammation with remissionof the infiltration of inflammatory cells blank control groupshowed almost normal hepatic tissue as shown in Figure 1

32 Result for Liver Function Indexes Except for the blankcontrol group the liver function indexes were increased bydifferent amounts in all the other groups After treatment ofsteroids group and the different Chinese medicine groupsthe liver function indexes all witnessed decreases whichwere statistically significant when compared with the controlgroup (119875 gt 005) However the difference between thesteroids and Chinese medicine groups and the differencebetween the different Chinese medicine groups were notstatistically significant (119875 gt 005) This shows that theChinese medicine was able to protect the liver against AIHand the results were on par with steroids The results areshown in Table 1 and Figure 2 (Figure 2(a) for ALT levelsFigure 2(b) for AST levels and Figure 2(c) for AKP levels)

33 Analysis of Results for Hepatic Lymph Cell Treg(CD4+CD25+Foxp3+) Cell and Th17 (CD3+CD4+IL-17+IFN-120574minus) Cell Analysis results of FACS showed that theTreg cells levels in the hepatic lymph tissue of the modelgroup were lower than in the control group When eachtreatment group was compared to the model group the levelsof Treg cells were considerably increased and the differencewas statistically significant (119875 lt 005) Comparison ofthe control group and the Chinese medicine high dosegroup to the model group showed a statistically significantdifference (119875 lt 001) The levels of Th17 cells in the modelgroup were higher than in the blank control group and inthe treatment groups and the difference was found to bestatistically significant (119875 lt 005) The TregTh17 ratio wasdecreased in the model group when compared to the blankcontrol group After the use of therapeutic drugs the ratioof TregTh17 was found to be increased When comparedto the model group the steroids group Chinese medicinemoderate dose group and Chinesemedicine high dose groupall had showed increases that were statistically significant(119875 lt 005) However there was no statistical differencebetween the Chinese and steroids groups or betweenthe different Chinese medicine groups Furthermore theChinese medicine treatment group had a dose-dependent

Table 2 Each group hepatic tissues Treg Th17 cell levels ()

Group (119899 = 8) Foxp3(+) Treg TH17 (IL17+IFN120574minus)K 132 plusmn 049 071 plusmn 040

M 053 plusmn 018 163 plusmn 078X 108 plusmn 044 095 plusmn 049

D 100 plusmn 056 107 plusmn 059

Z 102 plusmn 042 084 plusmn 039

G 113 plusmn 058 083 plusmn 039When compared to M 119875 lt 005 when compared to M 119875 lt 001

Table 3 TregTh17 cell ratio for each group

Group (119899 = 8) TregTH17 Mean rank When comparedto M group 119885 ratio

K 277 plusmn 257 3438 337M 040 plusmn 024 838 mdashX 164 plusmn 135 265 295D 141 plusmn 126 2363 190Z 158 plusmn 135 2638 274G 172 plusmn 137 2775 263When compared to M 119875 lt 00083

Table 4 Analysis results for cytokines in hepatic tissues for eachgroup

Group(119899 = 10) TGF-1205731 (pgmL) IL-10 (pgmL) IL-17 (pgmL)

K 17936 plusmn 5357 22447 plusmn 4959 14523 plusmn 5249

M 29382 plusmn 10218 645 plusmn 2025 29871 plusmn 7950X 28179 plusmn 6470 10245 plusmn 2480 19229 plusmn 10756

D 27714 plusmn 6486 7255 plusmn 1873 21995 plusmn 11076

Z 22871 plusmn 8321 9937 plusmn 4163 21342 plusmn 9850

G 25136 plusmn 8511 14168 plusmn 2160 15474 plusmn 6296

When compared with group M 119875 lt 005

trend These results are shown in Figures 3 4 and 5 andTables 2 3 and 4

34 Analysis of Results for Hepatic Tissue Cytokines for EachGroup Analysis of TGF-1205731 in hepatic tissues showed thatin the model group TGF-1205731 was increased when comparedto the control group but there was no statistical differencebetween each treatment group and the model group (119875 gt005) Analysis of IL-10 showed that model group IL-10levels were lower when compared to blank control groupand when compared to the model group IL-10 for eachtreatment group was increased with the difference beingstatistically significant (119875 lt 005) Analysis results of IL-17showed that IL-17 of themodel groupwas considerably higherthan that of the blank control group and of each treatmentgroup The difference was statistically significant (119875 lt 005)Furthermore there was no statistical difference between theChinese medicine and steroids groups or between each ofthe Chinese medicine groups (119875 gt 005) Chinese medicine

Evidence-Based Complementary and Alternative Medicine 5

XK

M G

Figure 1 The figure shows the histological analysis for control group (K) model group (M) steroids group (X) and Chinese medicine highdose group (G) Histological analysis for each group

Table 5 Results for the analysis Foxp3 mRNA for each group (2-ΔΔCT)

Group(119899 = 8)

RQ(2-ΔΔCT)median

RQ(2-ΔΔCT)

Q25

RQ(2-ΔΔCT)

Q75119885 ratio 119875 ratio

K 01887 01167 19460 2731 0006

M 00529 00121 01038 mdash mdash

X 00729 00332 01886 1470 0141

D 00842 00549 01391 1470 0141

Z 01152 00432 04352 1575 0115

G 00642 00181 02824 0840 0401When compared to group M 119875 lt 005

treatment groups showed a dose-dependent trend Results areshown in Table 4 and Figure 6

35 Analysis Results for Liver Specific Transcription FactorsFoxp3 and ROR120574t Real-time RT-PCR analysis showed thatthe difference between each group hepatic tissue transcrip-tion factor Foxp3 mRNAwas not statistically significant (119875 lt005) The model group ROR120574t mRNA was higher than thatof the control group and treatment groups the difference wasstatistically significance (119875 lt 005 119875 lt 001) The results areshown in Figures 7 and 8 and Tables 5 and 6

Table 6 Results for the analysis of ROR120574t mRNA for each group(2-ΔΔCT)

Group (119899 = 8) RQ (2-ΔΔCT) 119875 ratioK 0110 plusmn 0062 001

M 0393 plusmn 0282 mdashX 0201 plusmn 0146 0020

D 0134 plusmn 0075 0002

Z 0161 plusmn 0122 0005

G 0165 plusmn 0085 0006

When compared to group M 119875 lt 005 and 119875 lt 001

36 Results for the Analysis of Hepatic Foxp3 and ROR120574tProteins For each group western blot testing showed thatFoxp3 protein expression in the model group was muchlower than in the control group In all treatment groupsthe expression of the Foxp3 protein was increased whencompared to the model group with the moderate doseChinese medicine group showing a much higher increasewhen compared to the steroids group the difference wasstatistically significant (119875 lt 005) The expression of ROR120574tprotein in the model group was much higher than in thecontrol group For each treatment group the ROR120574t proteinexpressionwas considerably decreasedwhen compared to themodel group and the difference was statistically significant(119875 lt 005) These results are shown in Figures 9 and 10 andTable 7

6 Evidence-Based Complementary and Alternative Medicine

KMX

DZG

0

100

200

300

400A

LT (U

L)

K M X D Z G

lowastlowast

lowastlowast

lowast

(a) ALT between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AST

(UL

)

lowast

(b) AST between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AKP

(UL

)

lowast

lowast

lowast

lowastlowast

(c) AKP between six groups

Figure 2 The figure shows the level of ALT AST and AKP for each of the six groups

Table 7 Foxp3 ROR120574t proteins for each liver group

Group (119899 = 8) ROR120574t protein(IOD ratio)

Foxp3 protein(IOD ratio)

K 097 plusmn 011 168 plusmn 016

M 192 plusmn 015 067 plusmn 005X 136 plusmn 006 084 plusmn 003

D 099 plusmn 013Δ 099 plusmn 051

Z 071 plusmn 007Δamp

106 plusmn 010Δ

G 137 plusmn 002amp 095 plusmn 009When compared with group M 119875 lt 005 and

119875 lt 001 Δwhen comparedwith group X 119875 lt 005 ampwhen compared with group D 119875 lt 001

4 Discussion

Autoimmune hepatitis is a type of autoimmune liver diseasewhich has an undefined etiology and its pathological charac-teristic ismainly interface hepatitis with infiltration of plasmacells in the portal vein which is accompanied by serumhypergammaglobulinemia and positive serum antibodies[5 6] The pathophysiology of AIH is relatively complexwith factors such as genetics autoantigens environmentalfactors and immune dysfunction commonly mentioned asbeing responsible [7] Previous studies on the subject mainlyfocused on liver cell apoptosis expression of inflammatorymediators gene polymorphism and immune system regula-tion [8ndash14] Autoimmune T cells and B cells activation are

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

azathioprine Furthermore the Chinese medicine protocolwas better apt at controlling the symptoms of the diseaseFollowing the new developments in the study of AIH animalmodel this study made use of 120572-Galcer to induce C57BL6mice AIH animal model 120572-Galcer is able to cause NKTinduced liver damage and infiltration of CD4+ lymphocytesin liver tissues as seen in classic presentations of AIH ashas been previously reported in other studies by Biburgerand Tiegs and by Matsumoto et al [3 4] At the sametime modeling is rendered simple and time for modeling isrelatively short 120572-Galcer hepatitis model has differences inindividual susceptibility gene C57BL6 mice showed higher120572-Galcer hepatitis susceptibility We therefore combinedwhat we already know about themechanism of AIH and earlystages of clinical studies to investigate the function of T cellsespecially the role of regulatory T cells (Treg)T helper cells-17 (Th17) abnormalities in the etiology of AIH with especialemphasis on the mechanism of the Bu Xu Hua Yu treatmentmethod

2 Materials and Methods

This study was approved by the Institutional Animal Careand Use Committee of Shanghai University of TraditionalChineseMedicineThe registration number for our approvedexperiment was 11007 and the following measures were takento ameliorate animal suffering

(1) The person taking care of the experimental animalsduring the study is very experienced in this type ofwork has very good animal handling skills and tookvery good care of all animals involved in this study ona daily basis

(2) All experiments of the animals were done after induc-ing anesthesia so as to decrease any pain that theanimals might have felt

(3) All animals were euthanized after the experiment wascarried out

(4) After they were introduced in our laboratory settingsall animals were given individual cages and the cageswere cleaned on a very regular basis

21 Choice of Experimental Animal and Grouping Sixty maleC57BL6 mice of SPF grade and aged between 4 and 6 weekswere chosen The mice were obtained from Shanghai Si LaiKe Experimental Animal Limited Liability Company (licensenumber SCXK 2007-0005 Shanghai China) The animalswere bred at Shanghai University of Traditional ChineseMedicineAffiliated LongHuaHospitalrsquos SPF grade laboratoryanimal room We made use of randomized block designto randomly separate the 60 mice into control group (K)model group (M) steroids group (X) Chinese medicine losedose group (D) Chinese medicine moderate dose group (Z)and Chinese medicine high dose group (G) with each grouphaving 10 mice A preexperiment was used by the samegroup of researchers to study the viability of this study beforeengaging in a larger scale study with more mice

22 Main Materials and Reagents FACS flow cytometry wasproducts of BD company (Becton Dickinson and CompanyEast Rutherford New Jersey United States) CD4+CD25+regulatory T cell mice staining kit mainly contains 3types of antibody namely FTTC-anti-mouse-CD4+ APC-anti-mouse-CD25 and PE-Cy5-anti-mouse-Foxp3TheTh17cell staining kit (PE Rat Anti-Mouse IL-17A APC IFN-120574Anti-Mouse FTTC-anti-mouse-CD3e and PerCP Rat Anti-Mouse CD4) was obtained from BD Company The TGF-1205731IL-10 and IL-17 Elisa reagent kit was obtained from ShanghaiAo Biological Company (Shanghai China) the antibodieswere obtained from RampD systems (Minneapolis USA) TheTrizol reagent was bought from Invitrogen Company (Cal-ifornia USA) Foxp3 ROR120574t and the reference GAPDHprimers were synthesized by Shanghai ShineGene Biotech-nology Companies (Shanghai China) cDNA synthesis kitand PCR amplification kit were provided by TaKaRa CoBiological Engineering (Dalian China) protein extractionkit was provided by the Bestbio Biotechnology Companies(Shanghai China) Mouse Anti-Human Foxp3 (2A11GG)polyclonal antibody and Rabbit Anti-Human ROR120574t (H-190)polyclonal antibody were purchased from Santa-Cruz Com-pany (Santa Cruz Biotechnology Inc) Reference antibodyHistoneH31 (Ab-10) was obtained fromSignalWayAntibody(Maryland USA)

23 Experiment Method

231 Drug Intervention and Preparation of AIH Mice Model

Bu Xu Hua Yu Drug Preparation A mouse (20 g) is more orless equivalent to a human adult (approximately 60Kg) Forthe normal daily dosage the following are required for theBu Xu Hua Yu recipe dried Rehmannia roots 005 g Angelica004 g Astragalus 005 g red peony root 005 g Chuanxiong003 g and Sedum sarmentosum 010 g

Dosage Dosage was calculated according to the ldquoEstimatingthe Safe Starting Dose in Clinical Trials for Therapeuticsin Adult Healthy Volunteersrdquo guidelines Low dose groupreceived a dose equivalent to 10 times the normal clinicaldose of the normal adult and one time the dose for micemediumdose group received a dose equivalent to 20 times thenormal clinical dose of a normal adult and 2 times the dosefor mice high dose group received a dose equivalent to 40times the normal clinical dose of a normal adult and 4 timesthe dose for mice steroid control group received prednisone01mg(micesdotday) Mice in the model control group and inthe blank control group were given isovolumetric normalsaline solution 02mLmice

Each group was first given the respective medications for7 days and on the 7th day except for the control group thefive other groups all received an intra-abdominal injection of2 120583g10 g 120572-Galcer (Enzo Life Sciences)

Histological Analysis of the Liver of Mice with AIH The liveris fixed with 10 neutral formalin and embedded in paraffinwax and slicedThe slides were stained with hematoxylin andeosin The degree of inflammation of the liver was graded as

Evidence-Based Complementary and Alternative Medicine 3

follows grade 0 = normal liver tissue grade 1 = mild infil-tration of inflammatory cells with rare hepatic cells necrosisgrade 2 = medium damage of hepatic cells accompanied byinfiltration of inflammatory cells and regional hepatic cellnecrosis grade 3 = wide range inflammatory cells infiltrationin portal area and hepatic lobules accompanied with widerange hepatic cells necrosis

Biochemical Indexes Ten to twelve hours after injection of themodeling agent blood was drawn from the micersquos eyeballwas kept at 4∘C for 2 hours and was centrifuged at 3000 rpmfor 15mins After centrifugation the serum sample was col-lected and was analyzed for alanine aminotransferase (ALT)aspartate aminotransferase (AST) and alkaline phosphatase(AKP) All experiments were done strictly following theinstructions of Nanjing Jian Cheng Ke Ji (Nanjing China)who provided us with the liver function test kit

24 Flow Cytometry Was Used to Detect the Number of TregCells and Th17 Cells in the Hepatic Tissue

241 Treg Cell We used monocytes extracted from lym-phocytes of the liver the cell concentration was set at 1 times107mL A 100 120583L hepatic monocytes suspension was addedto 025 120583L FITC anti-mice CD4 monoclonal antibody 03 120583LAPC-anti-mice CD25 +monoclonal antibody and 25 120583L PE-anti mice Foxp3monoclonal antibody incubation FACS flowcytometry was used to detect the number of Treg cells and thecontent of Foxp3 in liver

242 Th17 Cell We used monocytes extracted from lym-phocytes of the liver the cell concentration was set at 1 times107mLA 100120583Lhepaticmonocytes suspensionwas added toPerCP labeled anti-CD4 CD3e after incubation with brokenmembrane agent intracellular cytokine staining and FITClabeled IL-17A 05 120583g incubation FACS flow cytometry wasused to detect Th17 cytokines IL-17 and IFN-120574 which aresecreted by CD4+ cells

25 ELISA Method for the Detection of Liver Homogenate IL-10 IL-17 and TGF-1205731 Levels The experiment was carried outaccording to the strict instructions of the ELISA kit

26 RT-PCR Was Used to Analyze the Expression of Foxp3mRNA and ROR120574t mRNA (1) Primer synthesis the specificsequence is as follows

Foxp3 Forward Prime 51015840-ACCCAGGAAAGACAG-CAACC-31015840 Reverse Primer 51015840-CTCGAAGACCTT-CTCACAACCA-31015840 length of products 107 bpROR120574t Forward Primer 51015840-CCATTGACCGAA-CCAGCC-31015840 Reverse Primer 51015840-TCTGCTTCTTGG-ACATTCGG-31015840 length of products 111 bpGapdh Forward Primer 51015840-TGTGTCCGTCGT-GGATCTGA-31015840 Reverse Primer 51015840-TCTGCTTCT-TGGACATTCGG-31015840 length of products 77 bp

(2) Extraction of total RNA and RT-PCR procedure 50ndash100mg tissue was collected from each group Trizol kit was

used to extract total RNA after using the spectrophotometerat 260280 nm in the examination of the quality and purityof total RNA Using the cDNA synthesis kit we performedtranscription of mRNA to cDNA We then used cDNAcorresponding specific primers for PCR amplification withthe real-time PCR 20120583L reaction volume includes PremixEx TaqTM (2x) 10 120583L upstream and downstream primers04 120583L Taq Probe 08 120583L Rox Reference Dye (50x) 04120583LcDNA 2120583L and DEPC water 6120583L PCR procedure was doneas follows 95∘C for 30 s after the initial hot-start followed by45 PCR cycles at 95∘C for 5 s annealing temperature of 60∘Cfor 30 s

(3) Extraction of 10 120583L PCR reaction product 10 120583L PCRreaction product was extracted and placed on a 2 agarosegel electrophoresis for 50min and the image acquisition isdone by image analyzer Using glyceraldehyde-3-phosphatedehydrogenase (Gapdh) reference as the benchmark weperformed semiquantitative analysis Real-time PCR resultsare shown as follows 2-ΔΔCt (2-ΔΔCt = RQ) represents thetarget genes relative to GAPDH Higher values for 2-ΔΔCtcorrespond to higher levels of gene expression and smallervalues correspond to lesser levels of gene expression

27 Western Blot Technique Used to Measure the Expressionof Foxp3 and ROR120574t Protein in Hepatic Tissue After 50ndash100mg of hepatic tissue was homogenized tissue proteinswere extracted BCAmethodwas used formeasuring the con-centration of protein levels and for dilution Sodium dodecylsulfate polyacrylamide gel electrophoresis was carried outand the protein was transferred to the PVDF film PonceauS staining was used for the examination of the strip film 1 timesTBST buffer skim milk closed 1 h 1 100 Foxp3 ROR120574t and1 200 reference antibody were separately added respectivelyand were incubated overnight at 4∘C phosphate bufferwas added after washing the membrane with horseradishperoxidase-labeled IgG electrochemiluminescence color wasobtained through X-ray exposure developing and fixing wasdone and Gel-Pro analysis software was used to locate andanalyze the strip

28 Statistical Analysis All data obtained in this study wasanalyzed using the SPSS160 software Data for each groupis represented in the form (119883 plusmn 119878) multisamples groupswere compared with single factor analysis of variance (one-way ANOVA) Skewness distribution was analyzed usingnonparametric test119875 lt 005was considered to be statisticallysignificant 119875 lt 001 was considered to be statistically highlysignificant

3 Results

31 Tissue Histology Examination of AIHMouse Model and ofDifferent Groups after Drug Usage Hepatic tissue of mouseafter hematoxylin-eosin staining shows that after inductionwith 120572-Galcer the C57BL6 mice showed varying degrees ofpathological changes of grade 1 to grade 2 inflammation theportal area and its surroundings showed obvious infiltrationof inflammatory cells when compared to the model group

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Liver function indexes for each group

Group(119899 = 10) ALT (UL) AST (UL) AKP (UL)

K 3032 plusmn 954 309 plusmn 807 3546 plusmn 1216

M 28616 plusmn 5288 25357 plusmn 3146 29002 plusmn 5923X 18687 plusmn 4265 16619 plusmn 3388 1473 plusmn 3825

D 19222 plusmn 4432 17063 plusmn 26 17986 plusmn 3785

Z 18605 plusmn 4079 16560 plusmn 2241 16889 plusmn 2025

G 15699 plusmn 3459 14764 plusmn 2583 13786 plusmn 4592

When compared to the model group 119875 lt 005

steroids group and the Chinese medicine moderate dosegroup showed signs of mild inflammation with remissionof the infiltration of inflammatory cells blank control groupshowed almost normal hepatic tissue as shown in Figure 1

32 Result for Liver Function Indexes Except for the blankcontrol group the liver function indexes were increased bydifferent amounts in all the other groups After treatment ofsteroids group and the different Chinese medicine groupsthe liver function indexes all witnessed decreases whichwere statistically significant when compared with the controlgroup (119875 gt 005) However the difference between thesteroids and Chinese medicine groups and the differencebetween the different Chinese medicine groups were notstatistically significant (119875 gt 005) This shows that theChinese medicine was able to protect the liver against AIHand the results were on par with steroids The results areshown in Table 1 and Figure 2 (Figure 2(a) for ALT levelsFigure 2(b) for AST levels and Figure 2(c) for AKP levels)

33 Analysis of Results for Hepatic Lymph Cell Treg(CD4+CD25+Foxp3+) Cell and Th17 (CD3+CD4+IL-17+IFN-120574minus) Cell Analysis results of FACS showed that theTreg cells levels in the hepatic lymph tissue of the modelgroup were lower than in the control group When eachtreatment group was compared to the model group the levelsof Treg cells were considerably increased and the differencewas statistically significant (119875 lt 005) Comparison ofthe control group and the Chinese medicine high dosegroup to the model group showed a statistically significantdifference (119875 lt 001) The levels of Th17 cells in the modelgroup were higher than in the blank control group and inthe treatment groups and the difference was found to bestatistically significant (119875 lt 005) The TregTh17 ratio wasdecreased in the model group when compared to the blankcontrol group After the use of therapeutic drugs the ratioof TregTh17 was found to be increased When comparedto the model group the steroids group Chinese medicinemoderate dose group and Chinesemedicine high dose groupall had showed increases that were statistically significant(119875 lt 005) However there was no statistical differencebetween the Chinese and steroids groups or betweenthe different Chinese medicine groups Furthermore theChinese medicine treatment group had a dose-dependent

Table 2 Each group hepatic tissues Treg Th17 cell levels ()

Group (119899 = 8) Foxp3(+) Treg TH17 (IL17+IFN120574minus)K 132 plusmn 049 071 plusmn 040

M 053 plusmn 018 163 plusmn 078X 108 plusmn 044 095 plusmn 049

D 100 plusmn 056 107 plusmn 059

Z 102 plusmn 042 084 plusmn 039

G 113 plusmn 058 083 plusmn 039When compared to M 119875 lt 005 when compared to M 119875 lt 001

Table 3 TregTh17 cell ratio for each group

Group (119899 = 8) TregTH17 Mean rank When comparedto M group 119885 ratio

K 277 plusmn 257 3438 337M 040 plusmn 024 838 mdashX 164 plusmn 135 265 295D 141 plusmn 126 2363 190Z 158 plusmn 135 2638 274G 172 plusmn 137 2775 263When compared to M 119875 lt 00083

Table 4 Analysis results for cytokines in hepatic tissues for eachgroup

Group(119899 = 10) TGF-1205731 (pgmL) IL-10 (pgmL) IL-17 (pgmL)

K 17936 plusmn 5357 22447 plusmn 4959 14523 plusmn 5249

M 29382 plusmn 10218 645 plusmn 2025 29871 plusmn 7950X 28179 plusmn 6470 10245 plusmn 2480 19229 plusmn 10756

D 27714 plusmn 6486 7255 plusmn 1873 21995 plusmn 11076

Z 22871 plusmn 8321 9937 plusmn 4163 21342 plusmn 9850

G 25136 plusmn 8511 14168 plusmn 2160 15474 plusmn 6296

When compared with group M 119875 lt 005

trend These results are shown in Figures 3 4 and 5 andTables 2 3 and 4

34 Analysis of Results for Hepatic Tissue Cytokines for EachGroup Analysis of TGF-1205731 in hepatic tissues showed thatin the model group TGF-1205731 was increased when comparedto the control group but there was no statistical differencebetween each treatment group and the model group (119875 gt005) Analysis of IL-10 showed that model group IL-10levels were lower when compared to blank control groupand when compared to the model group IL-10 for eachtreatment group was increased with the difference beingstatistically significant (119875 lt 005) Analysis results of IL-17showed that IL-17 of themodel groupwas considerably higherthan that of the blank control group and of each treatmentgroup The difference was statistically significant (119875 lt 005)Furthermore there was no statistical difference between theChinese medicine and steroids groups or between each ofthe Chinese medicine groups (119875 gt 005) Chinese medicine

Evidence-Based Complementary and Alternative Medicine 5

XK

M G

Figure 1 The figure shows the histological analysis for control group (K) model group (M) steroids group (X) and Chinese medicine highdose group (G) Histological analysis for each group

Table 5 Results for the analysis Foxp3 mRNA for each group (2-ΔΔCT)

Group(119899 = 8)

RQ(2-ΔΔCT)median

RQ(2-ΔΔCT)

Q25

RQ(2-ΔΔCT)

Q75119885 ratio 119875 ratio

K 01887 01167 19460 2731 0006

M 00529 00121 01038 mdash mdash

X 00729 00332 01886 1470 0141

D 00842 00549 01391 1470 0141

Z 01152 00432 04352 1575 0115

G 00642 00181 02824 0840 0401When compared to group M 119875 lt 005

treatment groups showed a dose-dependent trend Results areshown in Table 4 and Figure 6

35 Analysis Results for Liver Specific Transcription FactorsFoxp3 and ROR120574t Real-time RT-PCR analysis showed thatthe difference between each group hepatic tissue transcrip-tion factor Foxp3 mRNAwas not statistically significant (119875 lt005) The model group ROR120574t mRNA was higher than thatof the control group and treatment groups the difference wasstatistically significance (119875 lt 005 119875 lt 001) The results areshown in Figures 7 and 8 and Tables 5 and 6

Table 6 Results for the analysis of ROR120574t mRNA for each group(2-ΔΔCT)

Group (119899 = 8) RQ (2-ΔΔCT) 119875 ratioK 0110 plusmn 0062 001

M 0393 plusmn 0282 mdashX 0201 plusmn 0146 0020

D 0134 plusmn 0075 0002

Z 0161 plusmn 0122 0005

G 0165 plusmn 0085 0006

When compared to group M 119875 lt 005 and 119875 lt 001

36 Results for the Analysis of Hepatic Foxp3 and ROR120574tProteins For each group western blot testing showed thatFoxp3 protein expression in the model group was muchlower than in the control group In all treatment groupsthe expression of the Foxp3 protein was increased whencompared to the model group with the moderate doseChinese medicine group showing a much higher increasewhen compared to the steroids group the difference wasstatistically significant (119875 lt 005) The expression of ROR120574tprotein in the model group was much higher than in thecontrol group For each treatment group the ROR120574t proteinexpressionwas considerably decreasedwhen compared to themodel group and the difference was statistically significant(119875 lt 005) These results are shown in Figures 9 and 10 andTable 7

6 Evidence-Based Complementary and Alternative Medicine

KMX

DZG

0

100

200

300

400A

LT (U

L)

K M X D Z G

lowastlowast

lowastlowast

lowast

(a) ALT between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AST

(UL

)

lowast

(b) AST between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AKP

(UL

)

lowast

lowast

lowast

lowastlowast

(c) AKP between six groups

Figure 2 The figure shows the level of ALT AST and AKP for each of the six groups

Table 7 Foxp3 ROR120574t proteins for each liver group

Group (119899 = 8) ROR120574t protein(IOD ratio)

Foxp3 protein(IOD ratio)

K 097 plusmn 011 168 plusmn 016

M 192 plusmn 015 067 plusmn 005X 136 plusmn 006 084 plusmn 003

D 099 plusmn 013Δ 099 plusmn 051

Z 071 plusmn 007Δamp

106 plusmn 010Δ

G 137 plusmn 002amp 095 plusmn 009When compared with group M 119875 lt 005 and

119875 lt 001 Δwhen comparedwith group X 119875 lt 005 ampwhen compared with group D 119875 lt 001

4 Discussion

Autoimmune hepatitis is a type of autoimmune liver diseasewhich has an undefined etiology and its pathological charac-teristic ismainly interface hepatitis with infiltration of plasmacells in the portal vein which is accompanied by serumhypergammaglobulinemia and positive serum antibodies[5 6] The pathophysiology of AIH is relatively complexwith factors such as genetics autoantigens environmentalfactors and immune dysfunction commonly mentioned asbeing responsible [7] Previous studies on the subject mainlyfocused on liver cell apoptosis expression of inflammatorymediators gene polymorphism and immune system regula-tion [8ndash14] Autoimmune T cells and B cells activation are

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

follows grade 0 = normal liver tissue grade 1 = mild infil-tration of inflammatory cells with rare hepatic cells necrosisgrade 2 = medium damage of hepatic cells accompanied byinfiltration of inflammatory cells and regional hepatic cellnecrosis grade 3 = wide range inflammatory cells infiltrationin portal area and hepatic lobules accompanied with widerange hepatic cells necrosis

Biochemical Indexes Ten to twelve hours after injection of themodeling agent blood was drawn from the micersquos eyeballwas kept at 4∘C for 2 hours and was centrifuged at 3000 rpmfor 15mins After centrifugation the serum sample was col-lected and was analyzed for alanine aminotransferase (ALT)aspartate aminotransferase (AST) and alkaline phosphatase(AKP) All experiments were done strictly following theinstructions of Nanjing Jian Cheng Ke Ji (Nanjing China)who provided us with the liver function test kit

24 Flow Cytometry Was Used to Detect the Number of TregCells and Th17 Cells in the Hepatic Tissue

241 Treg Cell We used monocytes extracted from lym-phocytes of the liver the cell concentration was set at 1 times107mL A 100 120583L hepatic monocytes suspension was addedto 025 120583L FITC anti-mice CD4 monoclonal antibody 03 120583LAPC-anti-mice CD25 +monoclonal antibody and 25 120583L PE-anti mice Foxp3monoclonal antibody incubation FACS flowcytometry was used to detect the number of Treg cells and thecontent of Foxp3 in liver

242 Th17 Cell We used monocytes extracted from lym-phocytes of the liver the cell concentration was set at 1 times107mLA 100120583Lhepaticmonocytes suspensionwas added toPerCP labeled anti-CD4 CD3e after incubation with brokenmembrane agent intracellular cytokine staining and FITClabeled IL-17A 05 120583g incubation FACS flow cytometry wasused to detect Th17 cytokines IL-17 and IFN-120574 which aresecreted by CD4+ cells

25 ELISA Method for the Detection of Liver Homogenate IL-10 IL-17 and TGF-1205731 Levels The experiment was carried outaccording to the strict instructions of the ELISA kit

26 RT-PCR Was Used to Analyze the Expression of Foxp3mRNA and ROR120574t mRNA (1) Primer synthesis the specificsequence is as follows

Foxp3 Forward Prime 51015840-ACCCAGGAAAGACAG-CAACC-31015840 Reverse Primer 51015840-CTCGAAGACCTT-CTCACAACCA-31015840 length of products 107 bpROR120574t Forward Primer 51015840-CCATTGACCGAA-CCAGCC-31015840 Reverse Primer 51015840-TCTGCTTCTTGG-ACATTCGG-31015840 length of products 111 bpGapdh Forward Primer 51015840-TGTGTCCGTCGT-GGATCTGA-31015840 Reverse Primer 51015840-TCTGCTTCT-TGGACATTCGG-31015840 length of products 77 bp

(2) Extraction of total RNA and RT-PCR procedure 50ndash100mg tissue was collected from each group Trizol kit was

used to extract total RNA after using the spectrophotometerat 260280 nm in the examination of the quality and purityof total RNA Using the cDNA synthesis kit we performedtranscription of mRNA to cDNA We then used cDNAcorresponding specific primers for PCR amplification withthe real-time PCR 20120583L reaction volume includes PremixEx TaqTM (2x) 10 120583L upstream and downstream primers04 120583L Taq Probe 08 120583L Rox Reference Dye (50x) 04120583LcDNA 2120583L and DEPC water 6120583L PCR procedure was doneas follows 95∘C for 30 s after the initial hot-start followed by45 PCR cycles at 95∘C for 5 s annealing temperature of 60∘Cfor 30 s

(3) Extraction of 10 120583L PCR reaction product 10 120583L PCRreaction product was extracted and placed on a 2 agarosegel electrophoresis for 50min and the image acquisition isdone by image analyzer Using glyceraldehyde-3-phosphatedehydrogenase (Gapdh) reference as the benchmark weperformed semiquantitative analysis Real-time PCR resultsare shown as follows 2-ΔΔCt (2-ΔΔCt = RQ) represents thetarget genes relative to GAPDH Higher values for 2-ΔΔCtcorrespond to higher levels of gene expression and smallervalues correspond to lesser levels of gene expression

27 Western Blot Technique Used to Measure the Expressionof Foxp3 and ROR120574t Protein in Hepatic Tissue After 50ndash100mg of hepatic tissue was homogenized tissue proteinswere extracted BCAmethodwas used formeasuring the con-centration of protein levels and for dilution Sodium dodecylsulfate polyacrylamide gel electrophoresis was carried outand the protein was transferred to the PVDF film PonceauS staining was used for the examination of the strip film 1 timesTBST buffer skim milk closed 1 h 1 100 Foxp3 ROR120574t and1 200 reference antibody were separately added respectivelyand were incubated overnight at 4∘C phosphate bufferwas added after washing the membrane with horseradishperoxidase-labeled IgG electrochemiluminescence color wasobtained through X-ray exposure developing and fixing wasdone and Gel-Pro analysis software was used to locate andanalyze the strip

28 Statistical Analysis All data obtained in this study wasanalyzed using the SPSS160 software Data for each groupis represented in the form (119883 plusmn 119878) multisamples groupswere compared with single factor analysis of variance (one-way ANOVA) Skewness distribution was analyzed usingnonparametric test119875 lt 005was considered to be statisticallysignificant 119875 lt 001 was considered to be statistically highlysignificant

3 Results

31 Tissue Histology Examination of AIHMouse Model and ofDifferent Groups after Drug Usage Hepatic tissue of mouseafter hematoxylin-eosin staining shows that after inductionwith 120572-Galcer the C57BL6 mice showed varying degrees ofpathological changes of grade 1 to grade 2 inflammation theportal area and its surroundings showed obvious infiltrationof inflammatory cells when compared to the model group

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Liver function indexes for each group

Group(119899 = 10) ALT (UL) AST (UL) AKP (UL)

K 3032 plusmn 954 309 plusmn 807 3546 plusmn 1216

M 28616 plusmn 5288 25357 plusmn 3146 29002 plusmn 5923X 18687 plusmn 4265 16619 plusmn 3388 1473 plusmn 3825

D 19222 plusmn 4432 17063 plusmn 26 17986 plusmn 3785

Z 18605 plusmn 4079 16560 plusmn 2241 16889 plusmn 2025

G 15699 plusmn 3459 14764 plusmn 2583 13786 plusmn 4592

When compared to the model group 119875 lt 005

steroids group and the Chinese medicine moderate dosegroup showed signs of mild inflammation with remissionof the infiltration of inflammatory cells blank control groupshowed almost normal hepatic tissue as shown in Figure 1

32 Result for Liver Function Indexes Except for the blankcontrol group the liver function indexes were increased bydifferent amounts in all the other groups After treatment ofsteroids group and the different Chinese medicine groupsthe liver function indexes all witnessed decreases whichwere statistically significant when compared with the controlgroup (119875 gt 005) However the difference between thesteroids and Chinese medicine groups and the differencebetween the different Chinese medicine groups were notstatistically significant (119875 gt 005) This shows that theChinese medicine was able to protect the liver against AIHand the results were on par with steroids The results areshown in Table 1 and Figure 2 (Figure 2(a) for ALT levelsFigure 2(b) for AST levels and Figure 2(c) for AKP levels)

33 Analysis of Results for Hepatic Lymph Cell Treg(CD4+CD25+Foxp3+) Cell and Th17 (CD3+CD4+IL-17+IFN-120574minus) Cell Analysis results of FACS showed that theTreg cells levels in the hepatic lymph tissue of the modelgroup were lower than in the control group When eachtreatment group was compared to the model group the levelsof Treg cells were considerably increased and the differencewas statistically significant (119875 lt 005) Comparison ofthe control group and the Chinese medicine high dosegroup to the model group showed a statistically significantdifference (119875 lt 001) The levels of Th17 cells in the modelgroup were higher than in the blank control group and inthe treatment groups and the difference was found to bestatistically significant (119875 lt 005) The TregTh17 ratio wasdecreased in the model group when compared to the blankcontrol group After the use of therapeutic drugs the ratioof TregTh17 was found to be increased When comparedto the model group the steroids group Chinese medicinemoderate dose group and Chinesemedicine high dose groupall had showed increases that were statistically significant(119875 lt 005) However there was no statistical differencebetween the Chinese and steroids groups or betweenthe different Chinese medicine groups Furthermore theChinese medicine treatment group had a dose-dependent

Table 2 Each group hepatic tissues Treg Th17 cell levels ()

Group (119899 = 8) Foxp3(+) Treg TH17 (IL17+IFN120574minus)K 132 plusmn 049 071 plusmn 040

M 053 plusmn 018 163 plusmn 078X 108 plusmn 044 095 plusmn 049

D 100 plusmn 056 107 plusmn 059

Z 102 plusmn 042 084 plusmn 039

G 113 plusmn 058 083 plusmn 039When compared to M 119875 lt 005 when compared to M 119875 lt 001

Table 3 TregTh17 cell ratio for each group

Group (119899 = 8) TregTH17 Mean rank When comparedto M group 119885 ratio

K 277 plusmn 257 3438 337M 040 plusmn 024 838 mdashX 164 plusmn 135 265 295D 141 plusmn 126 2363 190Z 158 plusmn 135 2638 274G 172 plusmn 137 2775 263When compared to M 119875 lt 00083

Table 4 Analysis results for cytokines in hepatic tissues for eachgroup

Group(119899 = 10) TGF-1205731 (pgmL) IL-10 (pgmL) IL-17 (pgmL)

K 17936 plusmn 5357 22447 plusmn 4959 14523 plusmn 5249

M 29382 plusmn 10218 645 plusmn 2025 29871 plusmn 7950X 28179 plusmn 6470 10245 plusmn 2480 19229 plusmn 10756

D 27714 plusmn 6486 7255 plusmn 1873 21995 plusmn 11076

Z 22871 plusmn 8321 9937 plusmn 4163 21342 plusmn 9850

G 25136 plusmn 8511 14168 plusmn 2160 15474 plusmn 6296

When compared with group M 119875 lt 005

trend These results are shown in Figures 3 4 and 5 andTables 2 3 and 4

34 Analysis of Results for Hepatic Tissue Cytokines for EachGroup Analysis of TGF-1205731 in hepatic tissues showed thatin the model group TGF-1205731 was increased when comparedto the control group but there was no statistical differencebetween each treatment group and the model group (119875 gt005) Analysis of IL-10 showed that model group IL-10levels were lower when compared to blank control groupand when compared to the model group IL-10 for eachtreatment group was increased with the difference beingstatistically significant (119875 lt 005) Analysis results of IL-17showed that IL-17 of themodel groupwas considerably higherthan that of the blank control group and of each treatmentgroup The difference was statistically significant (119875 lt 005)Furthermore there was no statistical difference between theChinese medicine and steroids groups or between each ofthe Chinese medicine groups (119875 gt 005) Chinese medicine

Evidence-Based Complementary and Alternative Medicine 5

XK

M G

Figure 1 The figure shows the histological analysis for control group (K) model group (M) steroids group (X) and Chinese medicine highdose group (G) Histological analysis for each group

Table 5 Results for the analysis Foxp3 mRNA for each group (2-ΔΔCT)

Group(119899 = 8)

RQ(2-ΔΔCT)median

RQ(2-ΔΔCT)

Q25

RQ(2-ΔΔCT)

Q75119885 ratio 119875 ratio

K 01887 01167 19460 2731 0006

M 00529 00121 01038 mdash mdash

X 00729 00332 01886 1470 0141

D 00842 00549 01391 1470 0141

Z 01152 00432 04352 1575 0115

G 00642 00181 02824 0840 0401When compared to group M 119875 lt 005

treatment groups showed a dose-dependent trend Results areshown in Table 4 and Figure 6

35 Analysis Results for Liver Specific Transcription FactorsFoxp3 and ROR120574t Real-time RT-PCR analysis showed thatthe difference between each group hepatic tissue transcrip-tion factor Foxp3 mRNAwas not statistically significant (119875 lt005) The model group ROR120574t mRNA was higher than thatof the control group and treatment groups the difference wasstatistically significance (119875 lt 005 119875 lt 001) The results areshown in Figures 7 and 8 and Tables 5 and 6

Table 6 Results for the analysis of ROR120574t mRNA for each group(2-ΔΔCT)

Group (119899 = 8) RQ (2-ΔΔCT) 119875 ratioK 0110 plusmn 0062 001

M 0393 plusmn 0282 mdashX 0201 plusmn 0146 0020

D 0134 plusmn 0075 0002

Z 0161 plusmn 0122 0005

G 0165 plusmn 0085 0006

When compared to group M 119875 lt 005 and 119875 lt 001

36 Results for the Analysis of Hepatic Foxp3 and ROR120574tProteins For each group western blot testing showed thatFoxp3 protein expression in the model group was muchlower than in the control group In all treatment groupsthe expression of the Foxp3 protein was increased whencompared to the model group with the moderate doseChinese medicine group showing a much higher increasewhen compared to the steroids group the difference wasstatistically significant (119875 lt 005) The expression of ROR120574tprotein in the model group was much higher than in thecontrol group For each treatment group the ROR120574t proteinexpressionwas considerably decreasedwhen compared to themodel group and the difference was statistically significant(119875 lt 005) These results are shown in Figures 9 and 10 andTable 7

6 Evidence-Based Complementary and Alternative Medicine

KMX

DZG

0

100

200

300

400A

LT (U

L)

K M X D Z G

lowastlowast

lowastlowast

lowast

(a) ALT between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AST

(UL

)

lowast

(b) AST between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AKP

(UL

)

lowast

lowast

lowast

lowastlowast

(c) AKP between six groups

Figure 2 The figure shows the level of ALT AST and AKP for each of the six groups

Table 7 Foxp3 ROR120574t proteins for each liver group

Group (119899 = 8) ROR120574t protein(IOD ratio)

Foxp3 protein(IOD ratio)

K 097 plusmn 011 168 plusmn 016

M 192 plusmn 015 067 plusmn 005X 136 plusmn 006 084 plusmn 003

D 099 plusmn 013Δ 099 plusmn 051

Z 071 plusmn 007Δamp

106 plusmn 010Δ

G 137 plusmn 002amp 095 plusmn 009When compared with group M 119875 lt 005 and

119875 lt 001 Δwhen comparedwith group X 119875 lt 005 ampwhen compared with group D 119875 lt 001

4 Discussion

Autoimmune hepatitis is a type of autoimmune liver diseasewhich has an undefined etiology and its pathological charac-teristic ismainly interface hepatitis with infiltration of plasmacells in the portal vein which is accompanied by serumhypergammaglobulinemia and positive serum antibodies[5 6] The pathophysiology of AIH is relatively complexwith factors such as genetics autoantigens environmentalfactors and immune dysfunction commonly mentioned asbeing responsible [7] Previous studies on the subject mainlyfocused on liver cell apoptosis expression of inflammatorymediators gene polymorphism and immune system regula-tion [8ndash14] Autoimmune T cells and B cells activation are

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Liver function indexes for each group

Group(119899 = 10) ALT (UL) AST (UL) AKP (UL)

K 3032 plusmn 954 309 plusmn 807 3546 plusmn 1216

M 28616 plusmn 5288 25357 plusmn 3146 29002 plusmn 5923X 18687 plusmn 4265 16619 plusmn 3388 1473 plusmn 3825

D 19222 plusmn 4432 17063 plusmn 26 17986 plusmn 3785

Z 18605 plusmn 4079 16560 plusmn 2241 16889 plusmn 2025

G 15699 plusmn 3459 14764 plusmn 2583 13786 plusmn 4592

When compared to the model group 119875 lt 005

steroids group and the Chinese medicine moderate dosegroup showed signs of mild inflammation with remissionof the infiltration of inflammatory cells blank control groupshowed almost normal hepatic tissue as shown in Figure 1

32 Result for Liver Function Indexes Except for the blankcontrol group the liver function indexes were increased bydifferent amounts in all the other groups After treatment ofsteroids group and the different Chinese medicine groupsthe liver function indexes all witnessed decreases whichwere statistically significant when compared with the controlgroup (119875 gt 005) However the difference between thesteroids and Chinese medicine groups and the differencebetween the different Chinese medicine groups were notstatistically significant (119875 gt 005) This shows that theChinese medicine was able to protect the liver against AIHand the results were on par with steroids The results areshown in Table 1 and Figure 2 (Figure 2(a) for ALT levelsFigure 2(b) for AST levels and Figure 2(c) for AKP levels)

33 Analysis of Results for Hepatic Lymph Cell Treg(CD4+CD25+Foxp3+) Cell and Th17 (CD3+CD4+IL-17+IFN-120574minus) Cell Analysis results of FACS showed that theTreg cells levels in the hepatic lymph tissue of the modelgroup were lower than in the control group When eachtreatment group was compared to the model group the levelsof Treg cells were considerably increased and the differencewas statistically significant (119875 lt 005) Comparison ofthe control group and the Chinese medicine high dosegroup to the model group showed a statistically significantdifference (119875 lt 001) The levels of Th17 cells in the modelgroup were higher than in the blank control group and inthe treatment groups and the difference was found to bestatistically significant (119875 lt 005) The TregTh17 ratio wasdecreased in the model group when compared to the blankcontrol group After the use of therapeutic drugs the ratioof TregTh17 was found to be increased When comparedto the model group the steroids group Chinese medicinemoderate dose group and Chinesemedicine high dose groupall had showed increases that were statistically significant(119875 lt 005) However there was no statistical differencebetween the Chinese and steroids groups or betweenthe different Chinese medicine groups Furthermore theChinese medicine treatment group had a dose-dependent

Table 2 Each group hepatic tissues Treg Th17 cell levels ()

Group (119899 = 8) Foxp3(+) Treg TH17 (IL17+IFN120574minus)K 132 plusmn 049 071 plusmn 040

M 053 plusmn 018 163 plusmn 078X 108 plusmn 044 095 plusmn 049

D 100 plusmn 056 107 plusmn 059

Z 102 plusmn 042 084 plusmn 039

G 113 plusmn 058 083 plusmn 039When compared to M 119875 lt 005 when compared to M 119875 lt 001

Table 3 TregTh17 cell ratio for each group

Group (119899 = 8) TregTH17 Mean rank When comparedto M group 119885 ratio

K 277 plusmn 257 3438 337M 040 plusmn 024 838 mdashX 164 plusmn 135 265 295D 141 plusmn 126 2363 190Z 158 plusmn 135 2638 274G 172 plusmn 137 2775 263When compared to M 119875 lt 00083

Table 4 Analysis results for cytokines in hepatic tissues for eachgroup

Group(119899 = 10) TGF-1205731 (pgmL) IL-10 (pgmL) IL-17 (pgmL)

K 17936 plusmn 5357 22447 plusmn 4959 14523 plusmn 5249

M 29382 plusmn 10218 645 plusmn 2025 29871 plusmn 7950X 28179 plusmn 6470 10245 plusmn 2480 19229 plusmn 10756

D 27714 plusmn 6486 7255 plusmn 1873 21995 plusmn 11076

Z 22871 plusmn 8321 9937 plusmn 4163 21342 plusmn 9850

G 25136 plusmn 8511 14168 plusmn 2160 15474 plusmn 6296

When compared with group M 119875 lt 005

trend These results are shown in Figures 3 4 and 5 andTables 2 3 and 4

34 Analysis of Results for Hepatic Tissue Cytokines for EachGroup Analysis of TGF-1205731 in hepatic tissues showed thatin the model group TGF-1205731 was increased when comparedto the control group but there was no statistical differencebetween each treatment group and the model group (119875 gt005) Analysis of IL-10 showed that model group IL-10levels were lower when compared to blank control groupand when compared to the model group IL-10 for eachtreatment group was increased with the difference beingstatistically significant (119875 lt 005) Analysis results of IL-17showed that IL-17 of themodel groupwas considerably higherthan that of the blank control group and of each treatmentgroup The difference was statistically significant (119875 lt 005)Furthermore there was no statistical difference between theChinese medicine and steroids groups or between each ofthe Chinese medicine groups (119875 gt 005) Chinese medicine

Evidence-Based Complementary and Alternative Medicine 5

XK

M G

Figure 1 The figure shows the histological analysis for control group (K) model group (M) steroids group (X) and Chinese medicine highdose group (G) Histological analysis for each group

Table 5 Results for the analysis Foxp3 mRNA for each group (2-ΔΔCT)

Group(119899 = 8)

RQ(2-ΔΔCT)median

RQ(2-ΔΔCT)

Q25

RQ(2-ΔΔCT)

Q75119885 ratio 119875 ratio

K 01887 01167 19460 2731 0006

M 00529 00121 01038 mdash mdash

X 00729 00332 01886 1470 0141

D 00842 00549 01391 1470 0141

Z 01152 00432 04352 1575 0115

G 00642 00181 02824 0840 0401When compared to group M 119875 lt 005

treatment groups showed a dose-dependent trend Results areshown in Table 4 and Figure 6

35 Analysis Results for Liver Specific Transcription FactorsFoxp3 and ROR120574t Real-time RT-PCR analysis showed thatthe difference between each group hepatic tissue transcrip-tion factor Foxp3 mRNAwas not statistically significant (119875 lt005) The model group ROR120574t mRNA was higher than thatof the control group and treatment groups the difference wasstatistically significance (119875 lt 005 119875 lt 001) The results areshown in Figures 7 and 8 and Tables 5 and 6

Table 6 Results for the analysis of ROR120574t mRNA for each group(2-ΔΔCT)

Group (119899 = 8) RQ (2-ΔΔCT) 119875 ratioK 0110 plusmn 0062 001

M 0393 plusmn 0282 mdashX 0201 plusmn 0146 0020

D 0134 plusmn 0075 0002

Z 0161 plusmn 0122 0005

G 0165 plusmn 0085 0006

When compared to group M 119875 lt 005 and 119875 lt 001

36 Results for the Analysis of Hepatic Foxp3 and ROR120574tProteins For each group western blot testing showed thatFoxp3 protein expression in the model group was muchlower than in the control group In all treatment groupsthe expression of the Foxp3 protein was increased whencompared to the model group with the moderate doseChinese medicine group showing a much higher increasewhen compared to the steroids group the difference wasstatistically significant (119875 lt 005) The expression of ROR120574tprotein in the model group was much higher than in thecontrol group For each treatment group the ROR120574t proteinexpressionwas considerably decreasedwhen compared to themodel group and the difference was statistically significant(119875 lt 005) These results are shown in Figures 9 and 10 andTable 7

6 Evidence-Based Complementary and Alternative Medicine

KMX

DZG

0

100

200

300

400A

LT (U

L)

K M X D Z G

lowastlowast

lowastlowast

lowast

(a) ALT between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AST

(UL

)

lowast

(b) AST between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AKP

(UL

)

lowast

lowast

lowast

lowastlowast

(c) AKP between six groups

Figure 2 The figure shows the level of ALT AST and AKP for each of the six groups

Table 7 Foxp3 ROR120574t proteins for each liver group

Group (119899 = 8) ROR120574t protein(IOD ratio)

Foxp3 protein(IOD ratio)

K 097 plusmn 011 168 plusmn 016

M 192 plusmn 015 067 plusmn 005X 136 plusmn 006 084 plusmn 003

D 099 plusmn 013Δ 099 plusmn 051

Z 071 plusmn 007Δamp

106 plusmn 010Δ

G 137 plusmn 002amp 095 plusmn 009When compared with group M 119875 lt 005 and

119875 lt 001 Δwhen comparedwith group X 119875 lt 005 ampwhen compared with group D 119875 lt 001

4 Discussion

Autoimmune hepatitis is a type of autoimmune liver diseasewhich has an undefined etiology and its pathological charac-teristic ismainly interface hepatitis with infiltration of plasmacells in the portal vein which is accompanied by serumhypergammaglobulinemia and positive serum antibodies[5 6] The pathophysiology of AIH is relatively complexwith factors such as genetics autoantigens environmentalfactors and immune dysfunction commonly mentioned asbeing responsible [7] Previous studies on the subject mainlyfocused on liver cell apoptosis expression of inflammatorymediators gene polymorphism and immune system regula-tion [8ndash14] Autoimmune T cells and B cells activation are

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

XK

M G

Figure 1 The figure shows the histological analysis for control group (K) model group (M) steroids group (X) and Chinese medicine highdose group (G) Histological analysis for each group

Table 5 Results for the analysis Foxp3 mRNA for each group (2-ΔΔCT)

Group(119899 = 8)

RQ(2-ΔΔCT)median

RQ(2-ΔΔCT)

Q25

RQ(2-ΔΔCT)

Q75119885 ratio 119875 ratio

K 01887 01167 19460 2731 0006

M 00529 00121 01038 mdash mdash

X 00729 00332 01886 1470 0141

D 00842 00549 01391 1470 0141

Z 01152 00432 04352 1575 0115

G 00642 00181 02824 0840 0401When compared to group M 119875 lt 005

treatment groups showed a dose-dependent trend Results areshown in Table 4 and Figure 6

35 Analysis Results for Liver Specific Transcription FactorsFoxp3 and ROR120574t Real-time RT-PCR analysis showed thatthe difference between each group hepatic tissue transcrip-tion factor Foxp3 mRNAwas not statistically significant (119875 lt005) The model group ROR120574t mRNA was higher than thatof the control group and treatment groups the difference wasstatistically significance (119875 lt 005 119875 lt 001) The results areshown in Figures 7 and 8 and Tables 5 and 6

Table 6 Results for the analysis of ROR120574t mRNA for each group(2-ΔΔCT)

Group (119899 = 8) RQ (2-ΔΔCT) 119875 ratioK 0110 plusmn 0062 001

M 0393 plusmn 0282 mdashX 0201 plusmn 0146 0020

D 0134 plusmn 0075 0002

Z 0161 plusmn 0122 0005

G 0165 plusmn 0085 0006

When compared to group M 119875 lt 005 and 119875 lt 001

36 Results for the Analysis of Hepatic Foxp3 and ROR120574tProteins For each group western blot testing showed thatFoxp3 protein expression in the model group was muchlower than in the control group In all treatment groupsthe expression of the Foxp3 protein was increased whencompared to the model group with the moderate doseChinese medicine group showing a much higher increasewhen compared to the steroids group the difference wasstatistically significant (119875 lt 005) The expression of ROR120574tprotein in the model group was much higher than in thecontrol group For each treatment group the ROR120574t proteinexpressionwas considerably decreasedwhen compared to themodel group and the difference was statistically significant(119875 lt 005) These results are shown in Figures 9 and 10 andTable 7

6 Evidence-Based Complementary and Alternative Medicine

KMX

DZG

0

100

200

300

400A

LT (U

L)

K M X D Z G

lowastlowast

lowastlowast

lowast

(a) ALT between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AST

(UL

)

lowast

(b) AST between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AKP

(UL

)

lowast

lowast

lowast

lowastlowast

(c) AKP between six groups

Figure 2 The figure shows the level of ALT AST and AKP for each of the six groups

Table 7 Foxp3 ROR120574t proteins for each liver group

Group (119899 = 8) ROR120574t protein(IOD ratio)

Foxp3 protein(IOD ratio)

K 097 plusmn 011 168 plusmn 016

M 192 plusmn 015 067 plusmn 005X 136 plusmn 006 084 plusmn 003

D 099 plusmn 013Δ 099 plusmn 051

Z 071 plusmn 007Δamp

106 plusmn 010Δ

G 137 plusmn 002amp 095 plusmn 009When compared with group M 119875 lt 005 and

119875 lt 001 Δwhen comparedwith group X 119875 lt 005 ampwhen compared with group D 119875 lt 001

4 Discussion

Autoimmune hepatitis is a type of autoimmune liver diseasewhich has an undefined etiology and its pathological charac-teristic ismainly interface hepatitis with infiltration of plasmacells in the portal vein which is accompanied by serumhypergammaglobulinemia and positive serum antibodies[5 6] The pathophysiology of AIH is relatively complexwith factors such as genetics autoantigens environmentalfactors and immune dysfunction commonly mentioned asbeing responsible [7] Previous studies on the subject mainlyfocused on liver cell apoptosis expression of inflammatorymediators gene polymorphism and immune system regula-tion [8ndash14] Autoimmune T cells and B cells activation are

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

KMX

DZG

0

100

200

300

400A

LT (U

L)

K M X D Z G

lowastlowast

lowastlowast

lowast

(a) ALT between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AST

(UL

)

lowast

(b) AST between six groups

KMX

DZG

K M X D Z G0

100

200

300

400

AKP

(UL

)

lowast

lowast

lowast

lowastlowast

(c) AKP between six groups

Figure 2 The figure shows the level of ALT AST and AKP for each of the six groups

Table 7 Foxp3 ROR120574t proteins for each liver group

Group (119899 = 8) ROR120574t protein(IOD ratio)

Foxp3 protein(IOD ratio)

K 097 plusmn 011 168 plusmn 016

M 192 plusmn 015 067 plusmn 005X 136 plusmn 006 084 plusmn 003

D 099 plusmn 013Δ 099 plusmn 051

Z 071 plusmn 007Δamp

106 plusmn 010Δ

G 137 plusmn 002amp 095 plusmn 009When compared with group M 119875 lt 005 and

119875 lt 001 Δwhen comparedwith group X 119875 lt 005 ampwhen compared with group D 119875 lt 001

4 Discussion

Autoimmune hepatitis is a type of autoimmune liver diseasewhich has an undefined etiology and its pathological charac-teristic ismainly interface hepatitis with infiltration of plasmacells in the portal vein which is accompanied by serumhypergammaglobulinemia and positive serum antibodies[5 6] The pathophysiology of AIH is relatively complexwith factors such as genetics autoantigens environmentalfactors and immune dysfunction commonly mentioned asbeing responsible [7] Previous studies on the subject mainlyfocused on liver cell apoptosis expression of inflammatorymediators gene polymorphism and immune system regula-tion [8ndash14] Autoimmune T cells and B cells activation are

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

CD25

(PeC

Y5)

CD4 (PeCY5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

CD25

(PeC

Y5)

100

101

102

103

104

100 101 102 103 104

096 130163

KCD4 (PeCY5)

100 101 102 103 104

MCD4 (PeCY5)

100 101 102 103 104

X

CD25

(APC

)

100

101

102

103

104

100101102103104105

CD4 (PeCY5)

100 101 102 103 104

D

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

Z

CD25

(APC

)

100

101

102

103

104

CD4 (PeCY5)

100 101 102 103 104

G

119 140 321

Cou

nts

100101102103104105106

Cou

nts

100

102

103

104

105C

ount

s

Foxp3 (PE)

8075 6152 8043

100 101 102 103 104

K

100101102103104105

Cou

nts

Foxp3 (PE)

100 101 102 103 104

D

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

Z

100101102103104105106

Cou

nts

Foxp3 (PE)

100 101 102 103 104

G

Foxp3 (PE)

100 101 102 103 104

MFoxp3 (PE)

100 101 102 103 104

X

8715 7655 3780

Figure 3 Treg (CD4+CD25+Foxp3+) FACS hepatic tissue for each group

important steps in the pathophysiology of AIH while thedysfunction of the immunological regulation of the liver hasalso been shown to be a very important step in themechanismof the disease [15] Increasingly studies are centering theirinterest on the Treg cell and theTh17 cell and the relationshipwhich exists between these two cells It is currently thoughtthat loss of balance between these two cells can lead to theinduction of the mechanism of abnormal immunity seen inAIH

120572-Galcer is able to specifically activate NKT cells pro-ducing several different types of inflammatory cells acti-vate CD4+ cells and induce autoimmune hepatitis Afterthe animal model of the disease was successfully createdpathological analysis showed that the AIHmouse model pre-sented with damaged hepatic cells combined with infiltratedinflammatory cells and regional hepatic cell necrosis Theliver function test indexes ALTAST andAKP for each groupwere increased by different degrees and when comparing the

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

CD4

(Per

CP)

CD3 (FITC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

1829

1862 2795 1987

2237 1669

K

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

Cou

nts

100

101

102

103

104

100 101 102 103 104

D

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

CD4

(Per

CP)

100

101

102

103

104

100 101 102 103 104

D

CD3 (FITC)

100 101 102 103 104

ZCD3 (FITC)

100 101 102 103 104

G

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

M

CD4

(Per

CP)

CD3 (FITC)

100

101

102

103

104

100 101 102 103 104

X

429

595 270 378

627 521

IFN120574 (APC)

100 101 102 103 104

DIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

ZIFN120574 (APC)

Cou

nts

100

101

102

103

104

100 101 102 103 104

GIFN120574 (APC)

100 101 102 103 104

ZIFN120574 (APC)

100 101 102 103 104

GIFN120574 (APC)

Figure 4 Th17 (CD3+CD4+IL-17+IFN-120574minus) FACS hepatic tissue for each group

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

0

05

1

15

2

K M X D Z G

Treg

live

r (

)

lowast

lowast

lowast

KMX

ZG

D

(a) Treg () between six groups

0

1

2

3

Th17

liver

()

lowast

lowast

lowast

K M X D Z G

KMX

ZG

D

(b) Th17 () between six groups

K M X D Z G

Treg

Th17

cell

ratio

75

60

45

30

15

0

lowast

lowast

lowast

(c) TregTh17 between six groups

Figure 5 The percentage of Th17 cells and Treg cells and the ratio of the two types of cells

control group to the steroids group the difference was statis-tically significant (119875 lt 005) Pathological and biochemicalindexes showed that our model was able to replicate AIHAfter intervention with steroids or Bu Xu Hua Yu treatmenteach drug intervention groupwitnessed a significant decreasein the levels of ALT AST and AKP when compared to theAIH model group (119875 lt 005) Histological studies of hepatictissues showed different degrees of amelioration which goesto show that Bu Xu Hua Yu Chinese medicine method is ableto provide good protection to the liver

Treg Cell (Regulatory T Cell) and AIH Studies have shownthat Treg cells are influenced by IL-10 and TGF-1205731 duringtheir differentiation of which the forkheadwinged helixtranscription factor Foxp3 is the specific transcription factorof the Treg cell and being themain factor of development and

cell function effect it is specifically presented on the Treg cell[16] Treg cells by way of direct contact inhibit CD8+ T lymphcells proliferationTheir abnormal presentation is a commonfeature of several autoimmune diseases for example systemiclupus erythematosus rheumatoid arthritis or multiple scle-rosis [17ndash19] Furthermore these cells also play a role in thepathophysiology of hepatitis B hepatitis C and primary hep-atic cancer [20ndash22] The newest developments on the studyof adult type I AIH have shown that CD4+CD25+Treg cellsand NKT cells are considerably decreased when comparedto healthy subjects and their products such as IFN-120574 andIL-4 are also considerably reduced This phenomenon hasalso been observed in infants [23] In this study the Tregcell count IL-10 cytokine levels and expression of specifictranscription factor Foxp3 protein in the model group weredecreased when compared to other groups The expression

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

0

100

200

300

400

500TG

F-1205731

(pg

mL)

K M X D Z G

KMX

ZG

D

(a) TGF-1205731 between six groups

0

100

200

300

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

(b) IL-10 between six groups

0

100

200

300

400

IL-1

0 (p

gm

L)

K M X D Z G

KMX

ZG

D

lowast

(c) IL-17 between six groups

Figure 6 Analysis of hepatic tissue cytokines

K M X D Z G K M X D Z G

Foxp3

Gapdh

ROR120574t

Gapdh

Figure 7 Imaging of Foxp3 ROR120574t mRNA gel electrophoresis

of TGF-1205731 was elevated in the model group when comparedto other groups however the difference was not statisticallysignificant This could mean that Bu Xu Hua Yu method isable to positively regulate the secretion of IL-10 and Foxp3and therefore promote the proliferation of Treg cells

Th17 Cell and AIH Th17 cells following the synergisticaction of TGF-1205731 IL-6 or IL-21 are differentiated from the

natural precursor T cells [24] are regulated by specific tran-scription factor ROR120574t [25] and produce the characteristiccytokine interleukin IL-17 These cytokines are involved inhost defense inflammatory diseases cancer and transplantrejection reactions Several studies have reported on the roleof Th17 cells and IL-17 in the mechanism of other autoim-mune diseases Th17 cells have been shown to be increasedin patients of chronic hepatitis B when compared to healthy

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

Amplification plot Amplification plot Amplification plot

Cycle CycleFoxp3 amplification curve

10

2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 2 6 10 14 18 22 26 30 34 38 42 46 50

01

001

0001

00001

000001

0000001

Gapdh amplification curveCycle

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

10

01

001

0001

00001

000001

0000001

ΔRn

1 1 1

ROR120574t amplification curve

Figure 8 Foxp3 ROR120574t mRNA real-time PCR images for each group

K M X D Z G K M X D Z G

Foxp3

Histone31 Histone31

ROR120574t

Figure 9 The protein expression of Foxp3 and ROR120574t for each hepatic tissue group

people which goes to show that after infection with HBVTh17 has a prominent role after immunological activation[26] Furthermore in the mouse model for viral hepatitishigh levels of Th17 cells have also been shown to be present[27] Collagen-induced arthritis experimental autoimmuneencephalomyelitis rheumatoid arthritis inflammatory boweldisease and systemic lupus erythematosus also show highlevels of IL-17 [28ndash30] Some researches have discovered thatin Con A-induced AIH animal models the expression of IL-17a is considerably increased [8] and is mainly concentratedin lymphatic organs or in liver with lymphocytes infiltrationIn our study the Th17 cell count IL-17 cytokine levelsexpression of specific transcription factor ROR120574t gene andproteinwere all increased in themodel groupwhen comparedto the control group (119875 lt 005) and decreased after the use ofmedication

The balance between Treg cells and Th17 cells is nowknown as being the paradigm of the autoimmune field[31] Even though the exact relation between the loss ofbalance of the TregTh17 ratio in the mechanism of AIHhas as of now not been clearly determined however thisphenomenon has also been shown to occur in other diseasessuch as acute coronary syndrome myasthenia gravis non-Hodgkinrsquos lymphoma or arthritis [32ndash35] On the other handLonghi et al have already showed that the inhibition ofIL-17 can increase the immunosuppressive activity of Tregcells and thus the promotion of CD25-cells (ngTreg cells)differentiation while Zhao et al have shown that IL-17 actson the expression of hepatic IL-6 which has a direct role inthe cause of AIH and according to the same study there exists

an opposite relationship between growth differentiation andfunction ofTh17 cells andTreg cells [36] According to Longhiet al [37] transforming growth factor-120573 is an essential factorin the activation of the differentiation ofTh17 cells HoweverFantini et al have underlined that TGF-120573 and IL-6 are theessential factors for the activation of the differentiation ofTh17 cells [38]

This study mainly focuses on the role of the levels of Tregcell and Th17 cell and their function in the pathogenesis ofAIH and the loss of balance in the ratio of these two cells inthe pathophysiology of AIH The ratio of Treg cell to Th17cell was studied and we found that in the control group theratio was 277 and in themodel group it was 040 After use ofmedication the ratio increased When the model group wascompared separately to the control group and to the steroidsgroup the difference was statistically significant (119875 lt 005)Therefore it can be assumed from this study that a loss ofbalance of the TregTh17 cell ratio occurs in AIH and that thelevel of these two cells and the change in their function hasa role to play in the mechanism of AIH The Bu Xu Hua Yumethod can be said to have the same immune suppressionrole as prednisone

Bu Xu Hua Yu Method and AIH As of now the main treat-ment for AIH is the use of immunosuppressive medicationswhich is mainly prednisone or low dose of prednisone com-bined with azathioprine In addition to this other immuno-suppressants that can be used include cyclosporine A aza-thioprine and KF506 however these treatment methods donot usually provide the best results because of recurrence side

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

0

05

1

15

2

Foxp

3 pr

otei

n in

live

r

K M X D Z G

lowast

KMX

ZG

D

Δ

(a) The expression of Foxp3 protein between six groups

0

05

1

15

2

25

K M X D Z G

ROR120574

t pro

tein

in li

ver

KMX

ZG

D

Δ

Δ Δamp

amp

lowast lowastlowast

lowastlowast

(b) The expression of RoR120574t protein between six groups

Figure 10 Results for the analysis of hepatic Foxp3 and ROR120574t proteins

effects or incomplete response Use of western medicine inthe treatment of AIH can rarely attain the desired outcome[2]

In our study the Chinese medicine group was dividedinto three groups which were low dose moderate dose andhigh dose and we mainly observed the effect of the use ofmedication on the histology liver function test includingTreg cell and Th17 cell count and function The results ofthis study have shown that the Bu Xu Hua Yu treatment canpositively ameliorate AIH in animal models with decrease inthe levels of serumALT AST and AKP causing an alleviationof hepatic inflammation The results were on par with thoseobtained with steroids Different doses of this treatment canincrease the expression of IL-10 decrease the expression ofIL-17 and regulate the expression of TGF-120573 which causes thelevels of Treg cells to increase andTh17 cell levels to decreasethus normalizing the Treg to Th17 ratio In addition to thisthe Chinese medicine treatment was also shown to be able tosignificantly decrease the amount of ROR120574tmRNA in hepatictissues of AIH mice in all groups This therefore negativelyinfluences the role of ROR120574t protein on the inhibition of thedevelopment and multiplication of Th17 cells with increasein the expression of Foxp3 and decrease in the expressionof ROR120574t allowing for the regulation of the developmentand multiplication of Treg cells and Th17 cells This processleads to the regulation of the TregTh17 cell ratio whichhelps in reaching proper functions of immune regulation andimmunological protection

Our study has shown that Chinese traditional medicinecomponents such as Bu Xu Hua Yu method can be usedsuccessfully in the preventive treatment of autoimmunehepatitis and can potentially be better tolerated by patientsdue to Chinese medicine drugs having less side effects

However additional studies and clinical trials are required todetermine the long term effects of using Chinese medicine inthe treatment of AIH

Disclosure

First author is LeiWang Co-first author isHuihuiDu Secondauthor is Yibo Liu Third author is Lingtai Wang

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by National Natural Science Foun-dation Project no 81373860 National Natural Science Foun-dationYouth FundProject no 81302891 andNationalNaturalScience Foundation no 30973824

References

[1] J J Feld H Dinh T Arenovich V A Marcus I R Wanlessand E J Heathcote ldquoAutoimmune hepatitis effect of symptomsand cirrhosis on natural history and outcomerdquoHepatology vol42 no 1 pp 53ndash62 2005

[2] A J Czaja ldquoTreatment of autoimmune hepatitisrdquo Seminars inLiver Disease vol 22 no 4 pp 365ndash377 2002

[3] M Biburger and G Tiegs ldquo120572-galactosylceramide-induced liverinjury inmice ismediated byTNF-120572 but independent ofKupffercellsrdquoThe Journal of Immunology vol 175 no 3 pp 1540ndash15502005

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

[4] H Matsumoto T Kawamura T Kobayashi Y Kanda HKawamura and T Abo ldquoCoincidence of autoantibody pro-duction with the activation of natural killer T cells in 120572-galactosylceramide-mediated hepatic injuryrdquo Immunology vol133 no 1 pp 21ndash28 2011

[5] A J Czaja ldquoAutoimmune hepatitis Evolving concepts andtreatment strategiesrdquoDigestiveDiseases and Sciences vol 40 no2 pp 435ndash456 1995

[6] Y-X Gong and B-M Wang ldquoType I autoimmune hepatitis ananalysis of 102 casesrdquoWorld Chinese Journal of Digestology vol16 no 3 pp 322ndash325 2008

[7] E L Krawitt ldquoAutoimmune hepatitisrdquoTheNewEngland Journalof Medicine vol 354 no 1 pp 54ndash66 2006

[8] B HeW-D Gao G-Q Song C-CWang M-L Yang and Q-S Liu ldquoExpression of 24p3 and interleukin-17A in autoimmunehepatitisrdquo Chinese Journal of Hepatology vol 15 no 9 pp 709ndash710 2007

[9] H Kita I RMackay J Van deWater andM E Gershwin ldquoThelymphoid liver considerations on pathways to autoimmuneinjuryrdquo Gastroenterology vol 120 no 6 pp 1485ndash1501 2001

[10] M S Longhi M J Hussain R RMitry et al ldquoFunctional studyof CD4+CD25+ regulatory T cells in health and autoimmunehepatitisrdquoThe Journal of Immunology vol 176 no 7 pp 4484ndash4491 2006

[11] H Morikawa K Hachiya H Mizuhara et al ldquoSublobular veinsas the main site of lymphocyte adhesiontransmigration andadhesion molecule expression in the porto-sinusoidal-hepaticvenous system during concanavalin A-induced hepatitis inmicerdquo Hepatology vol 31 no 1 pp 83ndash94 2000

[12] Q De-kai and M A Xiong ldquoRelationship between type Iautoimmune hepatitis and alleles of HLA-DRB1 in Chinesepatients of Shanghai areardquo Chinese Journal of Hepatology vol10 no 5 pp 347ndash349 2002

[13] A Vogel H Liermann A Harms C P Strassburg M PManns and P Obermayer-Straub ldquoAutoimmune regulatorAIRE evidence for genetic differences between autoimmunehepatitis and hepatitis as part of the autoimmune polyglandularsyndrome type 1rdquoHepatology vol 33 no 5 pp 1047ndash1052 2001

[14] M Yachida K Kurokohchi K Arima and M NishiokaldquoIncreased bcl-2 expression in lymphocytes and its associationwith hepatocellular damage in patients with autoimmune hep-atitisrdquo Clinical and Experimental Immunology vol 116 no 1 pp140ndash145 1999

[15] E Bettelli Y Carrier W Gao et al ldquoReciprocal developmentalpathways for the generation of pathogenic effector T

119867

17 andregulatory T cellsrdquoNature vol 441 no 7090 pp 235ndash238 2006

[16] S Hori T Nomura and S Sakaguchi ldquoControl of regulatory Tcell development by the transcription factor Foxp3rdquo Science vol299 no 5609 pp 1057ndash1061 2003

[17] J C Crispin AMartınez and J Alcocer-Varela ldquoQuantificationof regulatory T cells in patients with systemic lupus erythemato-susrdquo Journal of Autoimmunity vol 21 no 3 pp 273ndash276 2003

[18] M R Ehrenstein J G Evans A Singh et al ldquoCompromisedfunction of regulatory T cells in rheumatoid arthritis andreversal by anti-TNF120572 therapyrdquo The Journal of ExperimentalMedicine vol 200 no 3 pp 277ndash285 2004

[19] V Viglietta C Baecher-Allan H L Weiner and D A HaflerldquoLoss of functional suppression by CD4+CD25+ regulatory Tcells in patients withmultiple sclerosisrdquo Journal of ExperimentalMedicine vol 199 no 7 pp 971ndash979 2004

[20] R Cabrera Z Tu Y Xu et al ldquoAn immunomodulatory rolefor CD4+CD25+ regulatory T lymphocytes in hepatitis C virusinfectionrdquo Hepatology vol 40 no 5 pp 1062ndash1071 2004

[21] J Fu D Xu Z Liu et al ldquoIncreased regulatory T cells correlatewithCD8T-cell impairment and poor survival in hepatocellularcarcinoma patientsrdquo Gastroenterology vol 132 no 7 pp 2328ndash2339 2007

[22] D Xu J Fu L Jin et al ldquoCirculating and liver residentCD4+CD25+ regulatory T cells actively influence the antiviralimmune response and disease progression in patients withhepatitis BrdquoThe Journal of Immunology vol 177 no 1 pp 739ndash747 2006

[23] S Ferri M S Longhi C de Molo et al ldquoA multifaceted imbal-ance of T cells with regulatory function characterizes type 1autoimmune hepatitisrdquoHepatology vol 52 no 3 pp 999ndash10072010

[24] R Nurieva X O Yang G Martinez et al ldquoEssential autocrineregulation by IL-21 in the generation of inflammatory T cellsrdquoNature vol 448 no 7152 pp 480ndash483 2007

[25] I I Ivanov B S McKenzie L Zhou et al ldquoThe orphannuclear receptor ROR120574t directs the differentiation program ofproinflammatory IL-17+ T helper cellsrdquo Cell vol 126 no 6 pp1121ndash1133 2006

[26] G L Zhang D Y Xie Y N Ye et al ldquoHigh level of IL-27positively correlated withTh17 cells may indicate liver injury inpatients infected with HBVrdquo Liver International vol 34 no 2pp 266ndash273 2014

[27] J-Y Zhang Z Zhang F Lin et al ldquoInterleukin-17-producingCD4+ T cells increase with severity of liver damage in patientswith chronic hepatitis Brdquo Hepatology vol 51 no 1 pp 81ndash912010

[28] M Fujimoto S Serada and T Naka ldquoRole of IL-6 in thedevelopment and pathogenesis of CIA and EAErdquo JapaneseJournal of Clinical Immunology vol 31 no 2 pp 78ndash84 2008

[29] C M Hedrich T Rauen K Kis-Toth V C Kyttaris and GC Tsokos ldquoCAMP-responsive element modulator 120572 (CREM120572)suppresses IL-17F protein expression in T lymphocytes frompatients with systemic lupus erythematosus (SLE)rdquoThe Journalof Biological Chemistry vol 287 no 7 pp 4715ndash4725 2012

[30] H Jyonouchi L Geng A Cushing-Ruby and I M MonteiroldquoAberrant responses to TLR agonists in pediatric IBD patientsthe possible association with increased production of Th1Th17cytokines in response to candida a luminal antigenrdquo PediatricAllergy and Immunology vol 21 no 4 part 2 pp e747ndashe7552010

[31] E M Eisenstein and C B Williams ldquoThe T(reg)Th17 cellbalance a new paradigm for autoimmunityrdquo Pediatric Researchvol 65 no 5 part 2 pp 26Rndash31R 2009

[32] X Cheng X Yu Y J Ding et al ldquoThe Th17Treg imbalance inpatients with acute coronary syndromerdquo Clinical Immunologyvol 127 no 1 pp 89ndash97 2008

[33] S Deng Y Xi H Wang et al ldquoRegulatory effect of vasoactiveintestinal peptide on the balance of Treg and Th17 in collagen-induced arthritisrdquoCellular Immunology vol 265 no 2 pp 105ndash110 2010

[34] Q-F Kong B Sun S-S Bai et al ldquoAdministration of bonemarrow stromal cells ameliorates experimental autoimmunemyasthenia gravis by altering the balance ofTh1Th2Th17Tregcell subsets through the secretion of TGF-120573rdquo Journal of Neu-roimmunology vol 207 no 1-2 pp 83ndash91 2009

[35] Z-Z Yang A J Novak S C Ziesmer T E Witzig and S MAnsell ldquoMalignant B cells skew the balance of regulatory T cells

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Evidence-Based Complementary and Alternative Medicine

and T H17 cells in B-cell non-Hodgkinrsquos lymphomardquo CancerResearch vol 69 no 13 pp 5522ndash5530 2009

[36] L Zhao Y Tang Z You et al ldquoInterleukin-17 contributes tothe pathogenesis of autoimmune hepatitis through inducinghepatic interleukin-6 expressionrdquo PLoS ONE vol 6 no 4Article ID e18909 2011

[37] M S Longhi R Liberal B Holder et al ldquoInhibition of inter-leukin-17 promotes differentiation of CD25minus cells into stableT regulatory cells in patients with autoimmune hepatitisrdquoGastroenterology vol 142 no 7 pp 1526ndash1535 2012

[38] M C Fantini A Rizzo D Fina et al ldquoIL-21 regulates experi-mental colitis bymodulating the balance betweenTreg andTh17cellsrdquo European Journal of Immunology vol 37 no 11 pp 3155ndash3163 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom