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

AIM OF THE STUDY: 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.

MATERIALS AND

METHODS

: 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).

RESULTS

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).

CONCLUSIONS

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.