The Role Of Autophagy In Free Fatty Acids Induced Non-alcoholic Fatty Liver Disease

Background/Aims: Nonalcoholic fatty liver disease(NAFLD)is a metabolic disorder commonly associated with lipid accumulation Recently,with the gradual improvement of the living standards of residents in China,the incidence of NAFLD is increasing year by year.Therefore,NAFLD has become a major health problem endamering the people's health.However,the mechanisms underlying the development of NAFLD remain unclear.Thus,searching for novel NAFLD associated molecules may broaden our understanding about the progression and development of NAFLD.Autophagy is a reparative life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments.Dysfunctions of autophagy play an important role in the progression of chronic liver diseases.The role of autophagy in the pathogenesis of non-alcoholic fatty liver disease(NAFLD)was still controversial.The current study was to study the role and molecular mechanisms of autophagy in the NAFLD.METHODS:Part one:Construction and indentification of NALFD cell model for investigationg the relationship bewteen NAFLD and autophagy.Objective:To construction NAFLD cell model,and then identify whether it can mimic the pathological features of human non-alcoholic fatty liver,eamine the relevance between autophagy and NAFLD.Methods:1.Cell model for investigating non-alcoholic fatty liver was induced by incubating LO2 cells and Huh7 cells with a free fatty acids(FFA)mixture(OA:PA=2:1)at a dose of 200,400,600 or 800 ?M for 24 hours.2.Cell viability was evaluated by MTT,the degree of lipid accumualtion was evaluated by H&E staining and flow cytomerty3.The autophagy-related genes were measured by western blotting analysis.RESULTS: 1.FFA induces a significant increment in the intracellular content of lipid droplets and the accumulation of fatty droplet is dose-dependent in L02 and Huh7 cells,which is observed by H&E staining and flow cytomerty.2.Triglyceride content of the model group was significantly increased than the normal(p<0.01).3.Cell viability,lipid preoxidation showed no significant differences between the normal and model group.4.The conversion of cytosolic LC3-I to membrane-bound LC3-II and the levels of BECN1 were increased,but the level of p62 was decreased in NAFL cell models.Conclusion: The NALF cell model,constructed by incubating Huh7 cells and LO2 cells with DMEM containing FFA mixture(OA:PA=2:1)for 24 hours,can replicate the pathological feature of human simple fatty liver.Autophagy is associated with NAFLD,Part two:The role of Autophagy in free fatty acids induced Non-alcoholic fatty liver diseaseObjective:To explore the causative role of autophagy in the free fatty acids induced nonalcoholic fatty liver disease.Methods:1.As described in the first part,we firstly incubate LO2 cells and Huh7 cells.Then we divined them into four groups:normal group,3-MA(5?M)group,FFA(600?M)group,3-MA(5?M)+FFA(600?M)group.2.The protein of four groups cells above were extracted,total protein was analyzed by western blot to delect the protein level of LC3 B make sure the function of 3-MA.3.The cells of four groups were processed by H&E staining,flow cytometry and triglyceride.Results:1.The 3-MA group's protein level of LC3-II was decreased,compared with normal group.2.the autophagy inhibitor 3-MA supplementation significantly alleviated the accumulation of fatty droplets in L02 and Huh7 cells.Conclusion: Our results suggest that autophagy has significant effect on hepatic lipid accumulation in NAFL cell models.Inhibition of autophagy impairs free fatty acids induced excess fat accumulation in the hepatocellular carcinoma and hepatic cells.Summary:1.Using FFA mixture(OA:PA)DMEM culture medium for 24 hours,the cell model of NAFLD can be constructed,replicate the pathological features of human NAFLD.2.inhibition of hepatocyte autophagy results in excessive liver lipid accumulation,and the autophagic function may be involved in the pathogenesis of NAFLD.3.Autophagy is a new mechanism,which might be translated its concept into the therapeutic.