Icaritin induces cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorates the development of liver fibrosis in rats

Abstract

Icaritin is an active ingredient extracted from the plant Herba Epimedium Sagittatum (Sieb. et Zucc.) Maxim. The purpose of this study is to investigate the effects and mechanisms of icaritin-induced cell death in activated hepatic stellate cells (HSCs) and ameliorating the development of liver fibrosis in rats. : In vitro, icaritin-induced cell death rates in HSC-T6 (rat) and LX-2 (human) HSC lines as well as normal hepatocyte cell lines HL-7702 (L02) and WRL-68 were assayed by MTT method, and the apoptotic ratios were detected by both flow cytometry and the Annexin-V-FITC Apoptosis Detection Kit. A Whole Rat Genome Microarray Kit was used to identify expression of interest genes through fold-change screening. In vivo study, experimental liver fibrosis models were built by carbon tetrachloride (CCl(4)) or common bile duct ligation (CBDL) in Wistar rats. Icaritin (1mg/kg/day, three days a week) was administered by gastric gavage for four weeks (n=6 per group). At the end of the study, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as the contents of hydroxyproline and collagen I in liver tissues were measured. Histopathological changes and the distribution of activated HSCs were observed in the liver tissues using hematoxyline-eosin (HE) staining and immunohistochemical staining for α-smooth muscle actin (α-SMA). Icaritin induced apoptosis in HSC-T6 and LX-2 in a concentration- and time-dependent manner with little toxicity to normal hepatocyte cell lines. The IC(50) of icaritin in HSC-T6 was 12.83 μM at 48 h. Apoptotic ratio of HSC-T6 treated with 24 μM icaritin was 20.19%, and the G2 phase of the cell cycle did not occur (P<0.05). Gene analysis showed that icaritin up-regulated Bak-1, Bmf and Bax expression while significantly down-regulated Bcl-2 expression (vs. control group, P<0.01). These results suggested that mitochondrial pathway played an important role in icaritin-induced apoptosis in activated HSCs. In vivo results showed that icaritin reduced the number of activated HSCs, and brought the elevated levels of AST, ALT, hydroxyproline and collagen I to normal or near normal values (vs. model group, P<0.05). Icaritin can induce cell death in activated HSCs through mitochondria-mediated apoptosis, ameliorate the progression of hepatic fibrosis in rats, and could be a promising drug for treating liver fibrosis.