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Curcuminpreventsand reverses irrhosisinducedby bile duct obstructionor CClain rats:role of TGF-BmodulationandoxidativestressKarina Reyes-Gordillo ',osSegoviab, inekoShibayama", ictorTsutsumi",PaulaVergarao,Mario G. Morenou,PabloMurielu*"Seccitt xtentade Farnncoloa, invstav-IPN., ptlo.Postal74-740, M,xico 7000, D.F. MxicobDepartumentode Fsiologn, olsica Neln'ocienci0.s,invstnu-lPN.,pdo.Postal74-740, Mxico07000, D,F,M:cico"DepLrrtamentlePatologa ).penrcntnl, invstnv-IPN.,pdo.Postal74-740, Mxico07000, D,F. Mxico

Keywordsbile duct ligation,ccl4,cirrhosis,cytokines,librosis,TGF-beta

Received3 November007;revised 1 f/arch2008;accepted tvlay 008

*Correspondenceand eprints:pamuriel@cinvestav.mx.

A B S T R A C TCurcumin is a phytophenolic compound, which is highly efficacious for treatingseveral nflammatory diseases. he aim o f this study was to eva luate he efficacyofcurcumin in preventingor reversing iver cirrhosis.A 4-weekbile duct ligation (BDL)rat modelwasused o test he ability of curcumin (100 mg/kg, p.o.,daily) to preventcirrhosis.To reverse irrhosis,CClawas administered hronically for 3 months, andthen it was withawryr and curcumin administered for 2 months. Alanine amino-transferase,y-glutamyl transpeptidase, iver histopathology, bilirubin, glycogen,reduced and oxidized glutathione, and TGF-B (mRNA and protein) Ievels wereassessed. urcumin preserved ormal valuesof markersof liver damage n BDL rats.Fibrosis, ssessedy measuringhydroxyproline evelsand histopathology, ncreasednearly fivefold after BDL and this effect was partially but significantly prevented bycurcumin. BDL ncreased ransforminggrowth factor-beta TGF-p)evels mRNA andproteins),while curcumin partially suppressedhis mediator of fibrosis.Curcuminalso partially reversed the fibrosis induced by CCIa. Curcumin was effective inpreventing and reversing cirrhosis, probably by its ability of reducing TGF-Fexpression.These data suggest hat curcumin might be an effective antifibrotic andlibrolitic drug in the treatment of chronic hepatic diseases.

I N T R O D U C T I ONCirrhosis,he end stageof progressiveibrosis,which iscausedby injury to the liver by a variety of etiologicalfactors, s a major health problemworldwide. Cirrhosisis characterizedby the accumulation of extracellularmatrix proteins(including collagens , III and IV), anddistortion fthe hepatic rchitecture1]. Thepresence fcirrhosis ndicates hat there is a perturbation n liverhomeostasiswith intracellular releaseof cytokines andsignaiing molecules. n particular, the fibrogenic cyto-kine transforminggrowth factor-beta TGF-P)s central

to the developmentof cirrhosis through its stimulatingeffecton matrix protein generationand inhibitory effecton matrix proteinremoval [2]. During hepatic ibrogen-esis,TGF-B laysa pivotal role in initiating,promotingand in the progression f the phenotypicalactivation ofhepatic stellate cells (HSC) o myofibroblasts.Concomi-tant with the increasedactivity of TGF-pduring fibro-genesis,HSC ncreases he production and depositionof collagen, leading to progressivescarring and lossof organ unction. TGF-p evel s elevatedn experimentaland human Iiver diseases,anging from hepatitis andcholestasiso cirrhosis [2-6]. Recently, he prospectof

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preventing and even reversing hepatic cirrhosis hasgenerated great interest because advances in basicscienceare being translated into promising new antiflb-rotic therapies; or example TGF-p downregulation maywell be an interesting therapeutic approach.Curcumin (diferuloylmethane), a derivative of thespice turmeric (Curcuma onga), s a pharmacologicallysafe agent [7,8], exhibiting strong antioxidant andantifibrotic activities [9]. In addition, curcumin blocksthe profibrotic actions ofTGF-B and has been reported tolimit the accumulation of collagen fibers in a TGF-B-driven model of fibrotic lung and kidney diseases 2,10].Recently, we demonstrated that curcumin providesprotection against inflammation and oxidative stress nCCla-inducedacute liver damage [11],It was found thatcurcumin prevents hioacetamide-inducedcirrhosis [1 2] ,but no mechanism was proposed to explain this effect.Therefore, n thi s study we evaluated whether curcuminplays a protective role in prolonged bile duct ligation(BDL) and whether it is capable of reversing CCla-induced cirrhosis, Furthermore, the antioxidant andimmunomodulatory properties of curcumin were evalu-ated as possibleaction mechanisms of the compound.Our results reveal that curcumin is highly eflective inboth preventing and reversing cirrhosis. Curcuminblocks serum markers of hepatic injury and possessesstrong antifibrotic effect. The mechanisms of action areprobably associated with curcumin's ability to blockoxidative stress ancl reduce the expression of the profib-rotic cytokine, TGF-P, nduced by BDL or chronic liverdamage induced by CCla n the rat.M A T E R I A L A N D M E T H O D SChemicalsCurcumin, carboxymethylcellulose, sodium thiosulfate,anthrone, thiobarbituric acid, chloramine-T, p-dimethyl-aminobenzaldehyde, y-glutamyl-p-nitroanilide, l-y-glut-amyl-p-nitroaniline, p-nitrophenyl phosphate, andbovine serum albumin were purchased from the SigmaChemical Company (St. Louis, MO, USA). Carbon tetra-chloride, sodium hydroxide, glacial acetic acid, hydro-chloric acid, sulphuric acid, ethanol, methanol, toluene,and formaldehyde were obtained from J.T. Baker (Xalos-toc, Mexico City, Mexico). All the reagents were ofanalytical quality.Study designMale Wistar rats were utilized in this study andmaintained on a standard rat chow diet with free access

K. Reyes-Gordloet aL

to drinhing water. Four or five animals were housed perpolycarbonate cage under controlled conditions(22 t 2'C, 50-60% relative humidity and 12-h light-dark cycles). The study complies with the institution'sguidelines and the Mexican oflicial regulation (NOM-062-200-7999) regarding technical specifications lbrproduction, care and use of laboratory animals.Prevention of cirrhosis induced by BD LA 4-week BDL rat model was utilized to produce hepaticdamage and to test the benefrcialpropertiesof curcumin.Four groups of rats (weighing initially 2OO-25O e;n = 8) were used. In the animals of group 1, the bileduct was identified, isolated, doubleJigated and sec-tioned. In group 2, the animals were sham-operated. Therats in group 3 were made to undergo BDL and receivedcurcumin (100 mg/ke, p.o., daily), suspended n car-boxymethylcellulose (0.7oln MC),after surgery. Animalsof group 4 were sham-operated and received onlycurcumin. All animals were killed 28 days after surgery.Reversion of cirrhosis after prolonged CCl4inject ionMale Wistar rats initially weighing 80 g were used.Cirrhosis was induced by intraperitoneal administrationof CCla(0.4 g/kg) dissolved n mineral oi l (linal volume,0.25 mL) three times per week for 3 months. Then, CCIaadministration was suspended and two groups of rats(n = 6) were ormed. Group 1 consistedof control animalsreceiving the vehicle curcumin only (1 mL of 0.7% CMC,p,o.,daily) for 2 month s. In group 2, curcumin (100 mg/kg, p.o., daily) was administered or 2 months.Biochemical estmationsAnimals were killed under Iight ether anesthesia;bloodsample was collected by cardiac puncture and the liverwas rapidly removed. Serum was obtained for determi-nation of liver damage by measuring the activities ofy-glutamyl transpeptidase y-GTP) [13], alanine amino-transferase ALT) [14] and bilirubin (kit: Bioxon, MexicoCity, Mexico).Glycogen determinationSmall liver pieces (0.5 g) were separated for glycogendetermination using the anthrone reagent, according toSeifteret al . [15].Collagen quantf icatonCollagen concentrations were determined by measur-ing the hydroxyproline content in fresh liver samples

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after digestion with acid [16] as described previously Image Acquisition and Analysis software (UVP), as[17], previously described 19].GlutathionedeterminationLiverpieces 250 mg) werehomogenized n iceusingapolytronhomogenizer. he solutionused or homogeni-zation consistedof 3.75mL of phosphate-ethylene-diaminetetraaceticacid (EDTA) buffer (pH = 8), and1 mL of 25% HjPO4, which was used as a proteinprecipitant.The total homogenatewas centrifugedat4 oCat 100 000 g for the assayof reduced GSH)andoxidizedGSSG)lutathione.Determinationsf GSHandGSSG ereperformed ccording o Hissinand Hilf [18].Western blot assaysThe TriPu re reagent(RocheDiagnostics,ndianapolis,IN, USA)wasused o isolate otalprotein romsamples fliver tissue,according o the manufacturer's nstruc-tions; 50 pg of protein,determined y the BCAproteinassay,was run per lane on a l2o/osodium dodecylsulfate-polyacrylamideel (SDS-PAGE),nd transferredto nitrocellulosemembranes BIO-RAD,Hercules,CA,tlSA). Blots were incubated with an alfinity-purifiedpolyclonal ntibodyagainst he cytokine soform,TGF-B(MAB1032 from Chemicon nt. Inc., Pemecula,CA,USA), diluted 1:2000. The secondaryantibody wasperoxidase-couplentimouse(Zymed,San Francisco,CA, USA)diluted 1:5000. Proteinsweredetected singenhanced hemiluminescenceNENLife Sciences rod-ucts, Elmer LAS, Inc., Boston,MA, USA).Membraneswere strippedof antibodiesby washing hem four times,5 min each with phosphate-saline uffer (pH = 7.4;0.015 u, 0.9% NaCl), immersed n stripping buffer(2-mercaptoethanol00 mu, SDS 2o/oand Tris-HCI62.5 mtvr, H 6.7),and then ncubatedwith a monoclo-nal antibodyagainstB-actin Imageswere digitalizedusing he BioDoc-It ystemUVP,Upland,CA, USA)andthen analyzed ensitometrically sing he Lab Worl

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Version).Diflerenceswere considered tatisticallysignif-i c a n t w h e n P < 0 . 0 5 .R E S U L T SPrevention of cirrhosis nduced by BDLThe activities of alanine aminotransferase (ALT),y-glutamyl ranspeptidasey-GTP) nd the concentrationof total bilirubin are shown n Table. Serummarkers nthe sham-operatedats remainedwithin control valuesdespite he treatment with curcumin, Serum levels ofALT (indicatorof necrosis) ncreased bout twofoldafterbile duct ligation when comparedwith the values insham-operatedats. As expected, -GTPand total biliru-bin were highly increasedafter 4 weeks oI BDL (22.5-and 7-fold,respectively) ecausehis is a model of liverdamagecharacterizedby cholestasis.Pharmacologicaltreatmentwith curcumin partially prevented P < 0.05)the increases f theseserummarker levels.Liver injury was also evaluated by a histologicalapproach(Figure1). Chronic iver damagewas accom-paniedby extended ecroticareas panelb), as comparedwith control (panela). Treatment of theBDl-group rats

K. Reyes-GordilIot al.

with curcumin (panel c) resulted in the absence ofnecrotic areas.As an indicator of oxidativestressat the hydrophilicIevel, we measured glutathione levels in the liver(Figure ). GSH,GSH/GSSGatio and otal(GSH+ GSSG)Iiverglutathionedecreasedigniflcantlyn the BDLgroup.In contrast,curcumin administrationncreased lutathi-one evels n BDL ats, and n rats administered urcuminalone.Glycogen,he main sourceof energy n the liver, is avery sensitive actor and thus a reliablemarker of liverdamage. This carbohydrate was depletedby biliaryobstruction for 4 weeks, while curcumin preserved twithin normalvalues Figure).Fibrosis,which is the final result of prolonged iverinjury, was quantifiedby hydroxyproline analysisandexpressed s liver collagen content (Figure4), Biliaryobstruction or 28 days ncreasedlbrosisnearly fivefold;this effectwas partially but significantlypreventedbycurcumin. Fibrosiswas also evaluatedhistologicallybyvsualizing ibers of collagen n sectionsof liver samplesstainedwith trichromic of Mallory (Figure5). Prolongedbiliary obstruction was accompaniedby a marked

Figure I Hematoxylin and eosin staining of liver sections rom: (a) sham-operated ats (SHAM); (b) bile duct-ligated rats (BDL); (c) BDL ratstreated with curcumin (BDL + Curc.) and (d) SHAM administeredwith curcumin (curcumin) (magn 100x)'

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Figure 2 Reduced GSH)and oxidized GSSG) lutathione,GSH/GSSGatio and total glutathione(GSH+GSSG)etermined n livers fromsham-operated ats (SHAM), bile duct-ligated rats (BDL),BDL treated with curcumin (BDL + Curc.),and SHAM administeredwith curcumin(curcumin). Each bar represents he mean value of experimentsperformed in duplicate assays+SE (n = 8). 'a' Mean values signilicantlydifferent rom control, P < 0,05. 'b ' Mean valuessignificantlydifferent rom CClagroup' P < 0'05.

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Figure 3 Glycogencontent determined n liver samples rom sham-operated ats (SHAM), bile duct-ligated rats (BDL),BDL treated withcurcumin IBDL+Curc.), and SHAM administeredwith curcumin(curcumin). Each bar represents he mean value of experimentsperformed n duplicateassays+S E n = 8) . 'a ' Mean valuessignificantlydiflerent rom control, P < 0.05. 'b ' Mean valuessignificantly different from CCIagroup, P < 0.05.increase in collagen deposition around the portal spacesand the group of hepatocyte-forming nodules(Figure 5b); therefore, the normal histological architec-ture was lost. Administration of curcumin to BDL ratsresultcd in a less scvcre or practically absent librosis(Figure 5c). The liver parenchyma of sham-operated atsand only curcumin-administered rats had a normalappearance Figure 5a,d).Numerous lines of evidence have shown that TGF-Ftreatment upregulates the expression of several pro-fibrotic genes n quiescent flbroblasts [2]. Therefore, weexamined the effect of curcumin on BDL rats' profibroticgene expressionby measuring TGF-B mRNA accumula-tion by RT-PCR. The results show increases in TGF-B

BDL+GurcuminCurc.

Figurc 4 Liver collagen, expressedas the hepatic hydroxyprolinecontent determined in liver samples rom sham-operated ats(SHAM), bile duct-ligated rats (BDL),BDL treated with curcumin(BDL+Curc.),and SHAM administered with curcumin (curcumin).Each bar represents he mean value of experiments performed induplicateassays SE (n = 8) . 'a'Mean valucssignilicantlydiffercntfrom control, P < 0.05. 'b' Mean values significantly different frornC C l a g r o u p , P < 0 . 0 5 .mRNA in livers derived from biliary obstructedrats;importantly, curcumintreatmentmarkedly nhibited hisincreaseFigure ). Inhibition of TGF-pgeneexpressionwas confirmedby measuringTGF-Bprotein levelsusingWestern blot analysis;TGF-B was elevated n BDLsurgery but significantly inhibited by curcumin(Fisure7).Reversionof cirrhosisafter CCla ntoxicationFigure8 shows that ALT enzyme activity increasedsignificantlyafter 3 months of chronic CClaadministra-tion as comparedwith vehicle-treatedats. Interestingly,

BD LDL+Curc. Curcumin BDL+Curc. Curcumin

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K. Reves-Gordilloet aL

Figurc5 Trichromicstainofl iversectionsfrom:a) sham-operatedrats(SHAM).Hepaticparenchymashowsanormalarchitecture.(b)Bileduct-ligateclrats (BDL).An increase deposil oi collagen is shown around a portal space.Hepatocyte nodules are also evident. (c) BDLtreated with curcumin (BDL+Curc.) shows a discrete deposit of collagen with areas of ischemia. (d) SHAM administered with curcumin(curcumin) showsan unaltered iver tissue magn 100x).

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380-pb w rtE ffi *h p-actinFigure 6 Analysis of TGF-p mRNA expressiondetermined by RT-PCR of liver RNA extracted from sham-operated ats (SHAM), bileduct-ligated rats (BDL),BDL treated with curcumin (BDL+Curc.).and SHAM administeredwith curcumin (curcumin). Lower panelshows the correspondingRT-PCRproducts of p-actin that was usedas a control. Total RNA was extracted,reverse-transcribedandamplified using speciflcTGF-p and P-actin primers.

discontinuation of the toxic agent or 2 months resulted na further and significant increase in serum ALT.Treatment with curcumin for 2 months of rats pretreatedwith CCla restored the levels of ALT enzyme activity tonormal values.Oxidative stress occurs on the CCla-induced liverdamage model; accordingly, in this study, a very impor-tant decrement in GSH, GSH/GSSG ratio and totalglutathione content caused by CCla administration for3 months was observed. Surprisingly, discontinuation ofCCI+ for 2 months increased oxidative stress at thehydrophilic level as GSH, GSH/GSSG atio and totalglutathione were lower than in rats treated with CCla for3 months, Importantly, curcumin administration for2 months resulted in normalization of glutathione levels(Figure 9) .Liver flbrosis occurs as a result of an imbalancebetween flbrogenesis and fibrolysis, and is a major factorcontributing to hepatic failure in cirrhosis. Figure 70shows that CCla intoxication for 3 months produced a

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Finally, liver glycogen content was found to bedecreasedbecauseof CCI ntoxication and discontinua-tion of CCla led to further decrement of glycogen.Interestingly, curcumin restored normal glycogen levels(Figure 77).Dtscuss toNThis study clearly shows that curcumin prevents thecirrhosis induced by BDL and reverses the fibrosis dueto 3 months of CCI ntoxication. This was assessed ybiochemical and histological methods. Furthermore, theantifibrotic and flbrolitic properties of curcumin wereassociatedwith its capacity to reduce TGF-p mRNA andprotein, a well*nown prolibrotic cytokine [21], and toits antioxidant actions.Prevention of cirrhosis induced by BD LLevels of ALT, y-GTP, bilirubin, collagen and glycogenwere increased in BDL rats, indicating hepatocellulariniury, fibrosis and dysfunction. The liver exhibitedoxidative stressmanifested by alterations of glutathioneIevels. n addition, increases n the profibrotic cytokine,TGF-0, were observed.Finally, histological examinationrevealed significant hepatic librosis and necrosis. Incontrast, BDL rats that received curcumin showed areduction in al l biochemical, histological and molecularindices of hepatic injury. Therefore, the protective effectof curcumin against hepatic cirrhosis may be mainlyassociatedwith the ability of this compound to attenuateoxidative stressand to downregulate TGF-p,Reduced glutathione, GSH/GSSG ratio and totalglutathione were significantly decreased after biliaryobstruction and curcumin prevented this effect. Theseresults are consistent with previous reports 122-24],indicating that curcumin acts as an antioxidant, Ourdata are also consistent with previous studies thatdemonstrate that curcumin induces de novo svnthesisof GSH 19,25-271.In this study we demonstrated both biochemicallyand histologically that curcumin significantly reducedlibrosis in BDL rats. In chronic cholestatic disorders (i.e.primary biliary cirrhosis), epithelial cellsstimulate portalaccumulation of myofibroblasts to initiate collagendeposition around damaged bile ducts [1]. Finally,changes in the composition of the ECM can directlystimulate flbrogenesis.Type IV collagen and fibrinogenstimulate HSCs by activating Iatent cytokines such as theprofibrotic factor TGF-B 6] . Therefore,we analyzed TGF-p as a target of curcumin to prevent librosis. Our results

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Figurc 7 Curcumin blockade of TGF-p protein in samplesof livertissue determined by Western blot analysis:SHAM-operated ats(SHAM), bile duct-ligated rats (BDL),BDL treated with curcumin(llDL+Curc.),an d SHAM administeredwith curcumin (curcumin).O-actinwas used as an internal control. Signal ntensitiesweredetermined by densitometric analysis of trealed blots and valuescalculatedas the ratio of TNI'-o/p-acti n.Each bar representshemean value of six rats *SU, a' Mean valuessignilicantlydilTerentfrom control P < 0.05.'b'Mean valuessignilicantlydifferent ro mC C l a g r o u p , P < 0 . 0 5 .

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Curcumin

0 3 5 llfonthsFigurc 8 Enzymatic aclivities of alanine aminotransferase,deter-mined in plasma from rats after chronic CCIaadministration for3 months (CCla),carboxymethylcelluloseadministration by2 months after discontinuation of the CCla reatment (vehicle),andcurcumin administration by 2 months after discontinuation of theCCI4 reatment (curcumin). Results are shown as the mean value ofsi x rats tSE. 'a ' Mean valuessignificantlydiffer:ent s. control (time0) , P < 0.05.

'b 'Mean valuessignificantll, ifferentvs. the groupreceivingCCIa or 3 months,P < 0.05. 'c'Mean valuessignificantly

different vs. the group receiving curcumin after discontinuation ofccl4, P < 0.05.

flvefold increase in liver collagen content. Discontinua-tion of CCl4 did not lead to fibrolJsis, but curcuminadministration resulted in a partial but significantreversionof CC+-inducedibrosis.

BDL+Curc. urcumin

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Figure l0 Liver collagen,expressedas the hepatic hydroxyprolinecontent, determined in livers from rats after chronic CCIadmin-istration during 3 months (CCl+),carboxymethylcelluloseadminis-tration by 2 months after discontinuation of the CCla reatment(vehicle), and curcumin administration by 2 months after discon-tinuation of the CCla reatment (curcumin). Resultsare shown asth e mean value of six rats +SE. a' Mean valuessignificantlydifferent s. control (time0) , P < 0.05. b' Mean valuessignificantlydifferent vs. the group receiving CCla or 3 months, P < 0.05. 'c'Mean valuessignilicantly differentvs' the group receiving curcuminafter discontinuationof CCla,P < 0.05.are consistentwith previous eports 2,9,28-30]' indi-cating that curcumin is capableof lowering the gener-ation of TGF-BmRNA and protein.Then, t is possible

K. Reyes-Gordllloet aL,

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Figure l1 Glycogencontent determined in liver samples rom ratsafter chronic CClaadministration for 3 months (CCIa),carboxy-methylcellulose administration by 2 months after discontinuationof the CCla reatment (vehicle), and curcumin administration by2 months after discontinuation of the CCla reatment (curcumin).Resultsare shown as the mean value of six rats *SE. 'a ' Meanvaluessignilicantlydillerentvs. control (time 0) , P < 0.05' "b "Mean values significantly different vs. the group receiving CCla or3 months, P < 0.05. 'c ' Mean valuessgnilicantlydifferentvs. thegroup receiving curcumin after discontinuation of CCla,P < 0.05.

that curcumin may modify the flbrotic phenotype offibroblastand thus modulate rbrous issue ormation inthe liver by altering collagensynthesisand deposition'

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Figurc 9 Reduced GSH) nctoxidized GSSG) lutathione,GSH/GSSGatio and total glutathione GSH+GSSG)eterminedn Iivers rom ratsafter chronic CClaadministration during 3 months (CCla),carboxymethylcelluloseadministration by 2 months after discontinuation of theCCla reatment (vehicle), and curcumin administration by 2 months after discontinuation of the CCla reatment (curcumin). Resultsareshovvnas the mean value of six rats +SB. a' Mean values significantly tlifferent vs. control (time 0), P < 0.05. 'b' Mean values significantlydifferent vs. the group receiving CCla or 3 months, P < 0.05, 'c' Mean valr,res ignilicantly dillerent vs. the group receiving cnrctlmin alterdiscontinuationof CCI+,P < 0.05.

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Bile duct ligation signiflcantl y decreasediver glycogencontent, but curcumin preserved almost completely thisparameter. One explanation is that curcumin preservesglycogen by its antiflbrotic mechanism because librosisdisrupts the blood flow to the liver not allowingabsorption of nutrients. Moreover, necrosis was inducedby BDL and this effect was partially prevented bycurcumin, and as necrotic cells do not store glycogen,the antinecrotic effect of curcumin may, in part, explainthe preservation of glycogen.The capacity of curcumin to attenuate oxidative stress,suppress he release of TGF-B, and the advantage of itbeing a non-toxic natural product (the pharmacologicalsafetyof curcumin is shown by the non-toxic consumptionof up to 100 mg/day in humans and up to 5 g/day in rats[7,8]) are important factors that make this compound aninteresting candidate for preventing and treating humanhepatic fibrtsisand cirrhosis.Reversion of cirrhosis after CCI-|ntoxicationThis is the first study to demonstrate that curcuminreverses iver cirrhosis. Advanced cirrhosis is generallyconsidered to be an irreversible process even afterremoval of the causative agent. Thus, the disease scharacterized by the incapacity of the injured liver toremodel the fibrotic matrix [31]. We observed thatprolonged CCla treatment (3 months vs. the 2 monthsnormally used n experimental protocols) was associatedwith progressive fibrogenesis,even after stopping admin-istration of CCla for 2 months. It is worth noting thatcurcumin was able to reverse all markers of liver damage.These effects were partial but significant and in somecases alues were near control values.It has been considered that the beneficial elfects ofcurcumin are mediated by its antioxidant defense abilityand the scavengingof free adicals;moreover, curcumin isat least 10 times more active as an antioxidant thanvitamin E 1321. CCla decreases the total antioxidantcapacity of the liver [33, this study], even after CCIdiscontinuation for 2 months, and here we show that theadministration of curcumin reversesoxidative stressat thehydrophilic level, Curcumin also demonstrated its abilityto restore glycogen levels previously depleted by liverdamage nduced by CClaadministration. Pari & Murugan[34] reported the capacity ofcurcumin to preservebloodglucose evels.They attributed this action to the ability ofcurcumin to restore the altered enzymatic activities of6-phosphate dehydrogenase,and glucose-6-phosphatasethat participate in gluconeogenesisand glucogenolysis.The sa me mechanism may apply in liver .

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Hepatic recovery from fibrosis involves the destructionof fibrous bands. Metalloproteinase enzymes (MMPs)especially pro-MMP-2 and pro-MMP-9 as well as theiractive forms are responsible for regulating most of theturnover of matrix proteins because these enzymesactivity together are capable of degrading basementmembrane proteins like gelatin, collagen IV, coll agen V,elastin, and fibronectin [35,36]. It has been reported thatcurcumin regulates the expression and activity of matrixpro-MMP-2 and pro-MMP-9 in human bronchial epithe-Iial cells, and during prevention and healing of indo-methacin-induced gastric ulcer [37*39]. Therefore, thereversion of fib rosis observed herein may be explainedthrough the modulation of MMPs.In conclusion, our results indicate that curcumin washighly effective in preventing and reversing hepaticcirrhosis of different etiologies. It blocks oxidative stress,the elevation of serum markers of hepatic injury andpossesses strong antifibrogenic and librolitic effects.Moreover, the ability of curcumin to downregulateTGF-p-induced ncrease expression of several genes andproteins (cclllagen , fibronectin, etc.) that contribute tothe accumulation of matrix proteins in chronic hepaticdisease and the antioxidant property of this compoundprovidesnovel insights into the mechanisms of curcuminto prevent and reverse hepatic cirrhosis. Previously,we demonstrated that curcumin is highly effective inpreventing CCla-acute nduced liver damage by blockingthe expression of proinflammatory cytokines and theactivation of NF-rB, and we have suggested hat agentsthat prevent the activation of the transcription factor NF-rB may suppress expression of a series of proinflamma-tory molecules and thereby prevent liver injury. Thus, thepreventing and reversing effectsofcurcumin are probablyassociatedwith its ability to reduce TGF-B (this study),maybe by inhibiting NF-rB activation as we previouslyshowed after acute CCla ntoxication [11]. On the otherhand, we cannot exclude other mechanism(s) o explainthe beneficial effectsof curcumin on liver injury becauseseveral intracellular signalling pathways are modulatedby this compound in HSCs, including c-Jun amino-terminal kinase, activator protein-l and peroxisomeproliferator-activated receptor-gamma [40-43].A C K N O W L E D G E M E N T SThe authors express their gratitude to Mr BenjamnSalinas Hernndez, Rubn Snchez Islas and Mr RamnHernndez, for their excellent technical assistance.Karina Reyes-Gordillo was a fellow of Conacyt

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(176585).This work was supportedn part by Conacytgrant 54756 (IS).R E F E R E N C E S1 BatallerR., Brenner D.A. Liver fibrosis. . Clin. Invest. 2005)l l 5 2 0 9 -2 1 8 .2 Gaedeke .,NoobleA.N., BorderA.W. Curcumi n blocksmultiplesitesof the TGF-psignalingcascade n renal cells.Kidney nt.(2004) 66 1r2-t20.3 Friedman S. Mechanisms f disease:Mechanisms f hepaticfibrosisand thcrapeutic mplcations.Nat. Clin. Pract.Castro-enterol.Hepatol. 2004) I 9l-105.4 Pinzani M., Marra F'.,Carloni V, Signal ransduction n hepaticstel late el ls, iver 1998) 18 2-13.5 Seyhan H., Hamzavi J., Wiercinska E. ct al. Livcr librogencsisdue 1o cholestasiss associated ith increasedSmadT expres-

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