Effect Of Curc-OEG On Rat Hepatic Stellate Cells In Vitro

Objective：To investigate the effects and mechanisms of Curc-OEG on theproliferation, activation and apoptosis of hepatic stellate cells（HSCs）.Methods（:1）The HSCs were isolated from male SD rats by collagenase IVand DNase digestion, and further purified by percoll gradient centrifugation.The cultured HSCs were identificated by immunostaining for GFAP,desmin, α-SMA, collagen I, vimentin and β-actin. The purity ofimmediately isolated and passaged HSCs was analyzed by flow cytometry.(2) Investigate the effects and mechanisms of Curc-OEG on theproliferation and activation of primary HSCs. Freshly isolated cells at day2were treated with Curc-OEG at concentrations of0,6.25and12.5μg/ml for7days. Morphology changes were photographed at1,4, and7days usinginverted metallurgical microscope. The expression of α-SMA, collagen I,TGF-β1and Smad2was confirmed by RT-PCR and Western blot.(3) Investigate the effects and mechanisms of Curc-OEG on theapoptosis of activated HSCs. Activated HSCs at day14were treated withCurc-OEG at concentrations of0,6.25,12.5,25,50,75μg/ml, andapoptosis was monitored by microscopy24h after treatment. Apoptosis of the cells was detected by flow cytometry with Annexin V FITC／PIdouble staining. The expression of albumin, Bax, Bcl-2, P53and othergenes associated with fibrogenesis including α-SMA, collagen I, collagenⅢ, TGF-β1, Smad2, Smad3, PDGF-βR, NF-κB, PPAR-γ, TIMP-1, TIMP-2and MMP-13was observed with RT-PCR.Results (1)The yield of isolated HSCs was about4.0-5.0×107per rat liver.Viability of freshly isolated HSCs was more than90%as measured bytrypan blue staining. Immunocytochemistry with specific antibodiesdemonstrated that the expression of desmin, α-SMA, collagenI, vimentinand β-actin increased during cultivation, whereas GFAP expressiondecreased. Flow cytometry analyzed the purity of immediately isolated andcultured cells at14days was56and96%, respectively.(2) Low concentrations of Curc-OEG significantly inhibited theproliferation and activation of primary HSCs. After7days cultivation,compared with control group, Curc-OEG caused a significantconcentration-dependent reduction in cell numbers of55and85%at6.25and12.5μg/ml, respectively. At12.5μg/ml, Curc-OEG reduced α-SMA,collagen I, TGF-β1and Smad2mRNA levels by83,77,85and75%（P<0.05）, and protein levels by94,82,92and73%, respectively（P<0.05）.(3) Curc-OEG induced apoptosis of activated HSCs. Comparedwith control group, flow cytometry analysis showed that the percentage of the early apoptotic and necrotic cells significantly increased with theupgrading of Curc-OEG concentration. At50μg/ml, Curc-OEGsignificantly upregulated the mRNA levels of the proapoptotic Bax by2.3-fold，downregulated the antiapoptotic Bcl-2by5.6-fold, while noapparent toxicity towards primary hepatocytes was observed atconcentrations up to50μg/ml. Moreover, Curc-OEG inhibited theproliferation of activated HSCs through regulation of the mRNA levels ofgenes involved in fibrogenesis including α-SMA, collagen I, collagen Ⅲ,TGF-β1, Smad2, Smad3, PDGF-βR, NF-κB, PPAR-γ, TIMP-1, TIMP-2.Conclusion: Curc-OEG not only significantly inhibited the proliferationand activation of primary HSCs, but also induced apoptosis of activatedHSCs and reduced ECM accumulation.