Etopside Induce Apoptosis In Activated Human Hepatic Stellate Cells Via ER Stress

BACKGROUND AND OBJECTIVES:Liver fibrosis is a wound-healing response to various types of injury factors.The improvement and reversal of liver fibrosis or even cirrhosis is the focus in the field of liver fibrosis research.The activation of hepatic stellate cells(HSCs)plays a vital role in the progression of liver fibrosis.HSCs are activated to obtain fibroblast phenotype,proliferating and secreting excess collagen,resulting in excessive deposition of extracellular matrix.Therefore,removal of activated HSCs is the key to reverse fibrosis.The resolution of fibrosis involves pathways that cause either HSCs apoptosis,senescence,or reversion to their quiescent stage,and induction of HSCs apoptosis was confirmed to attenuate or reverse fibrogenesis.The therapeutic effects of etoposide(VP-16),a widely used anticancer agent,on HSCs apoptosis and liver fibrosis resolution are still unclear.Our study was designed to invistigate the effect of VP-16 on LX-2 cells in vitro.METHOD:LX-2 cells were treated with different doses of VP-16,then cell viability was assessed by CCK-8 assay,mitochondrial membrane potential was detected by JC-1 staining,cell cycle and cell apoptosis level were measured by flow cytometry,caspase-3 activity was analyzed using a caspase-3 activity assay kit,western blotting was employed to detect the expression of the associated protein,quantitative real-time PCR was used to assesse the expression of ?-SMA and type I collagen.LX-2 cells were treated with different doses of VP-16,caspase inhibitor,JNK inhibitor,and their combinations,respectively.Then,cell viability was assessed by CCK-8 assay,cell apoptosis level was measured by flow cytometry,western blotting was employed to detect the expression of the associated protein.The normal hepatocytes LO-2 and QSG-7701 were incubated with the same concentrations of VP-16 and cell viability and cell apoptosis were detected using CCK-8 assay and flow cytometry,respectively.RESULT:VP-16 reduced the proliferation of LX-2 cells,interrupted the cell cycle at G2/M restriction point,and led to significantly high levels of apoptosis.VP-16 inhibits the proliferation of HSCs by inhibiting the phosphorylation of ERK in vitro,and the apoptosis rate can be partially reversed by the caspase inhibitor.Additionally,the unfolded protein response regulators CHOP,BIP,caspase-12 and JNK,which are considered endoplasmic reticulum(ER)stress markers,were upregulated by VP-16.Furthermore,JNK-mediated phosphorylation activates the proapoptotic protein Bim and Bax,while inhibiting the antiapoptotic protein Bcl-2.And treatment with JNK inhibitor can reverse the inhibitory effect of VP-16 on proliferation and apoptosis.Remarkably,VP-16 treatment decreased the expression of a-SMA and type I collagen and simultaneously increased the ratio of matrix metalloproteinases(MMPs)to tissue inhibitor of matrix metalloproteinases(TIMPs).In contrast,VP-16 induced significantly more apoptosis in HSCs than in normal hepatocytes.CONCLUSION:VP-16 exerts a proapoptotic effect on LX-2 cells via ER stress and has an antifibrogenic effect on collagen deposition,suggesting a new strategy for the treatment of liver fibrosis.