Effects Of Icaritin On Rat Liver Fibrosis And Inhibition Of Primary Rat Hepatic Stellate Cell Activation

[Objective]To establish a stable and reliable rat model of liver fibrosis, observe in rat by intra-gastric administration of Icaritin, research the changes in liver function and liver tissue ofα-SMA expression. To isolate and culture the primary hepatic stellate cells (HSCs) of rat and investigate the effects of Icaritin on inhibiting the activation of primary HSCs in rat.[Methods]In vivo: In order to explore the protection impacts of Icaritin on rat experimental liver fibrosis, rat's hepatic fibrosis was induced by common bile duct ligation (BDL) or intraperitoneal injections of carbon tetrachloride (CCl4). After generation of fibrosis, Icaritin was intra-gastric administration at a does of 1 mg/kg and treated for 4 weeks, 3 d/w. Then, rats(n=6 per group) were sacrified. Serum samples and liver tissue were prepared for liver function evaluations and immunohistochemical evaluations. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL) and albumin (ALB) were measured by an automatic chemistry analyzer. Haematoxylin & eosin (HE) and semi-quantitative analysis of immunohistochemical staining forα-SMA were used to analyze the distribution of activated HSCs. All data was expressed as x_±s.d. and performed by SPSS 16.0. Significance of the differences was evaluated by one-way ANOVA. A probability level of 0.05 or less was accepted as significant.In vitro: To isolate and perfuse the rat's liver with collagenase and obtain the liver cell suspension. Then primary HSCs were separated from other nonparenchymal cells by density gradient centrifugation of Nycodenz. The isolated cells were cultured and identification by immunohistochemical evaluations. Primary rat HSCs were treated with various concentration of Icaritin. The cell growth was assayed by MTT. The inhibitive effect of Icaritin on the proliferation of HSCs was examined by using tetrazolium blue test and the semi-quantitative analysis of immunohistochemical staining forα-SMA. All these experiments endeavor to identify the possible effect of Icaritin on inhibating the activation of primary HSCs in rats. All data was expressed as x~_±s.d., and performed by SPSS 16.0. Significance of the differences was evaluated by one-way ANOVA. A probability level of 0.05 or less was accepted as significant. [Results]In vivo: The liver of BDL rats was intumescent and brownish yellow, some of the rats had green abdominal dropsy. The liver tissue of BDL rats appeared to exhibit a marked increase in the number of bile ductules and an extensive deposition of collagen, which contributed to the formation of pseudolobule. The liver of CCl4-induced hepatic injury rats was greasy and dark red, some of the rats had light yellow abdominal dropsy. CCl4-induced liver injury and fibrosis exhibited extensive cellular swelling and steatosis. The inflammatory infiltration and lobular architecture with thin bands of reticulin joining central areas. Compared with model groups, the liver function index as AST, ALT and DBIL were all significantly decreased in Icaritin-treated groups (P<0.05). Activated HSCs were examined by determiningα-SMA positive areas in rat livers and research by semi-quantitative analysis of immunohistochemistry. 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).In vitro: Through the method of isolating and perfusing rat's liver with collagenase and density gradient centrifugation with Nycodenz, the yield rate of primary HSCs was 3-4x107 per rat. The cell viability was above 90% by Trypan blue dye test. The purity of HSCs was nearly 95% by immunohistochemical staining forα-SMA. Significant inhibition in primary HSCs can be observed in different concentrations of Icaritin, especially co-cultured in the concentration of 16μmol/L at 48h. In the concentration range of 16-48μmol/L, increased growth inhibition on HSCs was detected with the Icaritin concentration increasing. Through the semi-quantitative analysis and immunohistochemical staining forα-SMA after co-cultured at 48h, we observed the significantly decreased expression of theα-SMA positive staining area and the percentage of total area was 34.31±12.68, 36.72±10.68, 30.25±11.58 (vs control group 70.24±20.46, P<0.05 ). These results suggested that inhibiting the activation of primary HSCs played an important role in the anti-fibrotic by Icaritin.[Conclusions]1. Icaritin ameliorates the liver fibrosis progression induced by BDL or CCl4 in rats.2. Icaritin decreases the expression ofα-SMA in fibrotic liver tissue and inhibits the actvation of HSCs in vivo. 3. It is an important model to use primary cultured rat HSCs to investigate liver fibrosis in vitro.4. Icaritin inhibits the activation of HSCs in vitro that may be one of the mechanisms of Icaritin improves hepatic fibrosis.