Suppressive Effect Of Arctigenin On Hepatic Fibrosis And Hepatic Stellate Cells Proliferation

Objective: To investigate the suppressive effect of arctigenin（ATG）on carbon tetrachloride（CCl4）-induced hepatic fibrosis rats and platelet-derived growthfactor-BB（PDGF-BB）-activated HSCs proliferation.Methods: Hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride（CCl4）in rats for 8 weeks.ATG and colchicine were administrated once a day starting from the fifth week after the CCl4 treatment for subsequent 4 weeks.At the end of the study,liver index,liver fibrosis markers,as well as the contents of hydroxyproline（Hyp）in liver tissues were measured.Histopathological changes was observed in the liver tissue sections using hematoxyline-eosin（HE）and Masson’s trichrome staining.Fibrosis-related protein in liver tissue was examined by immunohistochemisty.The proliferation of hepatic stellate cells（HSCs）and expression of cell cycle-related proteins were assayed by indirect immunofluescence staining and Western blotting,respectively.The anti-proliferation potency of ATG on PDGF-BB-activated HSCs was evaluated using Roche Cell Proliferation ELISA.Flow cytometry was performed to analyze the effect of ATG on cell cycle progression.The expression of cell cycle-related proteins were assayed by Western blotting.The cyclin-CDKs complexes were immuno-precipitated with specific anti-CDKs antibodies.The activities of CDKs kinase were measured with histone H1（for CDK2）or bacterially synthesized GST-Rb fusion protein（for CDK4/6）as substrates.P21Cip1 and p27^kip1 proteins were also immuno-precipitated from the whole cell lysates and the interaction of p21Cip1 and p27^kip1 proteins with CDK2,CDK4 and CDK6 was investigated.To test the involvement of p27^kip1 in ATG-induced G0/G1 phase arrest and cell proliferation inhibition in PDGF-BB-activated HSCs,the effects of p27^kip1 knockdown by specific siRNA were examined.To delineate the molecular mechanisms underlying ATG-induced up-regulation of p27^kip1 and cell proliferation inhibition,we examined the effects of ATG on downstream signaling cascades induced by PDGF-BB in HSCs.including phosphorylation levels of Akt and FOXOs,the interaction of FOXO3a with 14-3-3,and FOXO3a nuclear translocation.Furthermore,we transfected FOXO3a siRNA to investigate the effect of knockdown of FOXO3a on ATG-mediated up-regulation of p27^kip1 expression.Results: We investigated the therapeutic effect of ATG on CCl4-induced liver fibrosis in rats.The results indicated that ATG 1 and 3 mg/kg improved the liver function by decreasing serum contention of aminotransferase（ALT）,aspartate aminotransferase（AST）,total bilirubin（TBIL）,hyaluronic acid（HA）,laminin（LN）,procollagen Ⅲ （PC Ⅲ）,and increasing serum contention of albumin（ALB）.Histological results indicated that ATG（1 and 3 mg/kg）alleviated liver damage and reduced the formation of fibrous septa.Moreover,ATG（1 and 3 mg/kg）significantly decreased liver HYP when compared with CCl4 model group.In addition,CCl4-induced proliferation of activated HSCs was inhibited by ATG（1 and 3 mg/kg）,and this was accompanied by down-regulation of cyclin D1,cyclin-dependent kinase CDK2,CDK4,and proliferating cell nuclear antigen（PCNA）,and up-regulation of p27^kip1 in activated HSCs.We investigated the anti-proliferative effect and possible molecular mechanisms of ATG in in a cell line of human HSCs（LX-2）activated by PDGF-BB.Our data indicated ATG inhibiting PDGF-BB-activated HSCs proliferation and arrested cell cycle at G0/G1 phase.The cyclin-dependent kinase（CDK）4/6 activities could be strongly inhibited by ATG through down-regulation of cyclinD1 and CDK4/6 expression in early G1 phase arrest.In the ATG-treated HSCs,the expression level of p27^kip1 and the formation of CDK2-p27^kip1 complex were also increased.P27Kip1 silencing significantly attenuated the effect of ATG,including cell cycle arrest and suppression of proliferation in activated HSCs.We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead boxO 3a（FOXO3a）,decreased binding of FOXO3a to 14-3-3 protein,and stimulated nuclear translocation of FOXO3a in activated HSCs.Furthermore,knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27^kip1 in activated HSCs.Conclusion: ATG can alleviate hepatic injury and fibrosis induced by CCl4,which is probably associated with increasing the levels of p27^kip1 protein through inhibition of PI3K/Akt and improvement of FOXO3a activity,which in turn inhibited the CDK2 kinase activity,and eventually caused an overall inhibition of HSCs proliferation.