Icaritin Induces The Apoptosis Of Hepatic Stellate Cells And Ameliorates The Development Of Liver Fibrosis In Rats

[Background and Objective]To investigate the effects and mechanisms of icaritin inducing the apoptosis of activated hepatic stellate cells and ameliorating the development of liver fibrosis in rats.[Methods]In vitro:Immortalized rat HSC lines, HSC-T6 (Rat) and LX-2 (Human), and four human hepatocyte lines (HL-7702 (L02), Chang Liver, WRL-68, THLE-3) were treated with various concentration of icaritin. The cell growth was assayed by MTT. Apoptotic cell ratios were examined by flow cytometry and the Annexin-V-FITC apoptotic detection kit. Then, the whole rat genome microarray kit, a tool for modeling human biology in the cell model organism, was used to identify the differentially expressed genes. Real-time PCR and Western blot were also utilized to determine the changes of apoptotic genes respectively at gene level and protein level. All these experiments endeavor to primarily identify the possible molecular mechanisms of icaritin induction apoptosis of HSCs.All data was evaluated by one-way ANOVA and between control groups with the PASW 18.0. A probability level of 0.05 or less was accepted as significant.In vivo:in order to study the protection effect of icaritin on rat experimental liver fibrosis, rats'hepatic fibrosis was induced by common bile duct ligation (CBDL) and CCl4. After generation of fibrosis, icaritin was intra-gastric administration at a does of 1 mg/kg icaritin given three times per week, followed by 4 weeks. Then, rats(n=6 per group) were sacrified. Liver and serum samples were prepared for immunohistochemical evaluations, liver function evaluations and hydroxypeolin content assay. ALT and AST were measured on an autodyne chemistry analyzer. Haematoxylin and eosin (HE), Western blot and Immunohistochemical staining were used to determine the relative content of collagen I in rats'livers. Staining for a-SMA was used to analyze the distribution of activated HSCs. All data was expressed as x±s.d. and performed by PASW 18.0. Significance of the differences was evaluated by one-way ANOVA and t-test analysis. A probability level of 0.05 or less was accepted as significant.[Results]Significant inhibition in hepatic stellate cells can be observed. The IC50 of icaritin on HSC-T6 was 12.83μmol/L at hour 48, and IC50 on LX-2 was 24.18μmol/L. In the concentration range of 8～32μmol/L, increased growth inhibition on HSCs were detected with the icaritin concentration increasing. However, icaritin caused little affections on hepatocyte strains. Then, we found that icaritin induced apoptosis of activated HSCs. It is supposed that apoptotic ratio of HSC-T6 was 20.19% when co-cultured with icaritin of 24μmol/L, besides the disappearance of Dip G2 in cell cycle. The apoptotic ratios were significantly increased compared to the HSC-T6 cultured under common conditions (P< 0.05). The Similar effects can be observed in LX-2. These data suggested that icaritin induced apoptosis of activated HSCs and inhibition HSCs proliferation through blocking the transform from phage S to phage G2. The whole rat genome oligo microarray kit detection suggested that, compared with control group, HSC-T6 co-cultured with icaritin had much difference on pathways related cell proliferation and apoptosis. Significant changes can be observed in many apoptotic genes, especially Bcl-2 family genes. The result showed that up-regulation genes included Bak-1, Bmf and Bax, while Bcl-2 was down-regulated significantly (vs. control group, P<0.01). In this experiment, BAX, caspase-3 and caspase-12 were up-regulated with the concentration of icaritin increased. BAX was activated after HSC-T6 co-cultured with 6μmol/L icaritin 48h, besides caspase-3 degraded from the zymogenic form (32 kD) to 17kD. These two apoptotic proteins was micro amount expression in control group (P<0.01). These results suggested that mitochondrion pathway and FasL signaling transductors played an important role in the apoptosis of activated HSCs caused by icaritin.In vivo study, we observed that a large mount of collagen deposit nearby the cholangioles with respect to shrinkage of the fibrotic area. While the pathological changes of liver in the group treated with icaritin (1 mg/kg, three times per week) were rather milder. And they show less fibrous tissue proliferation (vs. model group, P<0.05). Compared with model group, AST, ALT and DBIL were all significantly decreased (P< 0.05). Especially the concentration of hydroxyproline in liver tissue had significant difference with model group (P<0.01), and no difference was found between the icaritin group and normal group. Activated HSCs were examined by determiningα-SMA-positive areas in rat livers. In model groups, the distribution ofα-SMA positive areas was similar to that of inflammation areas. In icaritin-treated groups, the expression ofα-SMA was much lower than that in model groups (P<0.05). Western blot results show that higher concentration can be observed in model groups compared to control, and the concentration was decreased significantly after icaritin-treatment (P<0.05).[Conclusions]1,Icaritin could induce growth and proliferation suppression of HSC-T6 (rat hepatic stellate cell line) and LX-2 (human hepatic stellate cell line) in vitro.2,Icaritin can induce apoptosis of activated HSCs in vitro, but had no toxic to normal hepatocyte lines.3,Icaritin induces activated HSCs apoptosis may regulated by death receptor signaling pathway and mitochondrion pathway.4,Icaritin ameliorates the fibrosis progression induced by CBDL or CCl4 in rats.5,Icaritin plays anti-fibrotic roles through decrease the activated HSCs in cirrhotic liver tissue.