The Role And Mechanism Of Endoplasmic Reticulum Unfolded Protein EIF 2? In Collagen Production In Hepatic Stellate Cells

Objectives:Liver fibrosis caused by chronic viral hepatitis and other stimulators is a necessary common way to develop into liver cirrhosis.Reversing liver fibrosis or delaying the progression of liver fibrosis has become an important entry point for the prevention and treatment of cirrhosis.The occurrence of hepatic fibrosis is mainly related to the activation and proliferation and the secretion of extracellular matrix(extracellular matrix,ECM)in hepatic stellate cells(HSC).Therefore,the reversal of activated HSC or the promotion of apoptosis has become a key to the treatment of liver fibrosis.Endoplasmic reticulum stress can induce apoptosis of HSC and also affect autophagy of HSC,but the relationship between HSC apoptosis and autophagy is still unclear.This study aims to determine the role and mechanism of eIF2? in hepatic stellate cells collagen production based on the study of endoplasmic reticulum unfolded protein eIF2? overexpression and activation of HSC endoplasmic reticulum stress,in order to provide new ideas for reversing liver fibrosis.Methods:The experimental subjects were human immortalized hepatic stellate cells(HSC-LX2 cell line),which were overexpress eIF2a by plasmid transfection and promoted themselves activation and Type I collagen(COL1A2)expression by TGF-?1.According to the experimental intervention conditions they were divided into plasmid empty group(vehicle group),transforming growth factor-?1 intervention group(TGF?1 group),transforming growth factor-?1+overexpression eIF2a transfection group(eIF2a plasmid group).Western Blot was performed to analyze of LX2 cell activation protein ?-SMA,type Icollagen,autophagy-associated protein LC3?,P62,endoplasmic reticulum stress unfolded protein eIF2a,eIF2a phosphorylated functional protein(p-eIF2a),differential expression of proapoptotic protein CHOP and anti-apoptotic protein Bcl-2;qRT-PCR was used to detect the transfection efficiency of eIF2a plasmid;autophagosomes were observed by fluorescence microscopy,and apoptosis of HSC groups was analyzed by flow cytometry.Results:Western Blot results showed that the ?-SMA and COL1A2 proteins of HSC in TGF?1 group were significantly higher than those in Vehicle group,and the expression levels of a-SMA and COL1A2 in eIF2a plasmid group were lower than those in TGF?1 group.Compared with the Vehicle group,autophagy related protein P62 was decreased and LC3? was increased in the TGF?1 group,while P62 was increased and LC3? was decreased in the eIF2?plasmid group,further indicating that HSC autophagy was inhibited in the eIF2 plasmid group.Compared with Vehicle group,the functional protein(p-eIF2?)of endoplasmic reticulum stress protein eIF2a was decreased,the apoptosis promoting protein CHOP was decreased,and the antiapoptotic protein Bcl-2 was increased in TGF?1 group.Compared with TGF?1 group,the functional protein(p-eIF2a)of endoplasmic reticulum stress protein eIF2 a was increased,the apoptosis promoting protein CHOP was increased,and the antiapoptotic protein Bcl-2 was decreased in eIF2a plasmid group compared with TGF?1 group.Further analysis showed that the expression of eIF2a protein was negatively correlated with the expression of COL1A2 protein and negatively correlated with the expression of LC3II.qRT-PCR detected eIF2a plasmid transfection was significantly higher than that of the Vehicle group;Fluorescence microscopy showed an increase in autophagosomes in the TGF-?1 group compared with the Control group.Flow analysis showed that the apoptosis rate in the eIF2a plasmid group was significantly higher than that in the TGF-?1 group.Conclusions:Overexpression of eIF2a in HSC may trigger autophagy inhibition and apoptosis promotion,thereby reduce collagen production in these cells,which may provide a potential therapeutic strategy for hepatic fibrosis.