Oregano Essential Oil Improves Intestinal Morphology and

Research ArticleOregano Essential Oil Improves Intestinal Morphologyand Expression of Tight Junction Proteins Associated withModulation of Selected Intestinal Bacteria and ImmuneStatus in a Pig Model

Yi Zou12 Quanhang Xiang12 Jun Wang12 Jian Peng12 and Hongkui Wei12

1Department of Animal Nutrition and Feed Science College of Animal Science and Technology Huazhong Agricultural UniversityWuhan 430070 China2The Cooperative Innovation Center for Sustainable Pig Production Wuhan 430070 China

Correspondence should be addressed to Jian Peng pengjianmailhzaueducn and Hongkui Wei weihongkuimailhzaueducn

Received 1 March 2016 Revised 3 May 2016 Accepted 8 May 2016

Academic Editor Kai Wang

Copyright copy 2016 Yi Zou et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Oregano essential oil (OEO) has long been used to improve the health of animals particularly the health of intestine which isgenerally attributed to its antimicrobial and anti-inflammatory effects However howOEO acts in the intestine of pig is still unclearThis study was aimed at elucidating how OEO promotes the intestinal barrier integrity in a pig model Pigs were fed a control dietalone or one supplemented with 25mgkg of OEO for 4 weeksThe OEO-treated pigs showed decreased (119875 lt 005) endotoxin levelin serum and increased (119875 lt 005) villus height and expression of occludin and zonula occludens-1 (ZO-1) in the jejunum Theseresults demonstrated that the integrity of intestinal barrier was improved by OEO treatment The OEO-treated pigs had a lower(119875 lt 005) population of Escherichia coli in the jejunum ileum and colon than the control This is in accordance with the greaterinactivation (119875 lt 005) of inflammation which was reflected by the mitogen-activated protein kinase (MAPK) protein kinase B(Akt) and nuclear factor 120581B (NF-120581B) signaling pathways and expression of inflammatory cytokines in the jejunum Our resultsshow that OEO promotes intestinal barrier integrity probably through modulating intestinal bacteria and immune status in pigs

1 Introduction

The ability of the intestinal epithelium to function as abarrier between the external environment and the closelyregulated internal milieu is essential for human and pighealth [1 2] Increased intestinal permeability is a potentialfactor of gastrointestinal dysfunction and pathology includ-ing Crohnrsquos disease multiple organ dysfunction bacterialtranslocation food allergies and acute pancreatitis [3 4]Currently antibiotics are widely available and have a varietyof proposed beneficial effects to promote intestinal healthbut the application of these drugs is limited by their toxicityand side effects [5] Therefore it is urgent to find alternativetreatments with fewer side effects

Oregano (Origanum vulgare L) is an aromatic plantwidely distributed throughout the Mediterranean area and

Asia [6] Oregano essential oil (OEO) a volatile oil isconcentrated from natural plant products which containthe volatile aroma compounds These mixtures of volatilecompounds exert different biological actions such as antimi-crobial anti-inflammatory and antioxidative activities [7]There have been evidences showing the therapeutic effectsof OEO supplementation on barrier defects in the gut ofmammals including mouse rat and broiler models [8ndash10]However there has been no report about the use of OEOsupplementation to improve the intestinal barrier integrity ofpigs

Several studies have indicated that intestinal microbiotaand immune status are important factors that influencethe function of the intestinal barrier [11] Alteration ofthe microbial composition results in increased immunestimulation epithelial dysfunction or enhanced mucosal

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 5436738 11 pageshttpdxdoiorg10115520165436738

2 BioMed Research International

permeability [12] In the present study we hypothesized thatdietary OEO supplementation promotes intestinal barrierintegrity by regulating intestinal bacteria and inflammationWe thus would use the pig model to test this hypothesisand study the morphology and permeability of the intestinethe composition of the intestinal microbiota the activationof innate immunity and the expression of proinflammatorycytokines after OEO supplementation

2 Materials and Methods

All animal handling protocols were approved by theHuazhong Agricultural University Animal Care and UseCommittee guidelines

21 Animals Diets and Treatments A total of 170 pigs(Large White times Landrace) with an initial body weight(BW) of 72 kg (plusmn40 kg) were obtained from the same farm(Wuhan China Pork Co Ltd Wuhan China)The pens werelocated in a building at a temperature maintained between15 and 25∘C For the experiment the 170 pigs were splitinto two groups according to the diet (each group of 85pigs was further split into 5 replicate pens each holding 17pigs) (1) control treatment without supplementation and(2) supplementation of OEO (25mgkg of feed for 28 d as-fed basis) The composition of the control diet is shownin Table 1 The OEO was in the form of a powder calledPhytogen (Meritech Bioengineering Co Ltd GuangzhouChina) The components of OEO are shown in Supplemen-tary Table S1 (see Supplementary Material available onlineat httpdxdoiorg10115520165436738) Pigs were allowedfeed and water ad libitum over a period of 4 weeks

22 Sample Collection On the day of slaughter between900 and 1400 a total of 12 pigs (100 kg BW) with 6 pigsfrom each dietary treatment were transported in an opentruck to the slaughterhouse Blood samples were collectedafter electrical stunning and then quickly separated into fivetubes A 10mL sample was placed on ice immediately whichwas subsequently centrifuged at 1300timesg at 4∘C for 15min toobtain serum The serum samples were stored at minus80∘C forsubsequent analysis The digesta samples were immediatelyremoved from the jejunum ileum and colon of each pig andstored at minus80∘Cuntil further analysis Samples of the jejunumitself were removed from the middle jejunum segment andthen rinsed with ice-cold physiological saline One sectionwas snap-frozen in liquid nitrogen and then stored at minus80∘Cuntil further analysis Other sections of jejunum (3 cm) werekept in 4 neutral buffered formalin for gut morphologicalanalysis

23 Gut Morphological Analysis The digestive tract wasremoved from the jejunum and fixed in 10 phosphate-buffered formalin The samples were sectioned at 5mmthickness and stained with hematoxylin and eosin Villusheight villus width and villus crypt depth were measuredon the stained sections using a light microscope fitted withan image analyzer (Image Pro Plus 60 Media Cybernetics

Table 1 Composition and analysis of the basal diet

Basal dietlowast

Composition (gkg)Wheat 38000Corn grains 46410Soybean meal (46) 8900Monocalcium phosphate 1400Limestone 700Mycetes adsorbent 150Antimildew agent 050Salt 350Soybean oil 2000Ethoxyquin 025Probiotics 020Y402 premixdagger 2000AnalysisDagger

Dry matter DM () 8680Metabolism energy (MJkg) 1320Crude protein CP () 1390Crude fiber () 280Ash () 360Fat () 430Calcium () 060Phosphorus () 060lowastControl group (C) was fed with the above basal diet whereas the oreganoessential oil (OEO) group consumed the basal diet supplemented with25mgkg OEOdaggerPremix contained per kg 105 g Fe 14 gCu 85 gZn 4 gMn 75mg Se 30mgI 350 kIU of vitamin A 40 kIU of vitamin D3 15 kIU of vitamin E 50mg ofvitamin K3 50mg of vitamin B1 150mg of vitamin B2 100mg of vitaminB6 01mg of vitamin B12 864 g lysine 175 g methionine 25 g threonine 4 gphytase and 15 g choline (kIU 1000 international units)DaggerMetabolism energy was calculated from data provide by Feed Database inChina (1999)

Bethesda MD USA) Twenty villi and crypts were measuredfor each segment

24 Measurement of Serum Endotoxin Level Serum endo-toxin level was measured by a quantitative chromogenicend point tachypleus amebocyte lysate endotoxin detectionkit following the manufacturerrsquos instructions (Xiamen TALExperimental Plant Co Ltd China) Briefly serum sam-ples were diluted to 1 10 with waterTris-HCl buffer Aftercentrifugation at 1270timesg for 10min the supernatant wasremoved and incubated with limulus amebocyte lysate at37∘C for 10min followed by incubation with the providedchromogenic substance for 6min The absorbance at 545 nmwas measured after the addition of appropriate reagents

25 Extraction of Microbial DNA from the GastrointestinalTract Digesta Total DNA of jejunum ileum and colondigesta was extracted and purified from gastrointestinal tractdigesta using a QIAamp DNA Stool Kit (Qiagen Germany)according to the manufacturerrsquos instructions DNA concen-tration was determined by spectrophotometry (NanoDrop)

BioMed Research International 3

Table 2 Species and genus specific primers used for real-time PCR

Gene Primers (senseantisense 51015840-31015840) Size (bp) Annealing temperature (∘C)

Total bacteria F ACTCCTACGGGAGGCAGCAGR ATTACCGCGGCTGCTGG 175 60

Lactobacillus spp F CACCGCTACACATGGAGR TGGAAGATTCCCTACTGCT 341 58

Escherichia coli F CATGCCGCGTGTATGAAGAAR TTTGCTCATTGACGTTACCCG 96 60

Enterococcus faecalis F CCCTTATTGTTAGTTGCCATCATTR ACAATGGGAAGTACAACGAGT 144 61

TNF-120572 F CACCACGCTCTTCTGCCTACTGR TTGAGACGATGATCTGAGTCCTTGG 115 63

MCP-1 F GTCCTTGCCCAGCCAGATGR CGATGGTCTTGAAGATCACTGCT 148 60

IL-1120573 F AAAGGGGACTTGAAGAGAGR CTGCTTGAGAGGTGCTGATGT 286 58

IL-6 F AAGGTGATGCCACCTCAGACR TCTGCCAGTACCTCCTTGCT 151 60

INF-120574 F GAGCCAAATTGTCTCCTTCTACR CGAAGTCATTCAGTTTCCCAG 140 61

ZO-1 F GGCGCACGGCGAAGGTAATTR CTATCAAACTCAGGAGGCGGCACT 405 60

Occludin F GGAGTGATTCGGATTCTGTCTATGCTR CGCCTGGGCTGTTGGGTTGA 423 60

120573-actin F CCAGGTCATCACCATCGGR CCGTGTTGGCGTAGAGGT 158 60

26 Protein Immunoblot Analysis Briefly 100mg of frozentissue of jejunum was homogenized in 1mL RIPA lysis buffer(with 1mMPMSF and 10 120583LmL phosphatase inhibitors) Fornuclear p65 measurement the nuclear fractions were iso-lated using the NuclearCytosol Fractionation Kit (BestBioChina) Next they were centrifuged at 12000timesg at 4∘C for10min and the supernatants were collected for assay Afterthe protein concentration was determined by a standardBCA protein assay protein sample was loaded per laneand separated on SDS-PAGE The target protein was thenelectrophoretically transferred to nitrocellulose membraneswhich were blocked in TBST (5 nonfat milk 10mM Tris150mM NaCl and 005 Tween-20) for 2 h Next they wereincubated with first antibodies anti-Phospho-p38 MAPK(1 1000 Cell Signaling USA) anti-Phospho-JNK (1 1000Cell Signaling USA) anti-Phospho-ERK12 (1 1000 CellSignaling USA) anti-Phospho-Akt (1 1000 Cell SignalingUSA) anti-p38 MAPK (1 1000 Affbiotech USA) anti-JNK (1 1000 Affbiotech USA) anti-ERK12 (1 1000 Aff-biotech USA) anti-Akt (1 1000 Affbiotech USA) anti-ZO-1(1 1000AffbiotechUSA) anti-occludin (1 1000AffbiotechUSA) anti-NF-120581B p65 (1 1000 Cell Signaling USA) anti-PCNA (1 5000 BD Transduction Laboratories San DiegoCA) or anti-actin antibodies (1 1000 Cell Signaling USA)at 4∘C overnight After three washes with Tris-bufferedsaline containing 01 Tween-20 blots were incubated withthe HRP-conjugated secondary antibodies anti-rabbit IgG(1 15000 Jackson ImmunoResearch USA) or anti-mouseIgG (1 15000 Jackson ImmunoResearch USA) for 2 hand were washed again Chemiluminescence detection was

performed using the ECL reagent (Thermo Scientific Rock-ford IL USA) according to the manufacturerrsquos instructionsSpecific bands were detected and were analyzed and quanti-fied by Image J Software (NIH Bethesda MD USA)

27 Quantitative PCR Total RNA was extracted from sam-ples of jejunum using Trizol Reagent (Invitrogen USA)according to the manufacturerrsquos instructions The DNAobtained from the intestinal luminal content was used asthe template to analyze intestinal bacteria Primers (Table 2)used in this study were either synthesized according to ourprevious protocols or designed with Primer 50 according topig gene sequences Real-time PCRwas performed accordingto our previous study [10] The relative expression of genes inthe treatment group was normalized based on the values ofthe control group

28 Statistics Statistical analysis was performed using Prismsoftware (Prism 50 GraphPad Software La Jolla CA)Numbers (119899) used for statistics are noted in the figuresAll data were analyzed by 119905-test procedures of SAS (v 82SAS Inst Inc Cary NC) All the values were presented asmeans plusmn standard error of the mean (SEM) and those at119875 lt 005 were considered significant

3 Results

31 Morphology of the Jejunum Morphology is a goodindicator of the status of the intestine [13] As shown inFigure 1 although no significant differences were observed in


Control OEO0

200

400

600Vi

llus h

eigh

t (120583

m)

lowastlowast

(a)

Villu

s wid

th (120583

m)

Control OEO0

50

100

150

(b)

Cryp

t dep

th (120583

m)

Control OEO0

100

200

300

400

(c)Control OEO

00

05

10

15

20

Villu

s hei

ght

Cry

pt d

epth

ratio

(d)

Figure 1 Effect of OEO on villus height villus width crypt depth and the villus height crypt depth ratio in the jejunum of pig Values aremeans plusmn SEM 119899 = 6 lowastlowastSignificantly different (119875 lt 001) from the control group

Control OEO

Figure 2 Effect of OEO on jejunum morphology in pig The jejunum was cut off and fixed in 10 formaldehyde-phosphate buffer and thenstained with hematoxylin and eosin Hematoxylin and eosin staining with original magnification times100 Bars represent 200 120583m

villus width crypt depth and the ratio of villus height to cryptdepth between different treatments (119875 gt 005) the villusheight of the OEO-treated pigs was significantly higher thanthat of the control (119875 lt 005) As shown in Figure 2 the villiwere scattered and seriously desquamated in the jejunum ofthe control group while higher and intact villi were observedin the jejunum of OEO-treated pigs

32 Endotoxin Levels in the Serum Endotoxin level is auseful biomarker for evaluating the integrity of the gastroin-testinal tract [14] The effects of OEO supplementation onendotoxin level in the serum of pigs are shown in Figure 3The OEO-treated pigs showed a significantly lower (119875 lt005) concentration of endotoxin in the serum than thecontrol


Endotoxin

Control OEO0

1

2

3EU

(mL)

lowast

Figure 3 Effect of OEO on endotoxin levels in the serum of pigValues are means plusmn SEM 119899 = 6 lowastSignificantly different (119875 lt 005)from the control group

33 The Expression of Barrier Tight Junction Proteins in theJejunum To determine the effects of OEO supplementationon the intestinal mucosal tight junction the expression ofoccludin and zonula occludens-1 (ZO-1) was measured atmRNA and protein level As shown in Figure 4 the mRNAlevels of occludin and ZO-1 were significantly higher in theOEO-treated pigs than in the control group (119875 lt 001)Similarly OEO supplementation also increased (119875 lt 005)the abundance of occludin and ZO-1 protein in the jejunum

34 Major Microbiota in Different Regions of the IntestinalTract The effects of OEO supplementation on selectedmicrobial populations in different intestinal tracts are shownin Figure 5 Although there were no significant differences(119875 gt 005) in the total bacteria Lactobacillus and Enterococ-cus spp populations the OEO-treated pigs had a significantlylower population of Escherichia coli (E coli) in the jejunum(119875 lt 005) ileum (119875 lt 005) and colon (119875 lt 001) comparedwith the control

35 MAPKs Akt and NF-120581B Pathways in the Jejunum Toinvestigate the mechanism responsible for the protectiveeffect of OEO against inflammation in the jejunum of pigswe further studied the underlying signaling pathways theMAPKs Akt and NF-120581B pathways which are inducedby intestinal microorganisms and have been shown to beimplicated in the induction of proinflammatory genes [15] Asshown in Figure 6(a) in the jejunum OEO supplementationinhibited (119875 lt 005) the activation of JNK ERK12and Akt due to lower abundance of phosphorylated JNKERK12 and Akt proteins compared with the control groupSimilarly OEO supplementation decreased (119875 lt 005) theabundance of NF-120581B p65 protein in the nucleus of thejejunum (Figure 6(b)) In contrast OEO supplementationhad no effect on phosphorylated p38

36 The mRNA Levels of Proinflammatory Cytokines inthe Jejunum We examined the gene expression levels offour major inflammatory cytokines involved in mucosal

inflammation in the jejunum tissues interleukin-1120573 (IL-1120573) interleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572)monocyte chemotactic protein-1 (MCP-1) and interferongamma (INF-120574) The results are presented in Figure 7 TheOEO-treated pigs showed significantly decreased (119875 lt 005)levels of TNF-120572 IL-1120573 IL-6 MCP-1 and INF-120574 comparedwith the control group

4 Discussion

Defects in the intestinal barrier of animals can be induced byseveral types of ongoing environmentallife factors linked tophysiology psychology antigens and toxins [16] Intestinalbarrier defects are a serious problem in human as well asin pigs [17 18] Various aromatic plants and their productshave been reported to have beneficial effects for the intestineof animals [19] Furthermore in a previous study of ourlaboratory it was also observed that the intestinal barriercould be improved by OEO supplementation in rat models[10] However to our knowledge there has been no reportabout the effect of OEO on the intestinal barrier of finishingpigs Therefore in the present study we investigated whetherdietary supplementation of OEO could have a protectiveeffect on the intestinal barrier and whether OEO could beused as a feed additive for pigs

Intestinal mucosal permeability is directly related to theintegrity of the intestinal barrier [20] The function of theintestinal barrier can be commonly assessed bymany indexessuch as serum endotoxin level intestine morphology andintestinal tight junction proteins [14 21 22] In the presentstudy the height of villi in the jejunum of pigs was increasedafter treatment with OEO indicating that OEO may protectthe intestine against villous atrophy and epithelial cell necro-sis Consistently the endotoxin level in pig serum decreasedsignificantly after treatment with OEO These results were inagreement with previous findings which demonstrated thatusing an OEO supplemented diet improves the ileum villusheight and decreases serum endotoxin level in broilers [23]In addition we found that the expression of occludin andZO-1 the two major tight junction proteins in epithelia affectingthe organization and the stability of the tight junction [21]was significantly decreased by OEO treatment SimilarlyPlacha et al [9] also observed that broilers fed with OEOhad improved intestinal barrier integrity Our results indicatethat OEO supplementation can be a promising approach forprotecting the intestinal barrier in pigs

The intestinal microbiota plays critical roles in the main-tenance of mucosal homeostasis [11] A greater population ofE colimight affect the intestinal mucosa because they releasetoxins resulting in an intimate interaction between themicrobiota and the host enterocytes [24 25] The abundanceand composition of intestinal bacteria can be easily affectedby various dietary factors [26] In the present study thedietary consumption of OEO decreased the populations ofE coli in the jejunum ileum and colon These results areconsistent with those of Tan et al [27] and Sun et al [23]who reported that that population of E coli in intestinalis decreased in OEO-treated pigs and broilers respectivelyThymol and carvacrol the main active components of


ZO-1

Control OEO0

1

2

3

4Re

lativ

e mRN

A le

vels

lowast

(a)

Occludin

Control OEO00

05

10

15

20

25

Relat

ive m

RNA

leve

ls

lowastlowast

(b)

ZO-1

ZO-1

Control OEO00

05

10

15

20

lowastlowast

Fold

of c

ontro

l

120573-actin

(c)

Occludin

Control OEO00

05

10

15

20

Occludin

lowastlowast

Fold

of c

ontro

l120573-actin

(d)

Figure 4 Effect of OEO on the ZO-1 and occludin levels in the jejunum of pig (a) ZO-1 mRNA level (b) occludin mRNA level (c) ZO-1protein level and (d) occludin protein level Expression of the selected genes was quantified by quantitative reverse transcription-PCR Equalloading was assessed by 120573-actin immunoblotting Values are means plusmn SEM 119899 = 6 lowastSignificantly different (119875 lt 005) from the control grouplowastlowastSignificantly different (119875 lt 001) from the control group ZO-1 zonula occludens-1

essential oil derived from thyme and oregano [7] were alsodocumented to inhibit the proliferation of E coli in vitro [28]

Recently it has been recognized that the gut microbiotacan influence the immune function beyond the gut [26] Therelationship between the immune system and the commensalflora is a precarious one and perturbations in immune orepithelial homeostasis can lead to gut inflammation [29]The initial sensing between bacteria and the host occursthrough pattern recognition receptors [30] In the intestinethe activated pattern recognition receptors trigger intesti-nal immune responses through various downstream signaltransductions such asMAPKs (eg p38MAPK ERK12 andJNK) Akt andNF-120581B [31 32]TheAkt andMAPKs pathwaysmodulate intestinal innate immunity through regulating the

phosphorylation of inhibitory 120581B kinases to activate the NF-120581B pathway [33] The NF-120581B pathway which is activatedby intestinal microbes plays an important role in activatinghost proinflammatory responses [34] The activation of thesepathways is associated with the increased expression of TNF-120572 IL-1120573 IL-6MCP-1 and INF-120574 [35 36]The innate immunesystem is activated as a defense mechanism and is generallybeneficial [37] However if the inflammation is uncontrolledthe migration of innate immune cells such as neutrophilsmacrophages and dendritic cells into the target mucosaltissues will result inmucosal injury [32] Regulating intestinalinflammation is of great significance for intestinal health[15 38 39] OEO has been found to possess a significant anti-inflammatory effect in vitro and in vivo [8 40] The present


Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent

Tot Lacto Entero E coil

(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)

Figure 5 Effect of OEO on selected microbial populations in the jejunum ileum and colon tract of pig Values are means plusmn SEM 119899 = 6lowastSignificantly different (119875 lt 005) from the control group lowastlowastSignificantly different (119875 lt 001) from the control group Tot total bacteriaLacto Lactobacillus spp Entero Enterococcus faecalis E coli Escherichia coli Log

10

16S rRNA gene copiesg contents

study indicates that OEO inhibits the inflammation in theintestine and downregulates the expression of TNF-120572 IL-1120573IL-6 INF-120574 andMCP-1 in the jejunum Similarly it has beenpreviously shown that OEO effectively reduces the produc-tion of proinflammatory cytokine and thereby attenuates theTNBS-induced colitis in mice [41] As the major componentof OEO carvacrol was also reported to probably influencethe release andor synthesis of inflammatory mediators [41]As the mechanisms mediating the suppressive effects of OEOon inflammation are still unclear we can only speculate thatthere might be several potential modes of action based onour results One possibility could be the influence of OEO onNF-120581B p65 and phosphorylated MAPKs (ERK12 and JNK)and Akt pathways which can activate the expression of thegenes involved in immune and inflammatory responses suchas TNF-120572 IL-1120573 IL-6 INF-120574 andMCP-1 On the other handintestinal microbial disorder (such as increased number of

E coli) is a potent inducer of intestinal inflammation Thepresent study has shown that OEO can inhibit intestinal Ecoli in pigs

5 Conclusion

In conclusion our results indicate that dietary administrationof OEO can reduce the production of proinflammatorycytokines and promote the integrity of the intestinal barrierin pigs The protective effect of OEO on the intestine isassociated with the decrease of intestinal E coli populationand the inactivation of the JNK ERK12 Akt and NF-120581B signaling pathways These results will contribute to abetter understanding of the possible mechanisms by whichOEO promotes the integrity of the intestinal barrier inpigs


Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)

Figure 6 Effect of OEO on the levels of p-Akt p-ERK p-p38 p-JNK and NF-120581B p65 proteins in the jejunum of pig (a) Akt protein ERKprotein p38 protein and JNK protein levels Equal loading was assessed by 120573-actin immunoblotting (b) Nuclear p65 protein level Equalloading was assessed by PCNA immunoblotting Values are means plusmn SEM 119899 = 6 lowastSignificantly different (119875 lt 005) from the control groupERK12 extracellular signal-regulated kinases 12 JNK c-Jun N-terminal protein kinase Akt protein kinase B NF-120581B nuclear factor kappaB P phosphorylated T total

Competing Interests

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

Acknowledgments

This studywas supported financially by the International SampTCooperation Projects of China (Grant no 2013DFG32510)


Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)

Figure 7 Effect of OEO on the mRNA levels of proinflammatory cytokines in the jejunum of pig Expression of the selected genes wasquantified by quantitative reverse transcription-PCR (a) IL-1120573mRNA level (b) IL-6mRNA level (c) TNF-120572mRNA level (d)MCP-1mRNAlevel and (e) INF-120574mRNA level Values are means plusmn SEM 119899 = 6 lowastSignificantly different (119875 lt 005) from the control group lowastlowastSignificantlydifferent (119875 lt 001) from the control group IL-1120573 interleukin-1120573 IL-6 interleukin-6 TNF-120572 tumor necrosis factor-120572 MCP-1 monocytechemotactic protein-1 INF-120574 interferon gamma

and theKeyTechnologyResearch andDevelopment ProgramofHubei Province (no 2014ABB014)The authors are gratefulto the colleagues for their helpful discussions and other kindmembers of the near laboratory for assisting with the samplecollection

References

[1] M Camilleri K Madsen R Spiller B G Van Meerveldand G N Verne ldquoIntestinal barrier function in health andgastrointestinal diseaserdquo Neurogastroenterology amp Motility vol24 no 6 pp 503ndash512 2012


[2] I P Oswald ldquoRole of intestinal epithelial cells in the innateimmune defence of the pig intestinerdquo Veterinary Research vol37 no 3 pp 359ndash368 2006

[3] H T Hassoun B C Kone D W Mercer F G Moody N WWeisbrodt and F AMoore ldquoPost-injurymultiple organ failurethe role of the gutrdquo Shock vol 15 no 1 pp 1ndash10 2001

[4] O D Rotstein ldquoPathogenesis of multiple organ dysfunctionsyndrome gut origin protection and decontaminationrdquo Sur-gical Infections vol 1 no 3 pp 217ndash225 2000

[5] Y H Kho M O Pool F G A Jansman and J W HartingldquoPharmacotherapeutic options in inflammatory bowel diseasean updaterdquo PharmacyWorld and Science vol 23 no 1 pp 17ndash212001

[6] M Milos J Mastelic and I Jerkovic ldquoChemical compositionand antioxidant effect of glycosidically bound volatile com-pounds from oregano (Origanum vulgare L ssp hirtum)rdquo FoodChemistry vol 71 no 1 pp 79ndash83 2000

[7] F Bakkali S Averbeck D Averbeck and M Idaomar ldquoBio-logical effects of essential oilsmdasha reviewrdquo Food and ChemicalToxicology vol 46 no 2 pp 446ndash475 2008

[8] A Bukovska S Cikos S Juhas G Ilrsquokova P Rehak andJ Koppel ldquoEffects of a combination of thyme and oreganoessential oils on TNBS-induced colitis in micerdquo Mediators ofInflammation vol 2007 Article ID 23296 9 pages 2007

[9] I Placha J TakacovaM Ryzner et al ldquoEffect of thyme essentialoil and selenium on intestine integrity and antioxidant status ofbroilersrdquo British Poultry Science vol 55 no 1 pp 105ndash114 2014

[10] H-KWei G Chen R-JWang and J Peng ldquoOregano essentialoil decreased susceptibility to oxidative stress-induced dysfunc-tion of intestinal epithelial barrier in ratsrdquo Journal of FunctionalFoods vol 18 pp 1191ndash1199 2015

[11] I I Ivanov and D R Littman ldquoModulation of immunehomeostasis by commensal bacteriardquoCurrentOpinion inMicro-biology vol 14 no 1 pp 106ndash114 2011

[12] D R Littman and E G Pamer ldquoRole of the commensalmicrobiota in normal and pathogenic host immune responsesrdquoCell Host amp Microbe vol 10 no 4 pp 311ndash323 2011

[13] A Viveros S Chamorro M Pizarro I Arija C Centeno andA Brenes ldquoEffects of dietary polyphenol-rich grape productson intestinal microflora and gut morphology in broiler chicksrdquoPoultry Science vol 90 no 3 pp 566ndash578 2011

[14] C B Forsyth K M Shannon J H Kordower et al ldquoIncreasedintestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in earlyParkinsonrsquos diseaserdquo PLoSONE vol 6 no 12 Article ID e280322011

[15] S Qi Y Xin Y Guo et al ldquoAmpelopsin reduces endotoxicinflammation via repressing ROS-mediated activationof PI3KAktNF-120581B signaling pathwaysrdquo InternationalImmunopharmacology vol 12 no 1 pp 278ndash287 2012

[16] J D Soderholm andM H Perdue ldquoStress and intestinal barrierfunctionrdquoAmerican Journal of PhysiologymdashGastrointestinal andLiver Physiology vol 280 no 1 pp G7ndashG13 2001

[17] A J Moeser L B Borst B L Overman and J S PittmanldquoDefects in small intestinal epithelial barrier function andmorphology associated with peri-weaning failure to thrivesyndrome (PFTS) in swinerdquo Research in Veterinary Science vol93 no 2 pp 975ndash982 2012

[18] JMMullin N Agostino E Rendon-Huerta and J JThorntonldquoKeynote review epithelial and endothelial barriers in humandiseaserdquoDrugDiscovery Today vol 10 no 6 pp 395ndash408 2005

[19] I Giannenas E Bonos E Christaki and P Florou-PanerildquoEssential oils and their applications in animal nutritionrdquoMedicinal amp Aromatic Plants vol 2 no 6 pp 1ndash12 2013

[20] Y Ma S Semba M R I Khan et al ldquoFocal adhesion kinaseregulates intestinal epithelial barrier function via redistributionof tight junctionrdquo Biochimica et Biophysica ActamdashMolecularBasis of Disease vol 1832 no 1 pp 151ndash159 2013

[21] T Suzuki ldquoRegulation of intestinal epithelial permeability bytight junctionsrdquoCellular andMolecular Life Sciences vol 70 no4 pp 631ndash659 2013

[22] M A M Vente-Spreeuwenberg J M A J Verdonk A CBeynen andMWAVerstegen ldquoInterrelationship between gutmorphology and faeces consistency in newly weaned pigletsrdquoAnimal Science vol 77 no 1 pp 85ndash94 2003

[23] Q Sun D Liu S Guo Y Chen and Y Guo ldquoEffects of dietaryessential oil and enzyme supplementation on growth perfor-mance and gut health of broilers challenged by Clostridiumperfringensrdquo Animal Feed Science and Technology vol 207 pp234ndash244 2015

[24] A Finamore M Roselli A Imbinto J Seeboth I P Oswaldand E Mengheri ldquoLactobacillus amylovorus inhibits theTLR4 inflammatory signaling triggered by enterotoxigenicEscherichia coli via modulation of the negative regulators andinvolvement of TLR2 in intestinal Caco-2 cells andpig explantsrdquoPLoS ONE vol 9 no 4 Article ID e94891 2014

[25] R B Sartor ldquoMicrobial influences in inflammatory boweldiseasesrdquo Gastroenterology vol 134 no 2 pp 577ndash594 2008

[26] K M Maslowski and C R MacKay ldquoDiet gut microbiota andimmune responsesrdquo Nature Immunology vol 12 no 1 pp 5ndash92011

[27] C TanHWei H Sun et al ldquoEffects of dietary supplementationof oregano essential oil to sows on oxidative stress statuslactation feed intake of sows and piglet performancerdquo BioMedResearch International vol 2015 Article ID 525218 9 pages2015

[28] I M Helander H-L Alakomi K Latva-Kala et al ldquoCharac-terization of the action of selected essential oil componentson gram-negative bacteriardquo Journal of Agricultural and FoodChemistry vol 46 no 9 pp 3590ndash3595 1998

[29] T TMacDonald andGMonteleone ldquoImmunity inflammationand allergy in the gutrdquo Science vol 307 no 5717 pp 1920ndash19252005

[30] S Akira K Takeda and T Kaisho ldquoToll-like receptors crit-ical proteins linking innate and acquired immunityrdquo NatureImmunology vol 2 no 8 pp 675ndash680 2001

[31] W Ren S Chen J Yin et al ldquoDietary arginine supplementationof mice alters the microbial population and activates intestinalinnate immunityrdquo The Journal of Nutrition vol 144 no 6 pp988ndash995 2014

[32] R J Xavier and D K Podolsky ldquoUnravelling the pathogenesisof inflammatory bowel diseaserdquo Nature vol 448 no 7152 pp427ndash434 2007

[33] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 no 1 pp 49ndash62 2007

[34] W-K Ren J Yin X-P Zhu et al ldquoGlutamine on intestinalinflammation a mechanistic perspectiverdquo European Journal ofInflammation vol 11 no 2 pp 315ndash326 2013

[35] M F Neurath ldquoCytokines in inflammatory bowel diseaserdquoNature Reviews Immunology vol 14 no 5 pp 329ndash342 2014


[36] M G Vizoso Pinto M Rodriguez Gomez S Seifert B WatzlW H Holzapfel and C M A P Franz ldquoLactobacilli stimulatethe innate immune response and modulate the TLR expressionof HT29 intestinal epithelial cells in vitrordquo International Journalof Food Microbiology vol 133 no 1-2 pp 86ndash93 2009

[37] M Lamkanfi and V M Dixit ldquoInflammasomes and their rolesin health and diseaserdquoAnnual Review of Cell andDevelopmentalBiology vol 28 pp 137ndash161 2012

[38] Y J Feng andY Y Li ldquoThe role of p38mitogen-activated proteinkinase in the pathogenesis of inflammatory bowel diseaserdquoJournal of Digestive Diseases vol 12 no 5 pp 327ndash332 2011

[39] M Pasparakis ldquoRole of NF-120581B in epithelial biologyrdquo Immuno-logical Reviews vol 246 no 1 pp 346ndash358 2012

[40] A Ocana-Fuentes E Arranz-Gutierrez F J Senorans and GReglero ldquoSupercritical fluid extraction of oregano (Origanumvulgare) essentials oils anti-inflammatory properties based oncytokine response on THP-1 macrophagesrdquo Food and ChemicalToxicology vol 48 no 6 pp 1568ndash1575 2010

[41] F V Silva A G Guimaraes E R S Silva et al ldquoAnti-inflammatory and anti-ulcer activities of carvacrol a monoter-pene present in the essential oil of oreganordquo Journal ofMedicinalFood vol 15 no 11 pp 984ndash991 2012

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


permeability [12] In the present study we hypothesized thatdietary OEO supplementation promotes intestinal barrierintegrity by regulating intestinal bacteria and inflammationWe thus would use the pig model to test this hypothesisand study the morphology and permeability of the intestinethe composition of the intestinal microbiota the activationof innate immunity and the expression of proinflammatorycytokines after OEO supplementation

2 Materials and Methods

All animal handling protocols were approved by theHuazhong Agricultural University Animal Care and UseCommittee guidelines

21 Animals Diets and Treatments A total of 170 pigs(Large White times Landrace) with an initial body weight(BW) of 72 kg (plusmn40 kg) were obtained from the same farm(Wuhan China Pork Co Ltd Wuhan China)The pens werelocated in a building at a temperature maintained between15 and 25∘C For the experiment the 170 pigs were splitinto two groups according to the diet (each group of 85pigs was further split into 5 replicate pens each holding 17pigs) (1) control treatment without supplementation and(2) supplementation of OEO (25mgkg of feed for 28 d as-fed basis) The composition of the control diet is shownin Table 1 The OEO was in the form of a powder calledPhytogen (Meritech Bioengineering Co Ltd GuangzhouChina) The components of OEO are shown in Supplemen-tary Table S1 (see Supplementary Material available onlineat httpdxdoiorg10115520165436738) Pigs were allowedfeed and water ad libitum over a period of 4 weeks

22 Sample Collection On the day of slaughter between900 and 1400 a total of 12 pigs (100 kg BW) with 6 pigsfrom each dietary treatment were transported in an opentruck to the slaughterhouse Blood samples were collectedafter electrical stunning and then quickly separated into fivetubes A 10mL sample was placed on ice immediately whichwas subsequently centrifuged at 1300timesg at 4∘C for 15min toobtain serum The serum samples were stored at minus80∘C forsubsequent analysis The digesta samples were immediatelyremoved from the jejunum ileum and colon of each pig andstored at minus80∘Cuntil further analysis Samples of the jejunumitself were removed from the middle jejunum segment andthen rinsed with ice-cold physiological saline One sectionwas snap-frozen in liquid nitrogen and then stored at minus80∘Cuntil further analysis Other sections of jejunum (3 cm) werekept in 4 neutral buffered formalin for gut morphologicalanalysis

23 Gut Morphological Analysis The digestive tract wasremoved from the jejunum and fixed in 10 phosphate-buffered formalin The samples were sectioned at 5mmthickness and stained with hematoxylin and eosin Villusheight villus width and villus crypt depth were measuredon the stained sections using a light microscope fitted withan image analyzer (Image Pro Plus 60 Media Cybernetics

Table 1 Composition and analysis of the basal diet

Basal dietlowast

Composition (gkg)Wheat 38000Corn grains 46410Soybean meal (46) 8900Monocalcium phosphate 1400Limestone 700Mycetes adsorbent 150Antimildew agent 050Salt 350Soybean oil 2000Ethoxyquin 025Probiotics 020Y402 premixdagger 2000AnalysisDagger

Dry matter DM () 8680Metabolism energy (MJkg) 1320Crude protein CP () 1390Crude fiber () 280Ash () 360Fat () 430Calcium () 060Phosphorus () 060lowastControl group (C) was fed with the above basal diet whereas the oreganoessential oil (OEO) group consumed the basal diet supplemented with25mgkg OEOdaggerPremix contained per kg 105 g Fe 14 gCu 85 gZn 4 gMn 75mg Se 30mgI 350 kIU of vitamin A 40 kIU of vitamin D3 15 kIU of vitamin E 50mg ofvitamin K3 50mg of vitamin B1 150mg of vitamin B2 100mg of vitaminB6 01mg of vitamin B12 864 g lysine 175 g methionine 25 g threonine 4 gphytase and 15 g choline (kIU 1000 international units)DaggerMetabolism energy was calculated from data provide by Feed Database inChina (1999)

Bethesda MD USA) Twenty villi and crypts were measuredfor each segment

24 Measurement of Serum Endotoxin Level Serum endo-toxin level was measured by a quantitative chromogenicend point tachypleus amebocyte lysate endotoxin detectionkit following the manufacturerrsquos instructions (Xiamen TALExperimental Plant Co Ltd China) Briefly serum sam-ples were diluted to 1 10 with waterTris-HCl buffer Aftercentrifugation at 1270timesg for 10min the supernatant wasremoved and incubated with limulus amebocyte lysate at37∘C for 10min followed by incubation with the providedchromogenic substance for 6min The absorbance at 545 nmwas measured after the addition of appropriate reagents

25 Extraction of Microbial DNA from the GastrointestinalTract Digesta Total DNA of jejunum ileum and colondigesta was extracted and purified from gastrointestinal tractdigesta using a QIAamp DNA Stool Kit (Qiagen Germany)according to the manufacturerrsquos instructions DNA concen-tration was determined by spectrophotometry (NanoDrop)




















3 Results



Control OEO0

200

400

600Vi

llus h

eigh

t (120583

m)

lowastlowast

(a)

Villu

s wid

th (120583

m)

Control OEO0

50

100

150

(b)

Cryp

t dep

th (120583

m)

Control OEO0

100

200

300

400

(c)Control OEO

00

05

10

15

20

Villu

s hei

ght

Cry

pt d

epth

ratio

(d)


Control OEO





Endotoxin

Control OEO0

1

2

3EU

(mL)

lowast







4 Discussion





ZO-1

Control OEO0

1

2

3

4Re

lativ

e mRN

A le

vels

lowast

(a)

Occludin

Control OEO00

05

10

15

20

25

Relat

ive m

RNA

leve

ls

lowastlowast

(b)

ZO-1

ZO-1

Control OEO00

05

10

15

20

lowastlowast

Fold

of c

ontro

l

120573-actin

(c)

Occludin

Control OEO00

05

10

15

20

Occludin

lowastlowast

Fold

of c

ontro

l120573-actin

(d)






Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent


(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)


10




5 Conclusion



Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































3 Results



Control OEO0

200

400

600Vi

llus h

eigh

t (120583

m)

lowastlowast

(a)

Villu

s wid

th (120583

m)

Control OEO0

50

100

150

(b)

Cryp

t dep

th (120583

m)

Control OEO0

100

200

300

400

(c)Control OEO

00

05

10

15

20

Villu

s hei

ght

Cry

pt d

epth

ratio

(d)


Control OEO





Endotoxin

Control OEO0

1

2

3EU

(mL)

lowast







4 Discussion





ZO-1

Control OEO0

1

2

3

4Re

lativ

e mRN

A le

vels

lowast

(a)

Occludin

Control OEO00

05

10

15

20

25

Relat

ive m

RNA

leve

ls

lowastlowast

(b)

ZO-1

ZO-1

Control OEO00

05

10

15

20

lowastlowast

Fold

of c

ontro

l

120573-actin

(c)

Occludin

Control OEO00

05

10

15

20

Occludin

lowastlowast

Fold

of c

ontro

l120573-actin

(d)






Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent


(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)


10




5 Conclusion



Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control OEO0

200

400

600Vi

llus h

eigh

t (120583

m)

lowastlowast

(a)

Villu

s wid

th (120583

m)

Control OEO0

50

100

150

(b)

Cryp

t dep

th (120583

m)

Control OEO0

100

200

300

400

(c)Control OEO

00

05

10

15

20

Villu

s hei

ght

Cry

pt d

epth

ratio

(d)


Control OEO





Endotoxin

Control OEO0

1

2

3EU

(mL)

lowast







4 Discussion





ZO-1

Control OEO0

1

2

3

4Re

lativ

e mRN

A le

vels

lowast

(a)

Occludin

Control OEO00

05

10

15

20

25

Relat

ive m

RNA

leve

ls

lowastlowast

(b)

ZO-1

ZO-1

Control OEO00

05

10

15

20

lowastlowast

Fold

of c

ontro

l

120573-actin

(c)

Occludin

Control OEO00

05

10

15

20

Occludin

lowastlowast

Fold

of c

ontro

l120573-actin

(d)






Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent


(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)


10




5 Conclusion



Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Endotoxin

Control OEO0

1

2

3EU

(mL)

lowast







4 Discussion





ZO-1

Control OEO0

1

2

3

4Re

lativ

e mRN

A le

vels

lowast

(a)

Occludin

Control OEO00

05

10

15

20

25

Relat

ive m

RNA

leve

ls

lowastlowast

(b)

ZO-1

ZO-1

Control OEO00

05

10

15

20

lowastlowast

Fold

of c

ontro

l

120573-actin

(c)

Occludin

Control OEO00

05

10

15

20

Occludin

lowastlowast

Fold

of c

ontro

l120573-actin

(d)






Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent


(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)


10




5 Conclusion



Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ZO-1

Control OEO0

1

2

3

4Re

lativ

e mRN

A le

vels

lowast

(a)

Occludin

Control OEO00

05

10

15

20

25

Relat

ive m

RNA

leve

ls

lowastlowast

(b)

ZO-1

ZO-1

Control OEO00

05

10

15

20

lowastlowast

Fold

of c

ontro

l

120573-actin

(c)

Occludin

Control OEO00

05

10

15

20

Occludin

lowastlowast

Fold

of c

ontro

l120573-actin

(d)






Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent


(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)


10




5 Conclusion



Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Tot0

5

10

15

ControlOEO

Jejunum

Lacto Entero E coil

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent

(a)

0

5

10

15Ileum

lowast

ControlOEO

Log 1

0CF

Rg

inte

stina

l con

tent


(b)

ControlOEO

0

5

10

15 Colon

lowastlowast

Log 1

0CF

Rg

inte

stina

l con

tent


(c)


10




5 Conclusion



Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Fold

of c

ontro

l

p-p38p38

Control OEO00

05

10

15

P-JNK

T-JNK

P-ERK

T-ERK

120573-actin

P-p38

T-p38

P-Akt

T-Akt

120573-actin

p-ERKERK

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

Fold

of c

ontro

l

p-JNKJNK

Control OEO00

05

10

15

lowast

p-AKTAKT

Control OEO00

05

10

15

Fold

of c

ontro

l

lowast

(a)

Control OEO00

05

10

15 Nuclear p65

p65

PCNA

Fold

of c

ontro

l

lowast

(b)


Competing Interests


Acknowledgments



Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control OEO00

05

10

15Re

lativ

e mRN

A le

vels

lowastlowast

IL-1120573

(a)

Relat

ive m

RNA

leve

ls

IL-6

Control OEO00

05

10

15

lowastlowast

(b)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 TNF-120572

lowastlowast

(c)

Relat

ive m

RNA

leve

ls

MCP-1

Control OEO00

05

10

15

lowastlowast

(d)

Relat

ive m

RNA

leve

ls

Control OEO00

05

10

15 INF-120574

lowast

(e)



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





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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