Research Article Molecular Mechanism of Yisui Shengxue …downloads.hindawi.com/journals/ecam/2014/213782.pdf · () L of erythrocyte sedimentation was added to L deionized water and

Research ArticleMolecular Mechanism of Yisui Shengxue Granulea Complex Chinese Medicine on Thalassemia PatientsSuffering from Hemolysis and Anemia of Erythrocytes

Na-Li Chu1 Zhi-kui Wu1 Xin-Hua Zhang2 Su-Ping Fang1

Wen-Juan Wang3 and Yan-Ling Cheng1

1Guangrsquoanmen Hospital China Academy of Chinese Medical Sciences Beijing 100053 China2303 Hospital of Chinese Peoplersquos Liberation Army (PLA) Nanning 530000 China3The Capital Medical University Beijing 100069 China

Correspondence should be addressed to Zhi-kui Wu gamwuzhikuisinacom

Received 22 August 2014 Revised 19 November 2014 Accepted 19 November 2014 Published 10 December 2014

Academic Editor Yuping Tang

Copyright copy 2014 Na-Li Chu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The objective of this study was to investigate the therapeutic biological mechanism of Yisui Shengxue Granule (YSSXG) a complexChinese medicine on the hemolysis and anemia of erythrocytes from patient with thalassemia disease Sixteen patients withthalassemia (8 cases of 120572-thalassemia and 8 cases of 120573-thalassemia) disease were collected and treated with YSSXG for 3 monthsThe improvements of blood parameter demonstrated that YSSXG had a positive clinical effect on patients with thalassemia diseaseFor patients with 120572-thalassemia disease RT-PCR showed that YSSXG upregulated the relative mRNA expression level of 120572-globin to 120573-globin and downregulated DNMT1 DNMT3a and DNMT3b mRNA compared with pretreatment Western blottingshowed that YSSXG downregulated the expression of DNMT1 and DNMT3a For patients with 120573-thalassemia disease the relativeexpression level of A120574-globin to 120572-globin had an increasing trend and the level of BCL11A mRNA expression obviously increasedFor all patients RT-PCR showed that YSSXG upregulated mRNA expression of SPTA1 and SPTB Activities of SOD and GSH-Px significantly increased andMDA obviously reduced on erythrocyte and blood serum after YSSXG treatment TEM showed thatYSSXGdecreased the content of inclusion bodies Activities ofNa+K+-ATPtase andT-ATPtase of erythrocyte increased significantlyafter YSSXG treatmentThis study provides the basis formechanisms of YSSXGon thalassemia sufferingwith hemolysis and anemiaof erythrocytes from patient

1 Introduction

Thalassemia encompasses a spectrum of hereditary anemiascharacterized by reduced or absent production of one ormore globin chains [1] Normal human adult hemoglobin(Hb)A(HbA) consists of two pairs of globin chains 12057221205732 of which synthesis is normally tightly coordinated toensure equal production The molecular defect leads to animbalance of 120572120573-globin chains synthesis The excess globinchain depositing on the red cell membrane induces immuneand oxidant injury causes secondary enzymes and metabolicabnormalities and results in the decreasing deformabilityand mechanical stability of RBC which cause hemolysis andineffective hematopoiesis [2 3] Transfusion is the major

treatment for thalassemia which can cause splenomegalyand hyperthyroidism and aggravate the anemia and othercells damage Some people try to use an alkylating agentbutyrate and its derivatives Myleran and other drugs totreat thalassemia disease but these drugs have strong sideeffects which limit the application in clinical practice It isdifficult to popularize the therapy of bone marrow and stemcell transplantation and gene therapy which were reportedabout individual cases in clinical practice Based on theldquokidney essence marrowrdquo theory YSSXG which is a typicalprescription of kidney-nourishing and marrow-replenishingtherapy hasmade a positive effect on the treatment of two dif-ferent genotypes (120572- and 120573-type) thalassemia disease in highincidence area of Guangxi [4ndash7] To verify scientific effect

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 213782 11 pageshttpdxdoiorg1011552014213782

2 Evidence-Based Complementary and Alternative Medicine

of kidney-nourishing and marrow-replenishing therapy onthalassemia we collected 16 cases of thalassemia patients inNanning Guangxi (8 cases with 120572-thalassemia and 8 caseswith 120573-thalassemia) and explored the biological mechanismsof clinical effect based on pathological mechanism of hemol-ysis and anemia

2 Materials and Methods

21 Diagnostic Criteria The diagnostic criteria of westernmedicine for intermedia thalassemia were referred to ldquoCri-teria for Diagnosis and Therapeutic Effect on Hematopathyrdquoedited by Zhang [8]

22 Inclusion Exclusion andWithdrawal Criteria The inclu-sion criteria were (1) for the people who accord with thediagnostic criteria ofwesternmedicine (2)Theage of patientsranged from 4 to 40 years old (3) Patients volunteered toparticipate in this study and they signed informed consentforms

The exclusion criteria were as follows (1) the patients whohave immunologic deficiency or primary diseases of the liverkidney or blood system (2) the patients who are pregnant (3)the patients who are allergic to this drug ingredients and (4)the patients who took the antianemia drugs in the last twomonths

The withdrawal criteria were as follows (1) the patientswho did not take drugs following requirements of the studyprotocol quit by themselves or were lost to follow-up and (2)patients who took any antianemia drugs and received bloodtransfusion in the treatment were rejected

23 Study Population and General Data Sixteen outpatients(8 cases of 120572-thalassemia and 8 cases of 120573-thalassemia) wereenrolled from theDepartment ofHematology 303Hospital ofChinese Peoplersquos Liberation Army (PLA) during the periodfrom October to December in 2013 Sixteen patients were7 to 26 years old with an average of 1319 plusmn 547 yearsThe patients of Zhuang and Han nationalities were 8 casesrespectively Of the 16 patients 10 were males and 6 werefemales Cases of mild anemia were 3 and cases of moderateanemia were 13 Three genotypes of 120572-thalassemia weredetected 1 case of genotype ofmdashSEA

12057242 1 case of genotype

ofmdashSEA12057237 6 cases of genotype ofmdashSEA

120572CS120572 Six genotypes

of 120573-thalassemia were detected 2 cases of genotypes of 17282 cases of genotypes of 41-42-28 1 case of genotypes of 17E1 case of genotypes of 17N 1 case of genotypes of 28IVS1-1and 1 case of genotypes of 4317

24 Drugs and Interventions A self-control studywas carriedout The course of treatment was 3 months YSSXG was pro-duced byGuangrsquoanmenHospital Preparation Factory accord-ing to the protocol described in patent (number CN1872182batch number 20120516) The modified YSSXG consisted of11 Chinese herbalmedicinal components RhizomaKaempfe-riae Radix PolygoniMultiflori RadixRehmanniae PreparataRadix Astragali Radix Codonopsis Radix Angelicae Sinen-sis Fructus Psoraleae Colla Corii Asini Caulis SpatholobiCarapax Trionycis and Fructus Amomi A pack of granules

contains 10 g powder (1 g powder contains 2368 g crudedrug)

Patients aged 2 to 6 years old were instructed to take halfa pack of granules twice daily aged 6 to 10 years old a fullpack of granules twice daily and aged over 10 years old a packof granules thrice daily The granules are dissolved in warmwater and taken orally Patients were required to have noblood transfusion during the observation period and askedto insist on the treatment regimen

25 Blood Sample Collection Venous blood samples (5mL)were collected into EDTA tubes from 16 patients withthalassemia disease before and after YSSXG treatment Theblood mixed by horizontal shaker was centrifuged at1700 rmin for 10min The upper layer of the plasma wasdiscarded The lower layer of blood cells which was shoppedat lymphocyte separationmediumwere added saline to 5mLand then centrifuged at 2500 rmin for 20min Mononuclearcell layer which was located between the plasma and thelymphocyte separation liquid was drawn to 15mL centrifugetube then was added saline to 10mL and centrifuged at1800 rmin for 10min twiceThe abovemixturewas discardedafter centrifuging and the precipitation mononuclear cellswere added to 1mL TRIZOL reagent and stored in EP tubesfrozen at minus80∘C refrigerator for RNA extraction After theblood isolated by lymphocytes separationmedium red bloodcells were at the lowest layer The red blood cells were addedsaline to 10mLand centrifuged at 1700 rmin for 5minwhosesupernatant was discarded Pure red blood cells was storedat frozen pipes for minus80∘C refrigerator Venous blood samples(4mL) were collected into non-anticoagulant tube from 16patients with thalassemia disease before and after YSSXGtreatment which were centrifuged at 1700 rmin for 10minto obtain blood serum for detection

26 Indicators of Observation and Detection

261 Reagents Lymphocyte separation medium was pur-chased from Solarbio Co (China) (batch number P8610)Trizol reagent was purchased from Invitrogen USA (batchnumber 14105) Test kits for superoxide dismutase (SOD)malonaldehyde (MDA) and glutathione peroxidase (GSH-Px) were purchased from Nanjing Jiancheng Institute ofBioengineering (batch number 20140224) RNA Mini Kitwas purchased from Tianjin Co (China) (batch number139315390) RevertAid First Strand cDNA Synthesis Kit waspurchased from Fermentas LT (Lithuania) (batch num-ber 00146314) Power SYBR Green PCR Master Mix waspurchased from ABI USA (batch number 1402445) Theantibody against DNMT1 and DNMT3a was purchased fromCST (batch number 5119 2160) DNMT3b was purchasedfrom Abcam (batch number ab79822) and 120573-actin waspurchased fromBeijing ZhongshanGolden Bridge BiologicalTechnologyCo (China) (batch number TA-09) BCAProteinAssay Kit was purchased from Beijing ComWin BiotechCo Ltd (batch number 02912E) Test Kit for Mini ATPenzyme (Na+K+ Ca2+Mg2+ and T-ATP enzyme) of RBCwaspurchased from Nanjing Jiancheng Institute of Bioengineer-ing (batch number 20140606)

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer used for detecting the gene expression by reverse real-time PCR

Target gene Primer sequence 51015840-31015840 Predicted amplificationsegments (bp)Forward primer Reverse primer

120573-Actin GAG ACC TTC AAC ACC CCA GCC AAT GTC ACG CAC GAT TTC CC 263SPTA1 GAT CTT GAA GCC AAT GTC CA CAA CTC CCT CCA CTG GTG 250SPTB AAC CGG GAT AAG GTC TTG AGT C GGT GCT TTA GCC ATT TAT TGT GA 289EPB41 TTA TCC ACT CAC TCA CCC TTC C CTC ATC AGC AAT CTC GGT CTC C 369BCL11A CGC CAG AGG ATG ACG AT CAG GCG TGG GGA TTA GA 138120572-Globin CCC ACC ACC AAG ACC TAC TT GCT CAC AGA AGC CAG GAA CTT 291120573-Globin TAG CAA CCT CAA ACA GAC ACC A ATC ACT AAA GGC ACC GAC CAC T 243A120574-Globin ATC AAT AAG CTC CTA GTC CAG ACG CCA AAG CTG TCA AAG AAC CTC T 166

G120574-Globin AGG AGG ACA AGG CTA CTA TCA CAA CTT GAG ATC ATC CAG GTG CTT TAT 233

DNMT1 TAC CTG GAC GAC CCT GAC CTC CGT TGG CAT CAA AGA TGG ACA 103DNMT3a TAT TGA TGA GCG CAC AAG AGA GC GGG TGT TCC AGG GTA ACA TTG AG 111DNMT3b GGC AAG TTC TCC GAG GTC TCT G TGG TAC ATG GCT TTT CGA TAG GA 132

262 Detection of Blood Parameters Blood parameters HbRBC count and reticulocyte percent (Ret) of all patients weremeasured dynamically before and after treatment by using acell DYN 3700 automatic blood analyzer (USA) The param-eter of HbF was only measured in 120573-thalassemia patientsby the method of high-performance liquid chromatography(HPLC) through using Bio-Rad Variant II System (VariantBio-Rad Hercules CA USA)

263 Detection of SOD MDA and GSH-Px The contentsof SOD GSH-Px and MDA were detected by the digestivemethod SOD by the xanthine oxidase method GSH-Px bythe enzyme to catalyze the reaction of hydrogen peroxide(H2O2) and GSH using 551015840-dithiobis(2-nitrobenzoic acid

DTNB) to determine the quantity of remainder GSH andMDA by thiobarbituric acid method

The detection steps of erythrocyte SOD were as follows(1) 20120583L of erythrocyte sedimentation was added to 200 120583Ldeionized water and fully mixed (2) The above mixturewas added to 100 120583L 95 ethanol and fully shocked 30 s(3) The mixture in step 2 was added to 100 120583L chloroformand thoroughly mixed for 1min which was centrifuged at3500 rmin for 8min The supernatant was SOD extract (4)Next detection of SOD was measured strictly in accordancewith the procedure for SOD test kit

The detection steps of erythrocyte GSH-Px were asfollows (1) 20120583L of erythrocyte sedimentation was added to480 120583L deionized water and fully mixed for 5min until themixture shows a fully transparent state (2) Next detectionof GSH-Px was measured strictly in accordance with theprocedure for GSH-Px test kit

The detection steps of erythrocyte MDA were as follows(1) 20120583L of erythrocyte sedimentation was added to 180 120583L1times hypotonic solution (001M Tris-HCl PH = 74) and fullymixed which was centrifuged at 12000 rmin for 10min afterkeeping hemolysis at 4∘C for 30min The above mixturewas discarded the supernatant The above procedure wasrepeated for four times (2) Isolation of erythrocyte ghostmembranes was added to 30 120583L PIPA lysis buffer which

has been added to PMSF inhibitors The mixture should berepeated pipetting and recrackingwhichwas incubated on icefor 10min (3)Next protein concentrationwas determined bythe bicinchoninic acid (BCA)method (4) Detection ofMDAwas measured according to the procedure for MDA test kit

The serum contents of SOD GSH-Px and MDA weredetected according to the procedure for SOD GSH-Px andMDA test kit

264 RNA Isolation and RT-PCR Total RNA was isolatedfrom mononuclear cells using Trizol reagent according tomanufacturerrsquos instructions The total RNA concentrationwas quantified and total RNA (5120583g) was reverse-transcribedto cDNA using an RT-PCR kit Reverse transcription wasperformed at 42∘C for 60min followed by inactivation at 70∘Cfor 5minThe resulting cDNAwas further used as a templatefor polychain reaction (PCR) amplification immediately orstored at minus40∘C until use Primer pairs of genes were synthe-sized and the parameters are included in Table 1 Real-timePCR was performed according to the protocol of the QiagenSybr Green PCR Kit in the Optical 96-Well Reaction Plateproduced by applied biosystems (batch number 8010560) and120573-actin as the endogenous control The PCR conditions were95∘C for 10min 95∘C for 30 s annealing for 35 s 72∘C for50 s repeating for 10 cycles and 72∘C for 8minThe differentannealing temperatures were 120572- 120573- A

120574- G120574-globin 55∘C

SPTA1 SPTB EPB41 55∘C DNMT1 DNMT3a DNMT3b60∘CBCL11A 54∘CPCRproductswere analyzed using a 12agarose gel Relative gene expression was calculated using thecomparative threshold cycle (2minusΔΔCt)methodThe sequencesof gene-specific primers are summarized in Table 1

265 Western Blot Analysis Western blot analysis was usedto determine DNMT1 DNMT3a and DNMT3b content inthe whole blood for 120572-thalassemia Protein was extractedaccording to the manufacturerrsquos protocol The protein con-centration was determined by the bicinchoninic acid (BCA)method and equal amounts loaded on a 10 sodium


Table 2 Effect of YSSXG on globin chain ratio of 120572-thalassemia patients pre- and posttreatment (119909 plusmn 119904)

Therapy time Case 2minusΔct (120572)2minusΔct (120573) 2

minusΔct (A120574)2minusΔct (120573) 2minusΔct (G120574)2minusΔct (120573)

Pretreatment 8 080 plusmn 062 013 plusmn 009 014 plusmn 0173 month posttreatment 8 202 plusmn 098 102 plusmn 133 033 plusmn 0353 month posttreatment versuspretreatment (95CI)119875 value 0004 0097 0168

Notes 119875 lt 005 119875 lt 001

dodecyl sulfate- (SDS-) polyacrylamide gel for electrophore-sis Protein bands were then transferred onto polyvinylidenefluoride (PVDF)membranes which were stained by Ponceaustaining reagents after the completion of transfer membraneThe membrane was completely immersed in phosphate-buffered solution with Tween (TBST) containing 5 bovineserum albumin (BSA) for 1 hThemembranes were incubatedwith primary antibodies overnight at 4∘C The mouse mon-oclonal anti-DNMT1 antibody and anti-DNMT3a antibodywere diluted 1 1000 The rabbit monoclonal anti-DNMT3bantibody was diluted 1 11000 Following incubation mem-branes were washed three times in PBS with 01 Tween-20 for 10min once After that horseradish peroxidase-conjugated goat anti-mouse and rabbit antibody (diluted1 110000 in 5BSA-TBST)were applied to themembrane for40min Then membranes were washed three times in PBSwith 01 Tween-20 for 10min once Reactive proteins weredetected on film using a chemiluminescent solution obtainedfromMillipore USA And the bands were then quantified bydensitometry using Gelpro 32 software

266 TEM Observation of Inclusion Bodies in ErythrocytesThe procedure of measurement for inclusion bodies inerythrocytes was referred to the method of Wang et al [9]

267 Detection of Na+K+ Ca2+Mg2+ and T-ATP Enzymein Erythrocytes ATP enzymes can break down ATP togenerate ADP and inorganic phosphate which content canbe used to determine the level of ATP The detection stepsof Na+K+ Ca2+Mg2+ and T-ATPtase were as follows 50 120583Lof erythrocyte sedimentation was added to 450120583L deionizedwater and fully mixed until observing that it was transparentDetection of Na+K+ Ca2+Mg2+ and T-ATPtase activity wasmeasured strictly in accordance with the procedure for MiniATP enzyme test kit

27 Statistical Analysis Statistical analysis was performedusing SPSS170The resultswere presented asmeanplusmn standarddeviation Paired t-test was used in comparing pre- andposttreatment A119875 lt 005was considered as having statisticalsignificance

28 Medical Ethics This study was approved by EthicsCommittees of Guangrsquoanmen Hospital China Academy ofChinese Medical Sciences All patients have signed theinformed consents before entering trials comprehensivelyunderstanding the purpose procedures possible risks andbenefits on participating in this study

3 Results

All the 16 patients completed the whole observation withoutdropout

31 YSSXGCan Improve Levels of Blood Parameters of Patientswith Thalassemia Disease For 120572-thalassemia patients levelsof Hb concentrations and RBC counts from 1 to 3 monthswere higher than the levels of pretreatment Levels of Hb at 2and 3 months and RBC counts at 3 months were significantlyincreased compared with the measurement before treatment(119875 lt 001 and 119875 lt 005 resp Figures 1(a) and 1(b))The Ret concentrations markedly decreased in the 3-monthposttreatment (119875 lt 005 Figure 1(c))

For 120573-thalassemia patients levels of Hb concentrationsand RBC counts had kept on increasing in 3 months oftreatment while differences were not statistically significantcompared with the measurements of pretreatment (Figures1(a) and 1(b)) The measurement of Ret had an obviousincrease at 2 months of posttreatment (119875 lt 005 Figure 1(c))Levels of HbF after treatment from 1 to 3 months weresignificantly increased compared with the measurementsprior to treatment (119875 lt 001 and 119875 lt 005 Figure 1(d))

32 YSSXG Can Promote the Balance of Globin Chain Ratioof Patients with 120572-Thalassemia and 120573-Thalassemia DiseaseFor120572-thalassemia patients the relative expression of120572-globinto 120573-globin was markedly increased compared with that ofpretreatment (119875 lt 001) and relative expressions of A

120574 andG120574-globin to 120573-globin had no statistically change compared

with levels of pretreatment (Table 2)For 120573-thalassemia patients relative expressions of 120573-

globin and G120574-globin to 120572-globin had no statistically change

compared with that of pretreatment (119875 lt 001) and the rel-ative expressions of A120574-globin to 120572-globin had an increasingtrend compared with levels of pretreatment (Table 3)

33 YSSXG Can Downregulate the mRNA Expression andDecrease the Activity of DNA Methyltransferase of Patientswith 120572-Thalassemia Disease mRNA expressions of DNMT1DNMT3a and DNMT3b markedly decreased when com-pared with the level of pretreatment (119875 lt 005 Figure 2(a))Western blotting showed protein expression of DNMT1DNMT3a and DNMT3b (Figure 2(b)) The protein expres-sion of DNMT1 and DNMT3a significantly decreased afterthe treatment of YSSXG (119875 lt 001) The protein expressionof DNMT3b had a decreasing trend (Figure 2(c))

34 YSSXG Can Downregulate BCL11A mRNA Expression ofPatients with 120573-Thalassemia Disease The level of BCL11A


140

120

100

80

60

40

20

0

120572-Thalassemia 120573-Thalassemia

Con

tent

(gL

)Hb

lowastlowast

lowastlowast

(a)


lowast

6

5

4

3

2

1

0

RBC

Con

tent

(times1012L

)(b)


Ret12

10

8

6

4

2

0

Con

tent

()

lowast

lowast

Pretreatment1-month posttreatment

2-month posttreatment3-month posttreatment

(c)

120573-Thalassemia

HbF

Con

tent

()

100

90

80

70

60

50

40

30

20

10

0

lowastlowast

lowastlowast



(d)

Figure 1 Effect of YSSXG on blood parameters of patients with 120572-thalassemia and 120573-thalassemia disease at pre- and posttreatment (119899 = 8resp) lowast119875 lt 005 lowastlowast119875 lt 001 compared with pretreatment (a) The changes of Hb concentration (b) The changes of RBC counts (c) Thechanges of Ret level (d) The changes of HbF level of patients with 120573-thalassemia disease





Notes 119875 lt 005 119875 lt 001


DNMT1 DNMT3a DNMT3b

Pretreatment

12

1

08

06

04

02

0

DN

MTs

mRN

A ex

pres

sion

lowastlowast

lowastlowast

lowastlowast

3-month posttreatment

(a)

5 6 7 8 5 6 7 8

Pretreatment Posttreatment

DNMT3a

DNMT3b

DNMT1

120573-Actin

1 2 3 4 1 2 3 4


DNMT3a

DNMT3b

DNMT1

120573-Actin

(b)

04

035

03

025

02

015

01

005

0

DN

MTs

pro

tein

expr

essio

n

lowast

lowast

DNMT1 DNMT3a DNMT3b


(c)

Figure 2 Effect of YSSXG on themRNA expression and the protein activity of DNAmethyltransferase of patients with 120572-thalassemia disease(119899 = 8) lowast119875 lt 005 lowastlowast119875 lt 001 compared with pretreatment (a)The fold changes relative of DNMTsmRNA expression level to pretreatment(b) Electrophoresis of Western blot of DNMTs Note that the number of 1 to 8 represents the sample of 8 patients with 120572-thalassemia diseaserespectively (c) The changes of protein expression level of DNMTs

expression of pretreatment was significantly higher than thatof posttreatment (119875 lt 005 Figure 3)

35 YSSXG Can Improve the Pro- and Antioxidative SystemBalance of Erythrocyte and Blood Serum of Patients with

Thalassemia Disease After treatment with YSSXG SOD andGSH-Px activities in erythrocytes and blood serum wereincreased significantly (119875 lt 001 Figures 4(a) 4(b) 4(c)and 4(d)) and the MDA concentrations in RBCs and bloodserum were obviously decreased (119875 lt 001 Figures 4(e) and4(f))


12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast


Figure 3 Effect of YSSXG on the mRNA expression of BCL11A ofpatientswith120573-thalassemia disease (119899 = 8)The fold changes relativeof BCL11A mRNA expression level to pretreatment lowast119875 lt 005compared with pretreatment

Table 4 Effect of YSSXG on erythrocyte membrane skeletonprotein gene expression pre- and posttreatment (119909 plusmn 119904)

Therapy time Case SPTA1 SPTB EPB41Pretreatment 16 033 plusmn 040 012 plusmn 016 032 plusmn 0333 monthposttreatment 16 117 plusmn 150 041 plusmn 043 087 plusmn 134

3 monthposttreatmentversuspretreatment(95CI)119875 value

0046 0018 0069

Notes 119875 lt 005 119875 lt 001

36 YSSXG Can Upregulate the mRNA Expression of Erythro-cyte Membrane Skeleton Protein of Patients with ThalassemiaDisease The mRNA expression of SPTA1 and SPTB oferythrocyte membrane skeleton protein markedly increasedcompared with levels of pretreatment (119875 lt 005) The mRNAexpression of EPB41 had an increasing trend whereas thedifference had no statistical change compared to the level ofpretreatment (Table 4)

37 YSSXG Can Decrease the Content of Inclusion Bodies inErythroid Cells of Patients with Thalassemia Disease TEMimages of erythroid cells are shown in Figure 5 RBCs hadnumerous dark grains indicative of inclusion bodies formedby unmatched denatured 120573-globin chains with 120572-thalassemiapatients (Figure 5(a)) which were also observed on the 120573-thalassemia patients formed by unmatched denatured 120572-globin chains before treatment with YSSXG (Figure 5(c))

After treatment with YSSXG themount and volume of inclu-sion bodies decreased in two types of thalassemia (Figures5(b) and 5(d))

38 YSSXG May Increase Activities of Na+K+-ATPtase andT-ATPtase of Erythrocyte After treatment with YSSXGNa+K+-ATPtase andT-ATPtase activities of Erythrocytewereincreased significantly (119875 lt 005) and Ca2+Mg2+-ATPtaseactivity had no significant change (Figure 6)

4 Discussion

Thalassemia belongs to ldquoBlood Deficiencyrdquo or ldquoConsump-tionrdquo category in Chinese medicine Based on the investi-gation of etiology clinical manifestations TCM syndromesand genetic background the professor ofWuZhikui proposesthat ldquocongenital deficiency kidney marrow damage andblood metaplasia passiverdquo are the core of the pathogenesisof thalassemia and then establishes the therapeutic principlewhich is ldquokidney-nourishing and marrow-replenishingrdquo Thecomposition of Yisui Shengxue Granule is based on thetraditional Chinese medicine theory of ldquokidney-nourishingand marrow-replenishingrdquo and clinical practice which iscomposed of 11 herbs complexes The thalassemia syndromeis classified according to which of the globin chains 120572 or 120573is affected These 2 major groups 120572- and 120573-thalassemia aresubclassified according to absent (1205720 and 1205730) or reduced (120572+and 120573+) globin chain synthesis [10] Difference in the amountof fetal hemoglobin (HbF) that persists into adulthood affectsthe severity of 120573-thalassemia syndromes [11] 120574-globin (a 120573-globin-like molecule) which binds to 120572-chains to produceHbF addresses the imbalance in globin chains and this inturn reduces the occurrence of ineffective erythropoiesisdecreases hemolysis and increases total Hb [12]

As observed in this experiment the levels of Hb sig-nificantly increased and the Ret concentrations markedlydecreased in the 3-month posttreatment for 120572-thalassemiapatients which indicated that the degree of anemia andineffective hematopoiesis were markedly improved Hb leveldid not increase significantly but the level of HbF signif-icantly elevated after 3-month treatment Scores of symp-toms were significantly lower than those before treatmentwhich showed that the improvement of clinical symp-toms was consistent with levels of improvement in bloodparameters Hb level is one of the most important bloodparameters in patients with thalassemia which reflect theseverity of thalassemia disease condition By analyzing wefound that Hb levels of 120573-thalassemia patients were signif-icantly lower than 120572-thalassemia patients indicating that 120573-thalassemia patientrsquos condition is generally more serious than120572-thalassemia patients which may be partly explained bythe fact that the clinical efficacy of 120572-thalassemia was betterthan that of 120572-thalassemia patients by using Yisui ShengxueGranule but it needs a large sample of clinical trials by furtherverification

Human hemoglobin from embryonic (1205722 1205762) is convertedto the fetus (1205722 1205742) and then from the fetus (1205722 1205742) it is


9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)

SOD of RBClowastlowast

(a)

SOD of serumlowastlowast

Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0

GSH-Px of RBClowastlowast

Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)

Figure 4 Effect of YSSXG on biomarkers of pro- and antioxidative system with thalassemia disease (119899 = 16) (a) The SOD activity level ofRBC (b)The SOD activity level of serum (c)TheGSH-Px activity level of RBC (d)TheGSH-Px activity level of serum (e)TheMDA activitylevel of RBC (f) The MDA activity level of serum lowast119875 lt 005 lowastlowast119875 lt 001 compared with pretreatment


(a) (b)

(c) (d)

Figure 5 Effect of YSSXG on the mRNA expression of erythrocyte membrane skeleton protein of patients with thalassemia disease (a)TEM images of inclusion bodies of 120572-thalassemia patients of pretreatment (b) TEM images of inclusion bodies of 120572-thalassemia patientsof posttreatment (c) TEM images of inclusion bodies of 120573-thalassemia patients of pretreatment (d) TEM images of inclusion bodies of120573-thalassemia patients of posttreatment

transmitted to adult (1205722 1205732) which are two different develop-mental stages The 120572-globin genes which are surrounded bywidely expressed genes in a gene dense region of the genomeare silenced very early in development via recruitment of thePolycomb (PcG) complex [13] The PcG complex seems tobe recruited to the 120572-cluster by sequences within the CpGislands associated with their promoters [13] The promotersof the human 120572-globin genes lie within large CpG islandsCpG methylation is thought to be carried out by differentenzymes the ldquodenovordquo MTases Dnmt3a and Dnmt3b andldquomaintenancerdquo MTase Dnmt1 respectively [14] DNAmethy-lation plays an important role in transcriptional repression[14] DNA methylase may affect the expression of 120572-globinby adjusting the level of DNA methylation in gene promoterregion GpG islands BCL11A gene regulates hemoglobin geneconversion and directly inhibits 120574-globin gene transcriptionand then silences 120574-globin gene [15ndash17] The results of thisstudy showed that for 120572-thalassemia patients the relativeexpression of 120572-globin to 120573-globin markedly increased andlevels of mRNA expression and protein expression decreasedcompared to levels of pretreatment which indicated thatthe mechanism of clinical efficacy is partly attributed to

improvement of globin chain ratio by inhibiting the expres-sion of DNA methyltransferase For 120573-thalassemia patientsafter YSSXG treatment the relative expression of A 120574-globin to 120572-globin had an increasing trend and BCL11Aexpression level of posttreatment was significantly lower thanthat of pretreatment which stated that the clinical efficacyfor 120573-thalassemia patients partly accounted for increasingHbF level through reducing the expression of BCL11A Theincreasing extent of HbF content was consistent with thedecreasing extent of BCL11A expression

The tetramer of normal adult hemoglobin is synthesizedmainly by two 120572-globin chains and two 120573-globin chains(1205722 1205732) Human globin tetramer (1205722 1205732) in the bodyis stable but free 120572-chain (120573-thalassemia) or 120573-chain (120572-thalassemia) in the body is unstable The excess 120572-chain(120573-thalassemia) or 120573-chain (120572-thalassemia) form unstablehomotetramers that precipitate on RBCs as inclusion bodies[13] 120572-Homotetramers in 120573-thalassemia are more unstablethan 120573-homotetramers in 120572-thalassemia and therefore pre-cipitate earlier in the RBC life span causing marked RBCdamage and severe hemolysis associated with ineffective ery-thropoiesis and extramedullary hemolysis [18] Iron overload


120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast

Na+K+-ATPase T-ATPaseCa2+Mg2+-ATPase


Figure 6 Effect of YSSXG on activities of Na+K+-ATPtase Ca2+Mg2+-ATPtase and T-ATPtase on erythrocyte of patients withthalassemia disease (119899 = 16) lowast119875 lt 005 compared withpretreatment

precipitated globin chains and premature hemolysis of redcell are contributing causes of oxidative stress in thalassemicpatients [19ndash22] The balance between the prooxidant andantioxidant levels becomes impaired while a decrease occursin levels of antioxidant enzymes an increase occurs in levelsof MDA [23] The degree of oxidative stress as indirectlymeasured by the alteration of antioxidant enzymes such assuperoxide dismutase (SOD) glutathione peroxidase (GSH-Px) and catalase [24ndash26] or the products of lipid perox-idation such as malondialdehyde (MDA) content [27ndash29]The precipitation of globin chain [30] and oxidative damage[31] induced by 120572- or 120573-globin chains which are associatedwith the membrane skeleton have been found to interactand disrupt the RBC membrane damaging the cytoskeletonand resulting in differential membrane alterations Na+K+-ATP enzyme andCa+-Mg+-ATP enzyme onRBCsmembranecan maintain a stable ion concentration and a normalmorphology of RBC Oxygen free radicals produced by lipidperoxidation inhibit the activity of erythrocyte membraneproteins which play a role in ion pump such as Na+K+-ATP enzyme and Ca2+Mg2+-ATP enzyme increase RBCsmembrane permeability and result in cell swelling and adecline in RBCs deformation [32]

TEM showed that RBCs of 120572-thalassemia and 120573-thalassemia patients were distributed in large number ofdark re-dye particles before treatment and then dark re-dye particles significantly reduced after treatment whichindicated that relative excess unpaired globin chains obvi-ously reduced suggesting that globin chain ratio tendsto balance The activity of SOD and GSH-Px of RBCsand serum antioxidative damage indicators significantlyincreased when compared with those of pretreatment Andthe activity of MDA of RBCs and serum oxidative damageindicator significantly decreased when compared with that

of pretreatment Those suggest that the improvement ofpro- and antioxidative system balance is directly related tothe alleviation of hemolysis and anemia by Yisui ShengxueGranule treatment The mRNA expressions of SPTA1 SPTBand EPB41 mainly erythrocyte membrane skeleton proteinwere markedly increased after treatment and the activities ofNa+K+-ATPtase and T-ATPtase of erythrocyte were also sig-nificantly increased which are the causes of Yisui ShengxueGranule promoting the intact of erythrocytemorphology andthe recovery of erythrocyte function

5 Conclusion

Yisui Shengxue Granule to treat thalassemia disease has anaffirmative clinical efficacy Mechanisms of YSSXG improv-ing hemolysis and anemia of erythrocytes are as followspromoting a balanced ratio of globin chains inhibiting DNAmethyltransferase activity and BCL11A mRNA expressionimproving antioxidant ability of erythrocyte reducing inclu-sion content of erythrocyte and improving the structure andfunction of erythrocyte

Conflict of Interests

The authors declared no conflict of interests regarding thepublication of this paper

Acknowledgments

This research was supported by National Basic ResearchProgram of China (ldquo973rdquo Program) (no 2010CB530406) andNational Funds of Natural Sciences (no 81173167)

References

[1] M Al-Khabori S Bhandari M Al-Huneini K Al-Farsi VPanjwani and S Daar ldquoSide effects of deferasirox iron chelationin patients with beta thalassemia major or intermediardquo OmanMedical Journal vol 28 no 2 pp 121ndash124 2013

[2] S L Schrier E Rachmilewitz and N Mohandas ldquoCellularand membrane properties of alpha and beta thalassemic ery-throcytes are different Implication for differences in clinicalmanifestationsrdquo Blood vol 74 no 6 pp 2194ndash2202 1989

[3] M D Scott P Rouyer-Fessard M Soda Ba B H Lubin and YBeuzard ldquo120572- and120573-haemoglobin chain induced changes in nor-mal erythrocyte deformability cComparison to 120573 thalassaemiaintermedia and Hb H diseaserdquo British Journal of Haematologyvol 80 no 4 pp 519ndash526 1992

[4] W J Wang Z K Wu X H Zhang et al ldquoInvestigation ofsurvival situation of 197 thalassemic cases treated with BushenYisui methodrdquo Chinese Journal of Information on TraditionalChinese Medicine vol 15 no 3 pp 21ndash22 2008

[5] Z-K Wu Y-M Liu X-H Zhang et al ldquoTreatment of 120573-thalassemia with Bushen Yisui therapy a randomized con-trolled trialrdquo Journal of Chinese Integrative Medicine vol 5 no2 pp 137ndash140 2007

[6] Z-K Wu X-H Zhang and M Li ldquoClinical observation oneffects of Yisui Shengxue Granule in treating 156 patients with120573-thalassemiardquo Chinese Journal of Integrated Traditional andWestern Medicine vol 26 no 4 pp 352ndash354 2006


[7] W JWang Z KWu XH Zhang et al ldquoClinical observation ofYisui ShengxueGranule in treating 25 patientswith hemoglobinH diseaserdquo Journal of Chinese Integrative Medicine vol 6 no 2pp 153ndash156 2008

[8] Z N Zhang Criteria for Diagnosis and Therapeutic Effect onHematopathy Science Press Beijing China 2007

[9] W J Wang Z K Wu X H Zhang et al ldquoEffect of YisuiShengxue Granule on the oxidative damage of erythrocytesfrom patients with hemoglobin H diseaserdquo Chinese Journal ofIntegrative Medicine vol 18 no 9 pp 670ndash675 2012

[10] E A Rachmilewitz and P J Giardina ldquoHow I treat thalassemiardquoBlood vol 118 no 13 pp 3479ndash3488 2011

[11] V G Sankaran T F Menne J Xu et al ldquoHuman fetalhemoglobin expression is regulated by the developmental stage-specific repressor BCL11Ardquo Science vol 322 no 5909 pp 1839ndash1842 2008

[12] M D Cappellini V Viprakasit andA T Taher ldquoAn overview ofcurrent treatment strategies for 120573-thalassemiardquo Expert Opinionon Orphan Drugs vol 2 no 7 pp 665ndash679 2014

[13] D Garrick M de Gobbi V Samara et al ldquoThe role of thepolycomb complex in silencing 120572-globin gene expression innonerythroid cellsrdquo Blood vol 112 no 9 pp 3889ndash3899 2008

[14] M C Lorincz D Schubeler S R Hutchinson D R Dickersonand M Groudine ldquoDNA methylation density influences thestability of an epigenetic imprint and Dnmt3ab-independentde novo methylationrdquo Molecular and Cellular Biology vol 22no 21 pp 7572ndash7580 2002

[15] J ChinM Singh V Banzon et al ldquoTranscriptional activation ofthe 120574-globin gene in baboons treated with decitabine and in cul-tured erythroid progenitor cells involves differentmechanismsrdquoExperimental Hematology vol 37 no 10 pp 1131ndash1142 2009

[16] D E Bauer and S H Orkin ldquoUpdate on fetal hemoglobingene regulation in hemoglobinopathiesrdquo Current Opinion inPediatrics vol 23 no 1 pp 1ndash8 2011

[17] V Lulli P Romania O Morsilli et al ldquoMicroRNA-486-3pregulates 120574-globin expression in human erythroid cells bydirectly modulating BCL11Ardquo PLoS ONE vol 8 no 4 ArticleID e60436 2013

[18] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005

[19] P Roussou N J Tsagarakis D Kountouras S Livadas andE Diamanti-Kandarakis ldquoBeta-thalassemia major and femalefertility the role of iron and iron-induced oxidative stressrdquoAnemia vol 2013 Article ID 617204 9 pages 2013

[20] D Rund and E Rachmilewitz ldquoMedical progress 120573-thalasse-miardquoThe New England Journal of Medicine vol 353 no 11 pp1135ndash1146 2005

[21] E Fibach and E Rachmilewitz ldquoThe role of oxidative stress inhemolytic anemiardquoCurrentMolecularMedicine vol 8 no 7 pp609ndash619 2008

[22] H Duman D Canatan G Alanoglu et al ldquoThe antioxidanteffects of capparis ovata and deferasirox in patients withthalassemia majorrdquo Journal of Blood Disorders amp Transfusionvol 4 no 3 p 3 2013

[23] V Kukongviriyapan N Somparn L Senggunprai A PrawanU Kukongviriyapan andA Jetsrisuparb ldquoEndothelial dysfunc-tion and oxidant status in pediatric patients with hemoglobin E-120573 thalassemiardquo Pediatric Cardiology vol 29 no 1 pp 130ndash1352008

[24] S Prasartkaew A Bunyaratvej S Fucharoen and P WasildquoOxidative stress and antioxidative enzymes in hemoglobin H

diseaserdquo Birth Defects Original Article Series vol 23 no 5 pp193ndash198 1987

[25] P Yenchitsomanus and P Wasi ldquoIncreased erythrocyte super-oxide dismutase activities in 120573 0-thalassaemiahaemoglobin Eand in haemoglobin H diseasesrdquo Journal of Clinical Pathologyvol 36 no 3 pp 329ndash333 1983

[26] S Ong-Ajyooth K Suthipark D Shumnumsirivath ALikidlilid S Fucharoen and P Pootrakul ldquoOxidative stressand antioxidants in 120573-thalassaemiahaemoglobin Erdquo Journal ofthe Medical Association of Thailand vol 70 no 5 pp 270ndash2741987

[27] S Vatanavicharn M Anuwatanakulchai P Yenchitsomanu etal ldquoRelationship of serum vitamin E erythrocyte nonhemeiron and malonyldialdehyde (lipid membrane peroxidationproduct) in thalassemiardquo Birth Defects vol 23 no 5A pp 207ndash211 1987

[28] E A Rachmilewitz S B Shohet and B H Lubin ldquoLipidperoxidation in beta-thalassemia majorrdquo Blood vol 47 no 3pp 495ndash505 1976

[29] J Stocks E L Offerman C B Modell and T L DormandyldquoThe susceptibility to autoxidation of human red cell lipids inhealth and diseaserdquo British Journal of Haematology vol 23 no6 pp 713ndash724 1972

[30] S L Schrier and N Mohandas ldquoGlobin-chain specificity ofoxidation-induced changes in red blood cell membrane prop-ertiesrdquo Blood vol 79 no 6 pp 1586ndash1592 1992

[31] E Shinar and E A Rachmilewitz ldquoOxidative denaturation ofred blood cells in thalassemiardquo Seminars in Hematology vol 27no 1 pp 70ndash82 1990

[32] J Cui Z L Zhao and X Y Hong ldquoBio-antioxidants with illtherapyrdquo Journal of Tsinghua University (Science and Technol-ogy) vol 40 no 6 p 9 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of kidney-nourishing and marrow-replenishing therapy onthalassemia we collected 16 cases of thalassemia patients inNanning Guangxi (8 cases with 120572-thalassemia and 8 caseswith 120573-thalassemia) and explored the biological mechanismsof clinical effect based on pathological mechanism of hemol-ysis and anemia

2 Materials and Methods

21 Diagnostic Criteria The diagnostic criteria of westernmedicine for intermedia thalassemia were referred to ldquoCri-teria for Diagnosis and Therapeutic Effect on Hematopathyrdquoedited by Zhang [8]

22 Inclusion Exclusion andWithdrawal Criteria The inclu-sion criteria were (1) for the people who accord with thediagnostic criteria ofwesternmedicine (2)Theage of patientsranged from 4 to 40 years old (3) Patients volunteered toparticipate in this study and they signed informed consentforms

The exclusion criteria were as follows (1) the patients whohave immunologic deficiency or primary diseases of the liverkidney or blood system (2) the patients who are pregnant (3)the patients who are allergic to this drug ingredients and (4)the patients who took the antianemia drugs in the last twomonths

The withdrawal criteria were as follows (1) the patientswho did not take drugs following requirements of the studyprotocol quit by themselves or were lost to follow-up and (2)patients who took any antianemia drugs and received bloodtransfusion in the treatment were rejected

23 Study Population and General Data Sixteen outpatients(8 cases of 120572-thalassemia and 8 cases of 120573-thalassemia) wereenrolled from theDepartment ofHematology 303Hospital ofChinese Peoplersquos Liberation Army (PLA) during the periodfrom October to December in 2013 Sixteen patients were7 to 26 years old with an average of 1319 plusmn 547 yearsThe patients of Zhuang and Han nationalities were 8 casesrespectively Of the 16 patients 10 were males and 6 werefemales Cases of mild anemia were 3 and cases of moderateanemia were 13 Three genotypes of 120572-thalassemia weredetected 1 case of genotype ofmdashSEA

12057242 1 case of genotype

ofmdashSEA12057237 6 cases of genotype ofmdashSEA

120572CS120572 Six genotypes

of 120573-thalassemia were detected 2 cases of genotypes of 17282 cases of genotypes of 41-42-28 1 case of genotypes of 17E1 case of genotypes of 17N 1 case of genotypes of 28IVS1-1and 1 case of genotypes of 4317

24 Drugs and Interventions A self-control studywas carriedout The course of treatment was 3 months YSSXG was pro-duced byGuangrsquoanmenHospital Preparation Factory accord-ing to the protocol described in patent (number CN1872182batch number 20120516) The modified YSSXG consisted of11 Chinese herbalmedicinal components RhizomaKaempfe-riae Radix PolygoniMultiflori RadixRehmanniae PreparataRadix Astragali Radix Codonopsis Radix Angelicae Sinen-sis Fructus Psoraleae Colla Corii Asini Caulis SpatholobiCarapax Trionycis and Fructus Amomi A pack of granules

contains 10 g powder (1 g powder contains 2368 g crudedrug)

Patients aged 2 to 6 years old were instructed to take halfa pack of granules twice daily aged 6 to 10 years old a fullpack of granules twice daily and aged over 10 years old a packof granules thrice daily The granules are dissolved in warmwater and taken orally Patients were required to have noblood transfusion during the observation period and askedto insist on the treatment regimen

25 Blood Sample Collection Venous blood samples (5mL)were collected into EDTA tubes from 16 patients withthalassemia disease before and after YSSXG treatment Theblood mixed by horizontal shaker was centrifuged at1700 rmin for 10min The upper layer of the plasma wasdiscarded The lower layer of blood cells which was shoppedat lymphocyte separationmediumwere added saline to 5mLand then centrifuged at 2500 rmin for 20min Mononuclearcell layer which was located between the plasma and thelymphocyte separation liquid was drawn to 15mL centrifugetube then was added saline to 10mL and centrifuged at1800 rmin for 10min twiceThe abovemixturewas discardedafter centrifuging and the precipitation mononuclear cellswere added to 1mL TRIZOL reagent and stored in EP tubesfrozen at minus80∘C refrigerator for RNA extraction After theblood isolated by lymphocytes separationmedium red bloodcells were at the lowest layer The red blood cells were addedsaline to 10mLand centrifuged at 1700 rmin for 5minwhosesupernatant was discarded Pure red blood cells was storedat frozen pipes for minus80∘C refrigerator Venous blood samples(4mL) were collected into non-anticoagulant tube from 16patients with thalassemia disease before and after YSSXGtreatment which were centrifuged at 1700 rmin for 10minto obtain blood serum for detection

26 Indicators of Observation and Detection

261 Reagents Lymphocyte separation medium was pur-chased from Solarbio Co (China) (batch number P8610)Trizol reagent was purchased from Invitrogen USA (batchnumber 14105) Test kits for superoxide dismutase (SOD)malonaldehyde (MDA) and glutathione peroxidase (GSH-Px) were purchased from Nanjing Jiancheng Institute ofBioengineering (batch number 20140224) RNA Mini Kitwas purchased from Tianjin Co (China) (batch number139315390) RevertAid First Strand cDNA Synthesis Kit waspurchased from Fermentas LT (Lithuania) (batch num-ber 00146314) Power SYBR Green PCR Master Mix waspurchased from ABI USA (batch number 1402445) Theantibody against DNMT1 and DNMT3a was purchased fromCST (batch number 5119 2160) DNMT3b was purchasedfrom Abcam (batch number ab79822) and 120573-actin waspurchased fromBeijing ZhongshanGolden Bridge BiologicalTechnologyCo (China) (batch number TA-09) BCAProteinAssay Kit was purchased from Beijing ComWin BiotechCo Ltd (batch number 02912E) Test Kit for Mini ATPenzyme (Na+K+ Ca2+Mg2+ and T-ATP enzyme) of RBCwaspurchased from Nanjing Jiancheng Institute of Bioengineer-ing (batch number 20140606)
















120574- G120574-globin 55∘C








Notes 119875 lt 005 119875 lt 001






3 Results












140

120

100

80

60

40

20

0


Con

tent

(gL

)Hb

lowastlowast

lowastlowast

(a)


lowast

6

5

4

3

2

1

0

RBC

Con

tent

(times1012L

)(b)


Ret12

10

8

6

4

2

0

Con

tent

()

lowast

lowast



(c)

120573-Thalassemia

HbF

Con

tent

()

100

90

80

70

60

50

40

30

20

10

0

lowastlowast

lowastlowast



(d)






Notes 119875 lt 005 119875 lt 001


DNMT1 DNMT3a DNMT3b

Pretreatment

12

1

08

06

04

02

0

DN

MTs

mRN

A ex

pres

sion

lowastlowast

lowastlowast

lowastlowast


(a)

5 6 7 8 5 6 7 8


DNMT3a

DNMT3b

DNMT1

120573-Actin

1 2 3 4 1 2 3 4


DNMT3a

DNMT3b

DNMT1

120573-Actin

(b)

04

035

03

025

02

015

01

005

0

DN

MTs

pro

tein

expr

essio

n

lowast

lowast

DNMT1 DNMT3a DNMT3b


(c)






12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast






0046 0018 0069

Notes 119875 lt 005 119875 lt 001





4 Discussion





9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































120574- G120574-globin 55∘C








Notes 119875 lt 005 119875 lt 001






3 Results












140

120

100

80

60

40

20

0


Con

tent

(gL

)Hb

lowastlowast

lowastlowast

(a)


lowast

6

5

4

3

2

1

0

RBC

Con

tent

(times1012L

)(b)


Ret12

10

8

6

4

2

0

Con

tent

()

lowast

lowast



(c)

120573-Thalassemia

HbF

Con

tent

()

100

90

80

70

60

50

40

30

20

10

0

lowastlowast

lowastlowast



(d)






Notes 119875 lt 005 119875 lt 001


DNMT1 DNMT3a DNMT3b

Pretreatment

12

1

08

06

04

02

0

DN

MTs

mRN

A ex

pres

sion

lowastlowast

lowastlowast

lowastlowast


(a)

5 6 7 8 5 6 7 8


DNMT3a

DNMT3b

DNMT1

120573-Actin

1 2 3 4 1 2 3 4


DNMT3a

DNMT3b

DNMT1

120573-Actin

(b)

04

035

03

025

02

015

01

005

0

DN

MTs

pro

tein

expr

essio

n

lowast

lowast

DNMT1 DNMT3a DNMT3b


(c)






12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast






0046 0018 0069

Notes 119875 lt 005 119875 lt 001





4 Discussion





9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Notes 119875 lt 005 119875 lt 001






3 Results












140

120

100

80

60

40

20

0


Con

tent

(gL

)Hb

lowastlowast

lowastlowast

(a)


lowast

6

5

4

3

2

1

0

RBC

Con

tent

(times1012L

)(b)


Ret12

10

8

6

4

2

0

Con

tent

()

lowast

lowast



(c)

120573-Thalassemia

HbF

Con

tent

()

100

90

80

70

60

50

40

30

20

10

0

lowastlowast

lowastlowast



(d)






Notes 119875 lt 005 119875 lt 001


DNMT1 DNMT3a DNMT3b

Pretreatment

12

1

08

06

04

02

0

DN

MTs

mRN

A ex

pres

sion

lowastlowast

lowastlowast

lowastlowast


(a)

5 6 7 8 5 6 7 8


DNMT3a

DNMT3b

DNMT1

120573-Actin

1 2 3 4 1 2 3 4


DNMT3a

DNMT3b

DNMT1

120573-Actin

(b)

04

035

03

025

02

015

01

005

0

DN

MTs

pro

tein

expr

essio

n

lowast

lowast

DNMT1 DNMT3a DNMT3b


(c)






12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast






0046 0018 0069

Notes 119875 lt 005 119875 lt 001





4 Discussion





9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















140

120

100

80

60

40

20

0


Con

tent

(gL

)Hb

lowastlowast

lowastlowast

(a)


lowast

6

5

4

3

2

1

0

RBC

Con

tent

(times1012L

)(b)


Ret12

10

8

6

4

2

0

Con

tent

()

lowast

lowast



(c)

120573-Thalassemia

HbF

Con

tent

()

100

90

80

70

60

50

40

30

20

10

0

lowastlowast

lowastlowast



(d)






Notes 119875 lt 005 119875 lt 001


DNMT1 DNMT3a DNMT3b

Pretreatment

12

1

08

06

04

02

0

DN

MTs

mRN

A ex

pres

sion

lowastlowast

lowastlowast

lowastlowast


(a)

5 6 7 8 5 6 7 8


DNMT3a

DNMT3b

DNMT1

120573-Actin

1 2 3 4 1 2 3 4


DNMT3a

DNMT3b

DNMT1

120573-Actin

(b)

04

035

03

025

02

015

01

005

0

DN

MTs

pro

tein

expr

essio

n

lowast

lowast

DNMT1 DNMT3a DNMT3b


(c)






12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast






0046 0018 0069

Notes 119875 lt 005 119875 lt 001





4 Discussion





9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DNMT1 DNMT3a DNMT3b

Pretreatment

12

1

08

06

04

02

0

DN

MTs

mRN

A ex

pres

sion

lowastlowast

lowastlowast

lowastlowast


(a)

5 6 7 8 5 6 7 8


DNMT3a

DNMT3b

DNMT1

120573-Actin

1 2 3 4 1 2 3 4


DNMT3a

DNMT3b

DNMT1

120573-Actin

(b)

04

035

03

025

02

015

01

005

0

DN

MTs

pro

tein

expr

essio

n

lowast

lowast

DNMT1 DNMT3a DNMT3b


(c)






12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast






0046 0018 0069

Notes 119875 lt 005 119875 lt 001





4 Discussion





9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















12

1

08

06

04

02

0

BCL11

A m

RNA

expr

essio

n

lowast






0046 0018 0069

Notes 119875 lt 005 119875 lt 001





4 Discussion





9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















9000

8000

7000

6000

5000

4000

3000

2000

1000

0

Con

tent

(Ug

Hb)


(a)


Con

tent

(Ug

Hb)

180

160

140

120

100

80

60

40

20

0

(b)

200

180

160

140

120

100

80

60

40

20

0


Con

tent

(Ug

Hb)

(c)

1200

1000

800

600

400

200

0

GSH-Px of serum

lowastlowast

Con

tent

(Ug

Hb)

(d)

35

3

25

2

15

1

05

0

MDA of RBC

Con

tent

(nm

olm

g)


lowastlowast

(e)

6

5

4

3

2

1

0

MDA of serum

Con

tent

(nm

olm

g)

lowastlowast


(f)



(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)






120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120

100

80

60

40

20

0

Con

tent

(Ug

Hb)

lowast

lowast







5 Conclusion




Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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