The Role Of PERK-autophagy Pathway In The Pathogenesis Of Non-alcoholic Fatty Liver Disease

Objective Nonalcoholic fatty liver disease is one of the most common metabolic diseases at present.Protein kinase-R-like endoplasmic reticulum kinase(PERK)pathway is one of the three pathways of endoplasmic reticulum stress activation.The aim of this study is to explore the relationship between PERK pathway and autophagy and its role in the pathogenesis of nonalcoholic fatty liver disease(NAFLD).Methods Oleic acid-induced HepG2 and LO2 cells’ lipid accumulation were used to form a fatty liver in vitro cell model.HepG2 and LO2 cell models were treated with different concentrations of rapamycin to detect autophagy levels and lipids in each group.Autophagy level,lipid accumulation and apoptosis were detected after cells were treated with different concentrations of rapamycin,autophagy inhibitor chloroquine and PERK inhibitor GSK2606414,cell groups are as follows: oleic acid group,oleic acid + rapamycin group,oleic acid + rapamycin group;blank control group,oleic acid group,oleic acid + rapamycin group,oil acid + rapamycin + chloroquine group and oleic acid + rapamycin + GSK2606414 group.The expression levels of Beclin-1 and PERK were detected in cell models transfected with Beclin-1 si-RNA and PERK si-RNA,cell groups are as follows: oleic acid + negative control si-RNA,oleic acid + Beclin-1 si-RNA group and acid + PERK si-RNA group;autophagy levels,lipid deposition and apoptosis in each group were detected.Quantitative determination of cell lipid deposition were detected by oil red O(ORO)staining qualitative and triglyceride(TG)content detection kit;expression levels of PERK,BECLIN-1 and LC3 proteins were detected by Western blot;autophagic flow levels of cells were visualized by transient transfection of mRFP-GFP-LC3 plasmid;flow cytometry by FITC-Annexin-V / PI double staining were used to detected the apoptosis rate of each group.Results(1)Effects of different concentrations of rapamycin on autophagy level,intracellular lipid deposition and apoptosis in HepG2 and LO2 cell models is as follows: In low concentration of rapamycin and high concentration of rapamycin groups,the levels of LC3II/β actin and BECLIN-1 /β actin increased,and in high concentration of rapamycin group,LC3II/β actin and BECLIN-1 /β actin levels were higher than low-concentration rapamycin group;low-concentration rapamycin treatment of cells can reduce lipid deposition and apoptosis in cell models,and high-concentration rapamycin-treated cells can significantly reduce lipid deposition,while increased apoptosis.(2)The results in both HepG2 cells and LO2 cell models were as follows: In oleic acid + rapamycin + chloroquine group,LC3 II / β actin level was significantly higher than oleic acid + rapamycin group,BECLIN-1 /β-actin and autophagic flow levels were lower than oleic acid + rapamycin group;In oleic acid + rapamycin + GSK2606414 group,Beclin-1 /β-actin,LC3II/β-actin and autophagic flow were lower than oleic acid + rapamycin group,but PERK / β-actin level was increased;In oleic acid + rapamycin + chloroquine group and oleic acid + rapamycin + GSK2606414 group,lipid accumulation were higher than oleic acid + rapamycin group,while the apoptotic rate was lower than oleic acid + rapamycin group.(3)The results in both HepG2 and LO2 cell models were as follows: In oleic acid + Beclin-1 si-RNA group,Beclin-1 /β-actin and LC3II/β-actin levels were decreased compared with oleic acid + negative control si-RNA group,and lipid deposition increased,apoptosis rate decreased.In oleic acid + PERK si-RNA group,PERK / β-actin,Beclin-1 / β-actin and LC3 II / β-actin decreased,at the same time,lipid deposition increases and the rate of apoptosis decreases.Conclusion Our study found that autophagy has a certain effect on lipid deposition and apoptosis in NAFLD cell model,but excessive autophagy increases the apoptotic rate of cells.Inhibition of PERK can reduce autophagy levels and it will increase lipid deposition,but it can reduce the rate of apoptosis in NAFLD cell model,so this approach may become a new method for the treatment of NAFLD.