Research Article The Protective Effect of Resveratrol on ...downloads.hindawi.com/journals/grp/2015/506390.pdf · The Protective Effect of Resveratrol on Concanavalin-A-Induced

Research ArticleThe Protective Effect of Resveratrol on Concanavalin-A-InducedAcute Hepatic Injury in Mice

Yingqun Zhou1 Kan Chen1 Lei He1 Yujing Xia1 Weiqi Dai1 Fan Wang1

Jingjing Li1 Sainan Li1 Tong Liu1 Yuanyuan Zheng1 Jianrong Wang12 Wenxia Lu12

Qin Yin13 Yuqing Zhou13 Jie Lu1 Hongfei Teng14 and Chuanyong Guo1

1Department of Gastroenterology Shanghai Tenth Peoplersquos Hospital Tongji University School of Medicine Shanghai 200072 China2The First Clinical Medical College of Nanjing Medical University Nanjing 210029 China3The First Affiliated Hospital of Soochow University Suzhou 215006 China4Department of General Surgery Shanghai Tenth Peoplersquos Hospital Tongji University School of Medicine Shanghai 200072 China

Correspondence should be addressed to Hongfei Teng yybbjb945sohucom and Chuanyong Guo guochuanyonghotmailcom

Received 7 December 2014 Revised 1 April 2015 Accepted 2 May 2015

Academic Editor Fabio Marra

Copyright copy 2015 Yingqun Zhou 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

Pharmacologic RelevanceResveratrol an antioxidant derived fromgrapes has been reported tomodulate the inflammatory processIn this study we investigated the effects of resveratrol and its mechanism of protection on concanavalin-A- (ConA-) induced liverinjury in miceMaterials and Methods Acute autoimmune hepatitis was induced by ConA (20mgkg) in BalbC mice mice weretreated with resveratrol (10 20 and 30mgkg) daily by oral gavage for fourteen days prior to a single intravenous injection of ConAEight hours after injection histologic grading proinflammatory cytokine levels and hedgehog pathway activity were determinedResultsAfter ConA injection the cytokines IL-2 IL-6 and TNF-120572were increased and Sonic hedgehog (Shh) Glioblastoma- (Gli-)1 and Patched (Ptc) levels significantly increased Pretreatment with resveratrol ameliorated the pathologic effects of ConA-inducedautoimmune hepatitis and significantly inhibited IL-2 IL-6 TNF-120572 Shh Gli-1 and Ptc The effects of resveratrol on the hedgehogpathway were studied by western blotting and immunohistochemistry Resveratrol decreased Shh expression possibly by inhibitingShh expression in order to reduce Gli-1 and Ptc expression Conclusion Resveratrol protects against ConA-induced autoimmunehepatitis by decreasing cytokines expression in mice The decreases seen in Gli-1 and Ptc may correlate with the amelioration ofhedgehog pathway activity

1 Introduction

Liver disease remains a significant global health problemLiver impairment is multifactorial and may result from viralinfection chronic excess alcohol use hepatotoxins such asibuprofen an atherogenic high fat diet ischemia-reperfusioninjury transplant and surgicalmodels aswell as radiation [1]Indeed liver disease remains a significant cause of morbidityand mortality globally [2ndash4] Thus there is an urgent need todiscover hepatoprotective agents A number of compounds ofbotanical origin have been investigated for their ameliorativeeffect in liver injury disease in both experimental andclinical situations For example flavonoids and other naturalproducts have been described to possess beneficial hepaticeffects [5ndash7]

Resveratrol (3410158405-trihydroxystilbene) is present in morethan 70 plant species including grapes peanuts berriesand red wine and possesses beneficial antioxidant anti-inflammatory antimutagenic and anticarcinogenic proper-ties [8 9] The phytochemical resveratrol has shown consid-erable promise as a potential hepatoprotective agent [10] Sev-eral studies have investigated the use of resveratrol in chronicillnesses such as arthritis diabetes neoplastic and neurode-generative diseases [11ndash13] Resveratrol provides protectionagainst ongoing liver damage resulting from hepatotoxinssuch as acetaminophen ethanol and carbon tetrachloride(CCl4) [14ndash16] A recent study demonstrated that 2 weeks

of resveratrol administration in rats prevented CCl4-induced

lipid peroxidation as evidenced by decreased MDA content

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2015 Article ID 506390 11 pageshttpdxdoiorg1011552015506390

2 Gastroenterology Research and Practice

Thus the antioxidant and anti-inflammatory properties ofresveratrol play a prominent hepatoprotective role [17]

The hedgehog (Hh) signaling pathway plays a crucialrole in vertebrate embryogenesis by controlling cell fateproliferation survival and differentiation The level of Hhpathway activation appears to be proportional to the severityand duration of liver injury in both rodents and humans [18]Mature hepatocytes themselves do not express all of the Hhproteins and are not capable of responding toHh ligands [19]However many neighboring cells are Hh-responsive includ-ing resident hepatic cell populations that are most engagedin liver remodeling (eg liver myofibroblasts hepatic pro-genitors hepatic satellite cells immature cholangiocytesendothelial cells and T lymphocytes) [18ndash26] The proximityof various types of Hh producing and Hh responding celltypes in damaged livers suggests that Hh pathway activationcoordinates complex autocrine and paracrine signaling loopsthat help to orchestrate remodeling and reconstruction of theinjured liver Jung et al reported Hh signaling to be activatedin cells that participate in the ductular reaction that is elicitedby chronic alcohol-induced liver injury in mice and humansThe cumulative data indicate that Hh pathway activation is acommon feature of various types of liver injury that mobilizehepatic progenitor populations [20]

Accordingly this study was undertaken to examine thepotential protective effects of resveratrol against liver damagecaused by concanavalin-A (ConA) in mice using differentbiochemical parameters and histopathologic studies ConA-induced liver injury is a mouse model of immune-mediatedliver injury that resembles viral and autoimmune hepatitisin humans [12] Intravenous injection of ConA into miceactivates T cells which infiltrates the liver leading to the sub-sequent process of hepatocyte apoptosis and necrosis finallyincreases the level of plasma alanine aminotransferase (ALT)[27ndash29]The activation of T cells byConA results in increasedlevels of inflammatory cytokines including tumor necrosisfactor- (TNF-) 120572 interferon- (IFN-) 120574 and interleukin- (IL-)6 [13 28 29]

Clearly there is a critical need to explore novel andalternative approaches for the treatment of liver disease Thestructural determinants of the properties of resveratrol areobscure and its effects have not been tested in the model ofacute liver damage induced by ConA We aimed to inves-tigate whether resveratrol was capable of preventing ConA-induced liver injury as well as its molecular mechanism Wereport that resveratrol can show functional and morphologicimprovement by decreasing Gli-1 and Ptc expression as wellas inflammatory mediator expression in mice with ConA-induced liver injury

2 Materials and Methods

21 Reagents andDrugAdministration ConA and resveratrolwere purchased from Sigma-Aldrich (St Louis MO USA)and stored at 4∘C Resveratrol was suspended in 05 car-boxymethylcellulose solution and prepared separately at 12 and 3 gmL concentrations Resveratrol was thoroughlymixed before the mice were fed each mouse was fed

01mL10 g weight in the following concentrations 10 20and 30mgkg The antibodies used for immunoblotting andimmunohistochemical staining were anti-TNF-120572 anti-IL-2IL-6 CD4 F480 Shh Gli-1 and Ptc (Santa Cruz Biotech-nology Dallas TX USA) ConA was dissolved in pyrogen-free physiological saline and intravenously injected at a doseof 20mgkg to induce hepatitis as previously described [27ndash29] All other chemicals and biochemicals used in this studywere of high analytical grade

22 Experimental Animals Male Balbcmice (6ndash8weeks old20plusmn 2 g)were purchased from the Shanghai SLACLaboratoryAnimal Co Ltd (Shanghai China) The animals were housedin plastic cages with controlled light and dark cycles andfed a standard diet with water in a controlled temperature(25 plusmn 1∘C) and humidity (50 plusmn 5) environment Hepaticinjury was elicited in 6ndash8-week-old male mice by injectingConA (20mgkg body weight or bw) into the tail veinResveratrol was given according to dose into three groups(high medium and low dose) The mice were randomlydivided into six groups of ten mice as follows (1) salinecontrol group (2) resveratrol-alone group (3) ConA-inducedmodel group (4) low-dose resveratrol + ConA group (5)medium-dose resveratrol + ConA group and (6) high-doseresveratrol + ConA group For the first two weeks themice in the resveratrol group received 01mL10 gbwdayby oral administration The remaining mice received 05carboxymethyl cellulose solution at 01mL10 gbwday Atthe fourteenth day one hour after oral administrationconcanavalin-A (005mL10 g) was injected into the caudalveins of mice except for the saline and resveratrol-alonegroups which received saline (005mL10 g) After 8 hoursanimals were sacrificed to obtain eye blood The left hepaticlobes were stored at minus80∘C until the IL-2 IL-6 and TNF-120572 assays were performed The right hepatic lobes werefixed in 4 paraformaldehyde at 4∘C for hematoxylin-eosin(HE) and immunohistochemical staining Experiments wereperformed according to the National Institutes of HealthGuidelines for the care and use of laboratory animals andwere approved by the Committee on the Ethics of AnimalExperimentation of Hirosaki University

23 Biochemical Analysis After blood collection serum wasseparated by centrifugation at 3000 rpm for 15min at roomtemperature Serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) were tested using an auto-mated chemistry analyzer (Olympus AU 1000 OlympusCorporation Tokyo Japan)

24 Western Blot Protein aliquots were used to determineprotein concentration (Bio-Rad Dc Protein Assay Bio-Rad)Samples were mixed with 4x sample buffer and were elec-trophoresed on SDS-polyacrylamide gels and transferredelectrophoretically onto nitrocellulose paper Total proteinwas prepared using standard proceduresTheprotein concen-tration in the cleared lysates was determined by the methodof Bradford (Bradford 1976) An equal amount of proteinfor each treatment was loaded onto 12 polyacrylamide gels

Gastroenterology Research and Practice 3

Table 1 Effect of resveratrol on serum biomarkers and Knodell scoring

Parameter GroupSaline Res + saline ConA Res (10mgkg) + ConA Res (20mgkg) + ConA Res (30mgkg) + ConA

ALT (UL) 3040 plusmn 345lowast3234 plusmn 534

lowast38940 plusmn 2560

25373 plusmn 1438



lowast

AST (UL) 9060 plusmn 1576lowast 8860 plusmn 1354lowast 45074 plusmn 1969 25049 plusmn 1934lowast

25989 plusmn 1996lowast


Knodell scores 0 0 551 plusmn 192



30 plusmn 152lowast

Effects of resveratrol on serum parameters and Knodell scoring in mice with ConA-induced liver injury Values are expressed as the mean plusmn standard deviation(119899 = 10) ALT alanine aminotransferase AST aspartate aminotransferase Res resveratrol Statistical significance lowast119901 lt 001 119901 lt 001 and lowast119901 lt 001versus saline respectively Res alone ConA alone ConA + Res (10mgkg) ConA + Res (20mgkg) and ConA + Res (30mgkg)The ConA concentration was20mgkg resveratrol was administrated by oral gavage

after electrophoresis the proteins were transferred to nylonmembranes Antibodies were diluted in PBS containing 05BSA The membranes were later incubated with horseradishperoxidase-conjugated goat anti-rabbit antibody of goatanti-mouse antibody for 15 hours at room temperatureAfter the final wash membranes were developed with theOdyssey two-color infrared laser imaging system (LI-CORBiosciences Lincoln NE USA) [30]

25 Histopathologic Examination Livers from all mice wererinsed with ice-cold saline and a small cross section of theliver was obtained Liver specimens were fixed in 10 neutralbuffered formalin and paraffin embedded sectioned (4 or5 120583m) and stained with hematoxylin and eosin (HampE) Thetissues were examined under a microscope in random orderwithout knowledge of the animal or group

26 Immunohistochemistry Frozen tissue sections werewashed with PBS After being blocked with normal goatserum (Vector Laboratories) for 1 h sections were incubatedfor 1 h at room temperature in a dilution of 1 100 of Gli-1and Ptc antibody (Santa Cruz Biotechnology) Sectionswere washed in PBS developed with a diaminobenzidinetetrahydrochloride substrate for 15min and counterstainedwith hematoxylin Slides were then observed by opticalmicroscopy [5 30] F480 and CD4 in mouse liver tissuewere measured by immunohistochemistry (formalinPFA-fixed paraffin-embedded tissue sections) Sections were fixedin formaldehyde and subjected to heat-mediated antigenretrieval in citrate buffer (pH 60) prior to blocking with 1BSAPBS and incubated for 1 h 30min at 25∘C

27 Statistical Analysis Statistical analysis was performedusing one-way analysis of variance (ANOVA) followed byTukey-Kramer Test The values are presented as the means plusmnthe standard error of the mean (SEM) for ten mice in eachgroup A 119901 value of less than 005 was considered statisticallysignificant All statistical analyses were performed using SPSSversion 170 (SPSS Inc Chicago IL USA) statistical software

3 Results

31 Resveratrol Pretreatment Ameliorates ConA-Induced Hep-atitis We performed an assay using a ConA-induced hep-atitis model and assessed liver function by measuring serum

ALT and AST levels Mice liver tissue of each group wasassessed by pathologic examination and Knodell scoringALT and AST levels increased after injection of ConA andpeak levels decreased in the high-dose resveratrol groupAs shown in Table 1 ALT and AST levels clearly increasedafter ConA injection compared with the saline group at 8hours (119901 lt 005) resveratrol pretreatment significantlyattenuated the ConA injection-induced elevation of serumALT and AST (119901 lt 005) This same result was demon-strated in the histopathologic study and Knodell scoresAs shown in Table 1 the Knodell scores of the resveratrolgroup gradually decreased and there was statistical signif-icance demonstrated between the different concentrationsof resveratrol (119901 lt 001) As shown in Figure 1 we foundmassive areas of necrosis in the ConA-induced group Incontrast the resveratrol-treated group showed minor liverdamage indicating that resveratrol pretreatment significantlyreduced liver necrosis Resveratrol administered at 30mgkgwas more effective According to the results as analyzedwith Image pro Plus 60 statistical significance was clearlydemonstrated among groups Thus resveratrol pretreatmentwas shown to attenuate ConA-induced autoimmune hepatitisin mice

32 Resveratrol Pretreatment Inhibits the Release of Cytokinesduring ConA-Induced Hepatitis and Expression of Hh SignalPathway ConA-induced hepatitis is associated with changesin the levels of inflammatory cytokines We demonstratedwith immunoblotting that TNF-120572 IL-2 and IL-6 expres-sion was significantly increased in the ConA model groupcompared to the saline control group Resveratrol reducedTNF-120572 IL-2 and IL-6 production in the ConA-inducedmiceliver which is consistent with the degree of liver injury Nextwe examined the effect of resveratrol on Shh Gli-1 andPtc expression in the ConA-induced mice liver Resveratrolpretreatment significantly attenuated Gli-1 and Ptc expres-sion compared to the ConA model group (119901 lt 005)whichwas consistentwith changes in immunohistochemistry(Figure 2)

33 Immunohistochemistry Analysis We hypothesized thatthe level of Hh pathway activation would increase in par-allel with the severity of liver damage To assess potentialcorrelations between known histologic and clinical predic-tors of advanced liver disease and Hh pathway activationimmunohistochemistry was performed on liver biopsies


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(g)

Figure 1 Resveratrol pretreatment attenuates ConA-induced autoimmune hepatitis Liver histopathology in ConA-induced liver injuryLiver samples were taken from mice treated with ConA (20mgkg) and mice treated with ConA and resveratrol (10 20 and 30mgkg respdaily oral administration times 14 days) Sections were stained with HampE (a) Saline group (b) resveratrol-alone group (c) ConA group liversection from a mouse from the ConA-treated group showing extensive liver focal necrosis portal infiltration and interface hepatitis HampEmagnification times100 ((d)ndash(f)) ConA + Res (10mgkg) ConA + Res (20mgkg) ConA + Res (30mgkg) liver section from a mouse treatedwithConA and resveratrol and liver histology shows only aminimal grade of cellular infiltration and hepatocyte damageHampEmagnificationtimes100 (g) The necrotic areas were analyzed with Image-pro Plus 60 indicating statistical significance among groups

Shh Gli-1 and Ptc expression indicators of the activatedHh signal pathway was detected by immunohistochemistryIt is well known that Gli-1 is more strongly expressed afterConA-induced liver injury Meanwhile we found that Gli-1was mostly located in the nuclei with some located in thecytoplasm and positively stained brown Ptc protein waslocalized in the cytoplasm that showed a brown granular

appearance As shown in Figure 3 Gli-1 was clearly locatedin the nuclei and was expressed at low levels in the salinegroupHowever after ConAwas induced Gli-1 was expressedmore strongly in the nuclei and had partly translocated tothe cytoplasm In contrast pretreatment with resveratrolsignificantly reduced Gli-1 expression in both the nuclei andcytoplasm In addition the location and expression of Gli-1


Gli-1

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Figure 2 Continued


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trol

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trol

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lowastlowastlowast

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(h)

Figure 2 The western blot analysis of IL-2 IL-6 TNF-120572 Shh Gli-1 and Ptc after ConA injection in mice and the effects of low (10mgkg)medium (20mgkg) and high (30mgkg) dose resveratrol pretreatment groups at the same time

and Ptc by immunohistochemical staining were stronger inthe model group than in the saline group at 8 hours (119901 lt005) Increased Hh activity (evidenced by accumulation ofHh-ligand producing cells and Hh-responsive target cells)strongly correlated with portal inflammation ballooningand fibrosis stage (119901 lt 00001 for each) supporting arelationship betweenHhpathway activation and liver damage(Figure 3)

34 Resveratrol Reduced the Proliferative Response of T Lym-phocytes and Macrophages After injection of ConA serumIL-2 IL-6 and TNF-120572 levels increased dramatically Thissuggests that CD4+ T helper (Th) cells were involved in liverinjury It was reported that CD4+ levels were positive Thecells recognize the ConA-modified major histocompatibilitycomplex (MHC) structures ofmacrophages and become acti-vated followed by an inflammation reaction and the releaseof IL-1 and IL-2 [31] Cell activation increases the relevantcytokine levels which leads to liver injury Meanwhile wefound that resveratrol reduced the proliferative response ofT lymphocyte cells Consistent with this resveratrol alsoreduced cytokines such as IL-2 The infiltrate induced byConA is characterized by macrophages and lymphocytes Inour study we evaluated the expression of F480 as well asT-cell recruitment (ie CD4 immunostaining) in differentconcentrations of resveratrol pretreatment In the acuteConA model resveratrol may inhibit macrophages and Thelper cells from releasing inflammatory cytokines whichexplains why it plays an anti-inflammatory role in the acutemodel (Figure 4)

4 Discussion

Resveratrol has been shown to have a protective effect on liverinjury [32] However the effects of resveratrol a powerful

and effective antioxidant on liver injury have not been inves-tigated Our study is the first to explore the effects of res-veratrol on liver injury

In this study we usedConA to induce acute liver injury inorder to create a model in which to characterize CD4+ T-cellactivation subsequent inflammation the types of cell deathand liver failure Our results show that ConA-induced liverinjury was ameliorated in resveratrol-pretreated mice andwas accompanied by a reduction in the expression of inflam-matory cytokines and Hh signaling pathway activation

SerumALT and AST levels were markedly elevated in theConA-treated group compared with saline controls SerumALT and AST levels are elevated after hepatocyte deathwith the release of the transaminases into the blood Hep-atocyte death caused by inflammation was clearly observedwith HampE staining (Figure 1) Serum ALT and AST levelswere reduced in the resveratrol-pretreated groups relativeto those in the ConA-treated groups at 8 hours (Table 1)The areas of cell death in the resveratrol-pretreated groupwere clearly reduced (Figure 1) Therefore resveratrol pre-treatment clearly attenuated the acute liver injury inducedby ConA The modulatory function of resveratrol in inflam-mation was reflected in the downregulation of inflammatorycytokines such as IL-2 IL-6 and TNF-120572 The effect wasmarked at the protein level as shown in Figure 2 The anti-inflammatory effects of resveratrol were explicit and consis-tent with other studies of a variety of inflammatory diseases[5ndash7] Both IL-2 and TNF-120572 are produced after macrophageactivation and result in an endothelial activation processleading to an increase of adhesion molecule expression aswell as secretion of other cytokines and growth factors IL-2 and TNF-120572 produce stimulation of acute phase systemicmanifestations including somnolence fever and changes inliver protein synthesis [33] Resveratrol decreased the liver


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Figure 3 Immunohistochemistry used to detect the expression level of Gli-1 and Ptc 8 hours in all five groups (saline group ConA modelgroup ConA + 10mgkg Res ConA + 20mgkg Res ConA + 30mgkg Res) Gli-1 and Ptc showed a strong immunoreactivity in ConAsections but it was weak after Res application in different dose groups


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Figure 4 Immunohistochemistry used to detect the expression level of CD4 and F480 8 hours in all five groups (saline group ConAmodelgroup ConA + 10mgkg Res ConA + 20mgkg Res ConA + 30mgkg Res) CD4 and F480 showed strong immunoreactivity in ConAsections but it was weak after Res application in different dose groups

laboratory values and the corresponding liver damagemdashprobably as a result of the diminished release of proinflam-matory cytokines such as IL-2 -6 and TNF-120572 and a result ofT lymphocyte and Kupffer cell activation [33]

The molecular mechanisms by which resveratrol exertsits cytoprotective and anti-inflammatory effects have beenattributed to its ROS-scavenging activity the inhibition ofoxygen-free radical formation and lipid peroxidation as wellas its effects on nitric oxide modulation of inflammatory

cytokines and chemokines [34] In addition resveratrol hasbeen reported to enhance antioxidant enzymes in CCl

4-

induced liver fibrosis and to reduce ethanol-induced oxida-tive tissue damage in rats [35 36] Guy et al reported thatthe severity of liver damage parallels the level of Hh pathwayactivity and the Hh pathway regulates several key aspectsof liver repair including the liver progenitor populationshepatic recruitment of inflammatory cells and accumulationof liver myofibroblasts [37]


In the adult organism Hh signaling remains active and isinvolved in the regulation of tissue homeostasis regenerationand stem cell maintenance [38]This pathway plays vital rolesin tissue morphogenesis during fetal development It alsomodulates wound healing responses in a number of adulttissues including the liver [39ndash41]Hh signaling is initiated bya family of ligands (Sonic hedgehog Shh Indian hedgehogIhh desert hedgehog Dhh) which interact with a cell surfacereceptor (Patched Ptc) that is expressed on Hh responsivetarget cells This interaction depresses the activity of anothermolecule Smoothened (Smo) and permits the propagationof intracellular signals that culminate in the nuclear local-ization of Glioblastoma (Gli) family transcription factors(Gli1 Gli2 and Gli3) that in turn regulate the expressionof Gli-target genes [39] The anti-inflammatory effects ofresveratrol are attributed to its binding to members of theHh pathway that initiate Shh expression and then decreaseGli1 and Ptc expression (Figure 3) Gli-1 and Ptc showedstrong immunoreactivity in ConA sections but were weakafter resveratrol application in different dose groups

Immunohistochemistry for F480 was used in studiesto identify macrophages in mice [42] and since that timehas been shown to provide a valid marker of macrophagesthroughout the body and in a variety of species In theliver lymphocytes sinusoid endothelial cells (SECs) Kupffercells (KCs) and stellate cells are all involved in the immuneresponse [39] According to immunohistochemical resultswe see that CD4 and F480 expression were significantlyreducedwith different concentrations of resveratrol a specificinhibitor of the Hh signaling pathway this indicates thatresveratrol reduces inflammation and cell aggregation islikely to inhibit the Hh signaling pathway It is reported thatConA-induced hepatitis was attenuated by the resveratrolwhich inhibited leukocyte infiltration and the expression ofT-cell related cytokines and chemokines [43ndash45] The resultwas consistent with our findings (Figure 4)

Several studies have already reported that resveratrolshowed anti-inflammatory effects in vitro and in vivo andinhibits the release of inflammatory cytokines [33 46]Nevertheless the anti-inflammatory effect of resveratrol inConA-induced liver injury has not yet been reported In thisstudy we observed that resveratrol inhibited the expressionof inflammatory mediators such as TNF120572 IL-2 and IL-6These results indicated that resveratrol was able to improveConA-induced liver injury by suppressing the inflammatoryresponse in mice

5 Conclusion

We aimed to determine whether resveratrol could inhibitthe expression and release of cytokines such as TNF120572 IL-2 and IL-6 in ConA-induced autoimmune hepatitis Wefound the following (1) resveratrol attenuated ConA-inducedautoimmune hepatitis in mice (2) resveratrol decreasesTNF120572 IL-2 and IL-6 expression in vivo and (3) resveratrolmay inhibit the release of Gli-1 and Ptc through modulationof the hedgehog signal pathway Although there are furthermechanisms to be explored our study provides a newapproach for the treatment of acute hepatitis in the clinic

Abbreviations

ALT Alanine aminotransferaseAST Aspartate aminotransferaseTNF-120572 Tumor necrosis factor-120572IL-2 Interleukin-2IL-6 Interleukin-6Shh Sonic hedgehogGli-1 Glioblastoma family transcription factor 1Ptc Patched

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yingqun Zhou and Kan Chen contributed equally to thiswork

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (no 81270515) the China Foundationfor Hepatitis Prevention and Control WBN Liver DiseaseResearch Fund (nos WBN20100021 and CFHPC20131011)and the Shanghai Health Bureau Issues (nos 2011287 and2012107)

References

[1] A Bishayee A S Darvesh T Politis and R McGory ldquoResver-atrol and liver disease from bench to bedside and communityrdquoLiver International vol 30 no 8 pp 1103ndash1114 2010

[2] P Muriel and Y Rivera-Espinoza ldquoBeneficial drugs for liverdiseasesrdquo Journal of Applied Toxicology vol 28 no 2 pp 93ndash103 2008

[3] M Heron D L Hoyert S L Murphy et al Deaths Final Datafor 2006 National Vital Statistics Report National Center forHealth Statistics Hyattsville Md USA 2009

[4] American Gastroenterological Association The Burden ofGastrointestinal Diseases American Gastroenterological Dis-eases American Gastroenterological Association BethesdaMd USA 2001

[5] S-W Hong K H Jung H-M Zheng et al ldquoThe protec-tive effect of resveratrol on dimethylnitrosamine-induced liverfibrosis in ratsrdquo Archives of Pharmacal Research vol 33 no 4pp 601ndash609 2010

[6] G Oktem A Uysal O Oral et al ldquoResveratrol attenuates dox-orubicin-induced cellular damage by modulating nitric oxideand apoptosisrdquo Experimental and Toxicologic Pathology vol 64no 5 pp 471ndash479 2012

[7] J A Rubiolo G Mithieux and F V Vega ldquoResveratrol protectsprimary rat hepatocytes against oxidative stress damage activa-tion of the Nrf2 transcription factor and augmented activities ofantioxidant enzymesrdquo European Journal of Pharmacology vol591 no 1ndash3 pp 66ndash72 2008

[8] M Maccarrone T Lorenzon P Guerrieri and A F AgroldquoResveratrol prevents apoptosis in K562 cells by inhibiting


lipoxygenase and cyclooxygenase activityrdquo European Journal ofBiochemistry vol 265 no 1 pp 27ndash34 1999

[9] J Martinez and J J Moreno ldquoEffect of resveratrol a naturalpolyphenolic compound on reactive oxygen species and pros-taglandin productionrdquo Biochemical Pharmacology vol 59 no 7pp 865ndash870 2000

[10] S Shishodia and B B Aggarwal ldquoResveratrol a polyphenol forall seasonsrdquo in Resveratrolin Health and Disease B B Aggarwaland S Shishodia Eds pp 1ndash16 CRC Press Boca Raton FlaUSA 2006

[11] P Saiko A Szakmary W Jaeger and T Szekeres ldquoResveratroland its analogs defense against cancer coronary disease andneurodegenerative maladies or just a fadrdquo Mutation Researchvol 658 no 1-2 pp 68ndash94 2008

[12] A Bishayee ldquoCancer prevention and treatment with resvera-trol from rodent studies to clinical trialsrdquo Cancer PreventionResearch vol 2 no 5 pp 409ndash418 2009

[13] C D Mann C P Neal G Garcea M M Manson A RDennison and D P Berry ldquoPhytochemicals as potential chem-opreventive and chemotherapeutic agents in hepatocarcinogen-esisrdquo European Journal of Cancer Prevention vol 18 no 1 pp13ndash25 2009

[14] G Sener H Z Toklu A O Sehirli A Velioglu-Ogunc SCetinel and N Gedik ldquoProtective effects of resveratrol againstacetaminophen-induced toxicity in micerdquoHepatology Researchvol 35 no 1 pp 62ndash68 2006

[15] A Kasdallah-Grissa B Mornagui E Aouani et al ldquoProtectiveeffect of resveratrol on ethanol-induced lipid peroxidation inratsrdquo Alcohol and Alcoholism vol 41 no 3 pp 236ndash239 2006

[16] E Chavez K Reyes-Gordillo J Segovia et al ldquoResvera-trol prevents fibrosis NF-120581B activation and TGF-120573 increasesinduced by chronic CCl4 treatment in ratsrdquo Journal of AppliedToxicology vol 28 no 1 pp 35ndash43 2008

[17] P Vitaglione B Ottanelli S Milani F Morisco N Caporasoand V Fogliano ldquoDietary trans-resveratrol bioavailability andeffect onCCl 4-induced liver lipid peroxidationrdquo Journal of Gas-troenterology and Hepatology vol 24 no 4 pp 618ndash622 2009

[18] S V Fleig S S Choi L Yang et al ldquoHepatic accumulation ofHedgehog-reactive progenitors increases with severity of fattyliver damage in micerdquo Laboratory Investigation vol 87 no 12pp 1227ndash1239 2007

[19] J K Sicklick Y-X Li A Melhem et al ldquoHedgehog signalingmaintains resident hepatic progenitors throughout liferdquo TheAmerican Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 290 no 5 pp G859ndashG870 2006

[20] Y Jung K D Brown R P Witek et al ldquoAccumulation ofHedgehog-responsive progenitors parallels alcoholic liver dis-ease severity in mice and humansrdquo Gastroenterology vol 134no 5 pp 1532ndash1543 2008

[21] Y Jung S JMcCall Y-X Li andAMDiehl ldquoBile ductules andstromal cells express hedgehog ligands andor hedgehog targetgenes in primary biliary cirrhosisrdquoHepatology vol 45 no 5 pp1091ndash1096 2007

[22] J A Lowrey G A Stewart S Lindey et al ldquoSonic hedgehogpromotes cell cycle progression in activated peripheral CD4+ Tlymphocytesrdquo Journal of Immunology vol 169 no 4 pp 1869ndash1875 2002

[23] G A Stewart J A Lowrey S J Wakelin et al ldquoSonic hedgehogsignaling modulates activation of and cytokine production byhuman peripheral CD4+ T cellsrdquo Journal of Immunology vol169 no 10 pp 5451ndash5457 2002

[24] R P Witek L Yang R Liu et al ldquoLiver cell-derived micropar-ticles activate hedgehog signaling and alter gene expression inhepatic endothelial cellsrdquo Gastroenterology vol 136 no 1 pp320e2ndash330e2 2009

[25] L Yang YWang HMao et al ldquoSonic hedgehog is an autocrineviability factor for myofibroblastic hepatic stellate cellsrdquo Journalof Hepatology vol 48 no 1 pp 98ndash106 2008

[26] S S Choi A Omenetti R P Witek et al ldquoHedgehogpathway activation and epithelial-to-mesenchymal transitionsduring myofibroblastic transformation of rat hepatic cells inculture and cirrhosisrdquo The American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 297 no 6 pp G1093ndashG1106 2009

[27] Y ZhouW Dai C Lin et al ldquoProtective effects of necrostatin-1against concanavalin A-induced acute hepatic injury in micerdquoMediators of Inflammation vol 2013 Article ID 706156 15pages 2013

[28] M Shen J Lu P Cheng et al ldquoEthyl pyruvate pretreatmentattenuates concanavalin A-induced autoimmune hepatitis inmicerdquo PLoS ONE vol 9 no 2 Article ID e87977 2014

[29] K Chen J Li J Wang et al ldquo15-Deoxy-1205741214-prostaglandinJ2 reduces liver impairment in a model of ConA-induced acutehepatic inflammation by activation of PPAR120574 and reduction inNF-120581B activityrdquo PPAR Research vol 2014 Article ID 215631 10pages 2014

[30] P Cheng W Dai F Wang et al ldquoEthyl pyruvate inhibitsproliferation and induces apoptosis of hepatocellular carcinomavia regulation of the HMGB1-RAGE and AKT pathwaysrdquoBiochemical andBiophysical ResearchCommunications vol 443no 4 pp 1162ndash1168 2014

[31] A Varthaman J Khallou-Laschet M Clement et al ldquoControlof T cell reactivation by regulatory Qa-1-restricted CD8+ Tcellsrdquo Journal of Immunology vol 184 no 12 pp 6585ndash65912010

[32] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[33] L Bujanda M Garcıa-Barcina V Gutierrez-de Juan et alldquoEffect of resveratrol on alcohol-induced mortality and liverlesions in micerdquo BMC Gastroenterology vol 6 article 35 2006

[34] H Ajamieh and N C Teoh ldquoRed wine coming up roses forintestinal ischemia reperfusion injury role for resveratrolrdquoJournal of Gastroenterology and Hepatology vol 24 no 11 pp1701ndash1709 2009

[35] A Kasdallah-Grissa BMornagui E Aouani et al ldquoResveratrola red wine polyphenol attenuates ethanol-induced oxidativestress in rat liverrdquo Life Sciences vol 80 no 11 pp 1033ndash10392007

[36] L Bujanda E Hijona M Larzabal et al ldquoResveratrol inhibitsnonalcoholic fatty liver disease in ratsrdquo BMC Gastroenterologyvol 8 article 40 2008

[37] C D Guy A Suzuki M Zdanowicz et al ldquoHedgehog pathwayactivation parallels histologic severity of injury and fibrosis inhuman nonalcoholic fatty liver diseaserdquoHepatology vol 55 no6 pp 1711ndash1721 2012

[38] WMo X Xu L Xu et al ldquoResveratrol inhibits proliferation andinduces apoptosis through the hedgehog signaling pathway inpancreatic cancer cellrdquo Pancreatology vol 11 no 6 pp 601ndash6092012

[39] A Omenetti S Choi G Michelotti and A M Diehl ldquoHedge-hog signaling in the liverrdquo Journal of Hepatology vol 54 no 2pp 366ndash373 2011


[40] J E Hooper andM P Scott ldquoCommunicating with hedgehogsrdquoNature ReviewsMolecular Cell Biology vol 6 no 4 pp 306ndash3172005

[41] M Varjosalo S-P Li and J Taipale ldquoDivergence of hedgehogsignal transductionmechanism between Drosophila andmam-malsrdquo Developmental Cell vol 10 no 2 pp 177ndash186 2006

[42] Y H Feng W L Zhou Q L Wu X Y Li W M Zhao and JP Zou ldquoLow dose of resveratrol enhanced immune response ofmicerdquo Acta Pharmacologica Sinica vol 23 no 10 pp 893ndash8972002

[43] H-XWang M Liu S-YWeng et al ldquoImmunemechanisms ofConcanavalin a model of autoimmune hepatitisrdquoWorld Journalof Gastroenterology vol 18 no 2 pp 119ndash125 2012

[44] J M Austyn and S Gordon ldquoF480 a monoclonal antibodydirected specifically against the mouse macrophagerdquo EuropeanJournal of Immunology vol 11 no 10 pp 805ndash815 1981

[45] B G Lopez M S Tsai J L Baratta K J Longmuir and RT Robertson ldquoCharacterization of Kupffer cells in livers ofdeveloping micerdquo Comparative Hepatology vol 10 article 22011

[46] K Bisht K-H Wagner and A C Bulmer ldquoCurcumin resver-atrol and flavonoids as anti-inflammatory cyto- and DNA-pro-tective dietary compoundsrdquo Toxicology vol 278 no 1 pp 88ndash100 2010

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


Thus the antioxidant and anti-inflammatory properties ofresveratrol play a prominent hepatoprotective role [17]

The hedgehog (Hh) signaling pathway plays a crucialrole in vertebrate embryogenesis by controlling cell fateproliferation survival and differentiation The level of Hhpathway activation appears to be proportional to the severityand duration of liver injury in both rodents and humans [18]Mature hepatocytes themselves do not express all of the Hhproteins and are not capable of responding toHh ligands [19]However many neighboring cells are Hh-responsive includ-ing resident hepatic cell populations that are most engagedin liver remodeling (eg liver myofibroblasts hepatic pro-genitors hepatic satellite cells immature cholangiocytesendothelial cells and T lymphocytes) [18ndash26] The proximityof various types of Hh producing and Hh responding celltypes in damaged livers suggests that Hh pathway activationcoordinates complex autocrine and paracrine signaling loopsthat help to orchestrate remodeling and reconstruction of theinjured liver Jung et al reported Hh signaling to be activatedin cells that participate in the ductular reaction that is elicitedby chronic alcohol-induced liver injury in mice and humansThe cumulative data indicate that Hh pathway activation is acommon feature of various types of liver injury that mobilizehepatic progenitor populations [20]

Accordingly this study was undertaken to examine thepotential protective effects of resveratrol against liver damagecaused by concanavalin-A (ConA) in mice using differentbiochemical parameters and histopathologic studies ConA-induced liver injury is a mouse model of immune-mediatedliver injury that resembles viral and autoimmune hepatitisin humans [12] Intravenous injection of ConA into miceactivates T cells which infiltrates the liver leading to the sub-sequent process of hepatocyte apoptosis and necrosis finallyincreases the level of plasma alanine aminotransferase (ALT)[27ndash29]The activation of T cells byConA results in increasedlevels of inflammatory cytokines including tumor necrosisfactor- (TNF-) 120572 interferon- (IFN-) 120574 and interleukin- (IL-)6 [13 28 29]

Clearly there is a critical need to explore novel andalternative approaches for the treatment of liver disease Thestructural determinants of the properties of resveratrol areobscure and its effects have not been tested in the model ofacute liver damage induced by ConA We aimed to inves-tigate whether resveratrol was capable of preventing ConA-induced liver injury as well as its molecular mechanism Wereport that resveratrol can show functional and morphologicimprovement by decreasing Gli-1 and Ptc expression as wellas inflammatory mediator expression in mice with ConA-induced liver injury

2 Materials and Methods

21 Reagents andDrugAdministration ConA and resveratrolwere purchased from Sigma-Aldrich (St Louis MO USA)and stored at 4∘C Resveratrol was suspended in 05 car-boxymethylcellulose solution and prepared separately at 12 and 3 gmL concentrations Resveratrol was thoroughlymixed before the mice were fed each mouse was fed

01mL10 g weight in the following concentrations 10 20and 30mgkg The antibodies used for immunoblotting andimmunohistochemical staining were anti-TNF-120572 anti-IL-2IL-6 CD4 F480 Shh Gli-1 and Ptc (Santa Cruz Biotech-nology Dallas TX USA) ConA was dissolved in pyrogen-free physiological saline and intravenously injected at a doseof 20mgkg to induce hepatitis as previously described [27ndash29] All other chemicals and biochemicals used in this studywere of high analytical grade

22 Experimental Animals Male Balbcmice (6ndash8weeks old20plusmn 2 g)were purchased from the Shanghai SLACLaboratoryAnimal Co Ltd (Shanghai China) The animals were housedin plastic cages with controlled light and dark cycles andfed a standard diet with water in a controlled temperature(25 plusmn 1∘C) and humidity (50 plusmn 5) environment Hepaticinjury was elicited in 6ndash8-week-old male mice by injectingConA (20mgkg body weight or bw) into the tail veinResveratrol was given according to dose into three groups(high medium and low dose) The mice were randomlydivided into six groups of ten mice as follows (1) salinecontrol group (2) resveratrol-alone group (3) ConA-inducedmodel group (4) low-dose resveratrol + ConA group (5)medium-dose resveratrol + ConA group and (6) high-doseresveratrol + ConA group For the first two weeks themice in the resveratrol group received 01mL10 gbwdayby oral administration The remaining mice received 05carboxymethyl cellulose solution at 01mL10 gbwday Atthe fourteenth day one hour after oral administrationconcanavalin-A (005mL10 g) was injected into the caudalveins of mice except for the saline and resveratrol-alonegroups which received saline (005mL10 g) After 8 hoursanimals were sacrificed to obtain eye blood The left hepaticlobes were stored at minus80∘C until the IL-2 IL-6 and TNF-120572 assays were performed The right hepatic lobes werefixed in 4 paraformaldehyde at 4∘C for hematoxylin-eosin(HE) and immunohistochemical staining Experiments wereperformed according to the National Institutes of HealthGuidelines for the care and use of laboratory animals andwere approved by the Committee on the Ethics of AnimalExperimentation of Hirosaki University

23 Biochemical Analysis After blood collection serum wasseparated by centrifugation at 3000 rpm for 15min at roomtemperature Serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) were tested using an auto-mated chemistry analyzer (Olympus AU 1000 OlympusCorporation Tokyo Japan)

24 Western Blot Protein aliquots were used to determineprotein concentration (Bio-Rad Dc Protein Assay Bio-Rad)Samples were mixed with 4x sample buffer and were elec-trophoresed on SDS-polyacrylamide gels and transferredelectrophoretically onto nitrocellulose paper Total proteinwas prepared using standard proceduresTheprotein concen-tration in the cleared lysates was determined by the methodof Bradford (Bradford 1976) An equal amount of proteinfor each treatment was loaded onto 12 polyacrylamide gels






25373 plusmn 1438



lowast







30 plusmn 152lowast






3 Results






Saline

(a)

Res

(b)

ConA

(c)

ConA + Res 10

(d)

ConA + Res 20

(e)

ConA + Res 30

(f)

0

Aver

age n

ecro

tic ar

ea (

)

lowast

3

0

4

20

0

lowast

5

10

lowast

lowastlowast

lowast

0

Aver

age n

ecro

tic ar

ea (

)

50

40

30

20

10

0

lowastlowast

lowastlowast

lowastlowast


Salin

e

Con

A

Res

Con

A+

Res1

0

Con

A+

Res2

0

Con

A+

Res3

0

(g)





Gli-1

IL-2

IL-6

Nuclear-Gli-1

Ptc

Shh

TBP

120573-actin

TNF-120572

Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

Res1

0+

Con

A

(a)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-2

120573-a

ctin

inte

nsity


lowastlowast

lowastlowast

(b)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-6

120573-a

ctin

inte

nsity


lowastlowast

(c)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

16

14

12

10

08

06

04

02

00


lowastlowast

lowastlowast

Rela

tive T

NF-120572

120573-a

ctin

inte

nsity

(d)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive S

hh120573

-act

in in

tens

ity


lowastlowast

(e)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

10

08

06

04

02

00

Rela

tive G

li-1

120573-a

ctin

inte

nsity


lowast

lowastlowast

(f)

Figure 2 Continued


Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Rela

tive P

tc120573

-act

in in

tens

ity

lowastlowastlowast

lowastlowastRe

s10+

Con

A

(g)

Relat

ive n

ucle

ar-G

li-1 120573

-act

in in

tens

ity

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

lowastlowastlowast

lowastlowast

(h)




4 Discussion







Gli-1

Enlarge

Ptc

Enlarge

Res 10 + ConA

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Relat

ive G

li-1

inte

nsity

lowastlowast


lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive P

tc in

tens

ity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















25373 plusmn 1438



lowast







30 plusmn 152lowast






3 Results






Saline

(a)

Res

(b)

ConA

(c)

ConA + Res 10

(d)

ConA + Res 20

(e)

ConA + Res 30

(f)

0

Aver

age n

ecro

tic ar

ea (

)

lowast

3

0

4

20

0

lowast

5

10

lowast

lowastlowast

lowast

0

Aver

age n

ecro

tic ar

ea (

)

50

40

30

20

10

0

lowastlowast

lowastlowast

lowastlowast


Salin

e

Con

A

Res

Con

A+

Res1

0

Con

A+

Res2

0

Con

A+

Res3

0

(g)





Gli-1

IL-2

IL-6

Nuclear-Gli-1

Ptc

Shh

TBP

120573-actin

TNF-120572

Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

Res1

0+

Con

A

(a)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-2

120573-a

ctin

inte

nsity


lowastlowast

lowastlowast

(b)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-6

120573-a

ctin

inte

nsity


lowastlowast

(c)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

16

14

12

10

08

06

04

02

00


lowastlowast

lowastlowast

Rela

tive T

NF-120572

120573-a

ctin

inte

nsity

(d)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive S

hh120573

-act

in in

tens

ity


lowastlowast

(e)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

10

08

06

04

02

00

Rela

tive G

li-1

120573-a

ctin

inte

nsity


lowast

lowastlowast

(f)

Figure 2 Continued


Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Rela

tive P

tc120573

-act

in in

tens

ity

lowastlowastlowast

lowastlowastRe

s10+

Con

A

(g)

Relat

ive n

ucle

ar-G

li-1 120573

-act

in in

tens

ity

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

lowastlowastlowast

lowastlowast

(h)




4 Discussion







Gli-1

Enlarge

Ptc

Enlarge

Res 10 + ConA

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Relat

ive G

li-1

inte

nsity

lowastlowast


lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive P

tc in

tens

ity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Saline

(a)

Res

(b)

ConA

(c)

ConA + Res 10

(d)

ConA + Res 20

(e)

ConA + Res 30

(f)

0

Aver

age n

ecro

tic ar

ea (

)

lowast

3

0

4

20

0

lowast

5

10

lowast

lowastlowast

lowast

0

Aver

age n

ecro

tic ar

ea (

)

50

40

30

20

10

0

lowastlowast

lowastlowast

lowastlowast


Salin

e

Con

A

Res

Con

A+

Res1

0

Con

A+

Res2

0

Con

A+

Res3

0

(g)





Gli-1

IL-2

IL-6

Nuclear-Gli-1

Ptc

Shh

TBP

120573-actin

TNF-120572

Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

Res1

0+

Con

A

(a)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-2

120573-a

ctin

inte

nsity


lowastlowast

lowastlowast

(b)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-6

120573-a

ctin

inte

nsity


lowastlowast

(c)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

16

14

12

10

08

06

04

02

00


lowastlowast

lowastlowast

Rela

tive T

NF-120572

120573-a

ctin

inte

nsity

(d)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive S

hh120573

-act

in in

tens

ity


lowastlowast

(e)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

10

08

06

04

02

00

Rela

tive G

li-1

120573-a

ctin

inte

nsity


lowast

lowastlowast

(f)

Figure 2 Continued


Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Rela

tive P

tc120573

-act

in in

tens

ity

lowastlowastlowast

lowastlowastRe

s10+

Con

A

(g)

Relat

ive n

ucle

ar-G

li-1 120573

-act

in in

tens

ity

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

lowastlowastlowast

lowastlowast

(h)




4 Discussion







Gli-1

Enlarge

Ptc

Enlarge

Res 10 + ConA

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Relat

ive G

li-1

inte

nsity

lowastlowast


lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive P

tc in

tens

ity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Gli-1

IL-2

IL-6

Nuclear-Gli-1

Ptc

Shh

TBP

120573-actin

TNF-120572

Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

Res1

0+

Con

A

(a)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-2

120573-a

ctin

inte

nsity


lowastlowast

lowastlowast

(b)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive I

L-6

120573-a

ctin

inte

nsity


lowastlowast

(c)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

16

14

12

10

08

06

04

02

00


lowastlowast

lowastlowast

Rela

tive T

NF-120572

120573-a

ctin

inte

nsity

(d)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Rela

tive S

hh120573

-act

in in

tens

ity


lowastlowast

(e)

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

10

08

06

04

02

00

Rela

tive G

li-1

120573-a

ctin

inte

nsity


lowast

lowastlowast

(f)

Figure 2 Continued


Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Rela

tive P

tc120573

-act

in in

tens

ity

lowastlowastlowast

lowastlowastRe

s10+

Con

A

(g)

Relat

ive n

ucle

ar-G

li-1 120573

-act

in in

tens

ity

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

lowastlowastlowast

lowastlowast

(h)




4 Discussion







Gli-1

Enlarge

Ptc

Enlarge

Res 10 + ConA

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Relat

ive G

li-1

inte

nsity

lowastlowast


lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive P

tc in

tens

ity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Rela

tive P

tc120573

-act

in in

tens

ity

lowastlowastlowast

lowastlowastRe

s10+

Con

A

(g)

Relat

ive n

ucle

ar-G

li-1 120573

-act

in in

tens

ity

Con

trol

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

lowastlowastlowast

lowastlowast

(h)




4 Discussion







Gli-1

Enlarge

Ptc

Enlarge

Res 10 + ConA

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Relat

ive G

li-1

inte

nsity

lowastlowast


lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive P

tc in

tens

ity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Gli-1

Enlarge

Ptc

Enlarge

Res 10 + ConA

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

14

12

10

08

06

04

02

00

Relat

ive G

li-1

inte

nsity

lowastlowast


lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive P

tc in

tens

ity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)




CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















CD4

CD4

F480

F480

(times200)

(times200)

(times400)

(times400)

(a)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive C

D4

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Salin

e

Con

A

Res1

0+

Con

A

Res2

0+

Con

A

Res3

0+

Con

A

12

10

08

06

04

02

00

Relat

ive F

480

inte

nsity

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)





4-






5 Conclusion


Abbreviations






Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion


Abbreviations






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





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article The Protective Effect of Resveratrol on ...downloads.hindawi.com/journals/grp/2015/506390.pdf · The Protective Effect of Resveratrol on Concanavalin-A-Induced