Study The Molecular Mechanism Of Curcumin On Inhibite Hepatic Stellate Cells Activation

Hepatic fibrogenesis occurs as a wound-healing process after manyforms of chronic hepatic injury, characterized by production of the ECM(extracellular matrix) exceeds its degradation in the liver, Withouteffective treatment at an early stage, reversible hepatic fibrosis progressesto irreversible cirrhosis, which is a common pathological process of mosttypes of chronic liver diseases, hepatic stellate cells(HSC) are the mostrelevant cell type and the primary source of over-production of ECM forthe development of liver fibrosis. HSC normally reside in the space ofDisse in a quiescent, non-proliferative state. During hepatic injury, HSCbecome active and undergo profound phenotypic changes leading to amyofiboblast-like cell, including enhanced cell proliferation andexcessive production and deposition of ECM. Curcumin, the activeingredient of the rhizome of the plant turmeric (Curcuma longa Linn),possesses anti-proliferative, anti-oxidant, anti-inflammatory,anti-angiogenic and anti-tumor effects. Previous studies havedemonstrated that curcumin significantly inhibited HSC activation, including reducing cell proliferation, inducing apoptosis, and suppressingECM gene expression, The precise mechanisms of these actions remainlargely unknown which need to be defined, make it as a potentialanti-fibrogenic candidate drug which focus on HSC target in theprevention and treatment of hepatic fibrosis.PartⅠActivation of PPARγis required for curcumin toinduce apoptosis and to inhibit the expression ofextracellular matrix genes in hepatic stellate cells in vitroAIM: to evaluate the roles and molecular mechanisms of PPARγactivation in the induction of apoptosis and suppression of ECM geneexpression by curcumin in activated HSC,METHODES: passaged HSC were treated with curcumin and otherreagent according to experimental design at the indicated concentrationsand time, these assays were performed based on different objective:western blotting, Caspase 3 activity assays, Flow cytometric analyses ofapoptotic HSC,real-time PCR, Plasmids and transient transfection assays,TGF-β1 immunoassaysResults: Activation of PPARγmediates the suppression by curcumin ofcyclin D1 expression, which, in turn, facilitates the activation of PPARγ; activation of PPARγby curcumin was a necessary step, and contributed to the induction of HSC apoptosis by stimulating caspase 3activity, increasing the abundance of pro-apoptotic Bax and reducing thelevel of anti-apoptotic Bc1-2 in activated HSC in vitro. In addition,activation of PPARγwas required for curcumin to inhibit ECM geneexpression. Activation of PPARγmediated the blockade of the TGF-βsignalling pathway by curcumin, by suppressing TGF-βreceptors.Conclusion: curcumin stimulated PPARγactivity in activated HSC invitro,which was required for curcumin to reduce cell proliferation, induceapoptosis and suppress ECM gene expression.PartⅡCurcumin suppresses the expression ofextracellular matrix genes in activated hepatic stellate cellsby inhibiting gene expression of connective tissue growthfactorAIM: to elucidate the mechanisms by which curcumin suppressesαI(Ⅰ)collagen gene expression in activated HSC.METHODES: passaged HSC were treated with curcumin and otherreagent according to experimental design at the indicated concentrationsand time, these assays were performed based on different objective:western blotting, ,real-time PCR, Plasmids and transient transfectionassays, GSH immunoassays Results: curcumin significantly reduces the abundance of CTGF inpassaged HSC and suppresses its gene expression. Exogenous CTGFdose-dependently abrogates the inhibitory effect of curcumin. Activationof PPARγby curcumin results in interruption of TGF-βsignaling bysuppressing gene expression of TGF-γreceptors, leading to inhibitionof CTGF gene expression. The phyto-chemical shows its potentantioxidant property by significantly increasing the level of totalglutathione (GSH) and the ratio of GSH/GSSG in activated HSC. de novosynthesis of cellular GSH is a prerequisite for curcumin to interruptTGF-βsignaling and to inhibit gene expression of CTGF andαI(Ⅰ)collagen in activated HSC.Conclusion: inhibition ofαI(Ⅰ) collagen gene expression by curcuminin activated HSC results from suppression of CTGF gene expressionthrough increasing cellular GSH contents and interrupting TGF-βsignaling.PartⅢDisruption of transforming growth factor-betasignaling by curcumin induces gene expression ofperoxisome proliferator-activated receptor-gamma in rathepatic stellate cells AIM: to evaluate the molecular mechanisms of an antagonisticrelationship between PPARγactivation and TGF-βsignaling in HSC.METHODES: passaged HSC were treated with curcumin and otherreagent according to experimental design at the indicated concentrationsand time, these assays were performed based on different objective:western blotting, real-time PCR, Plasmids and transient transfectionassays, Site-directed mutageneses, Electrophoretic Mobility Shift Assay(EMSA)Results: exogenous TGF-β1 inhibits gene expression of PPARγinactivated HSC, which is eliminated by the pretreatment with curcuminlikely by interrupting TGF-βsignaling. Transfection assays furtherindicates that blocking TGF-βsignaling by dominant negative typeⅡTGF-βreceptor increases the promoter activity of PPARγgene.Promoter deletion assays, site-directed mutageneses and gel shift assayslocalize two Smad binding elements (SBEs) in the PPARγgenepromoter, acting as curcumin response elements and negatively regulatingthe promoter activity in passaged HSC. The Smad3/4 protein complexspecifically binds to the SBEs. Over-expression of Smad4dose-dependently eliminates the inhibitory effects of curcumin on thePPARγgene promoter and TGF-βsignaling.Conclusion: TGF-βsignaling might negatively regulate gene expressionof PPARγ, in activated HSC. The induction of gene expression of PPARγ by curcumin in activated HSC might result, at least partially, from thedisruption of TGF-βsignaling.