Role And Potential Mechanisms Of Caveolin1-mediated Autophagy Activation In Non-alcoholic Fatty Liver Disease

Objective Non-alcoholic fatty liver disease(NAFLD)starts from simple steatosis to more severe steatohepatitis,which may develop into cirrhosis and even liver cancer.Caveolin-1(CAV1),a scaffold protein,have been attributed multiple roles in cells besides caveolae formation,including endocytosis,autophagy,liver energy metabolism and lipid homeostasis.However,it is unclear whether CAV1 can affect liver lipid metabolism by regulating autophagy.The purpose of this study was to investigate the role of CAV1 in NAFLD and its possible molecular mechanism.Methods1.Experiment in vivo The biological function of CAV1 can be realized by CSD,the CAV1 scaffolding domain peptides(amino acids 82-101 of CAV1)which bind to a variety of signaling molecules.The mice were divided into 3 groups.The control group was fed with control diet,the other two groups were fed a high-fat diet(HFD).CSD treatment group mice were daily received intraperitoneal injections of CSD solution.HE and Oil red O staining were used to observe the pathological changes of liver tissues.Serum ALT,AST and TG levels were determined by the kit.Autophagosomes and lipid droplets were observed by transmission electron microscope.Western blot was used to detect the expression of CAV1,SREBP-1,LC3-II/I,Beclin1,p62,Akt and m TOR proteins.2.Experiment in vitro2.1 L02 cells were treated with alcohol and oleic acid(A/O)for 48 h to establish an in vitro model of hepatic steatosis.A/O treated cells were transfected with GV146-CAV1 and CAV1-si RNA to explore the effect of CAV1 on lipid metabolism and autophagy.2.2 To assess the effect of autophagy inhibition on CAV1-induced alleviative steatosis,CQ was treated in steatosis-cells after plasmid transfection experiment.2.3 To assess whether the m TOR pathway was involved in CAV1-si RNA-induced autophagy inhibition and lipid deposition,m TOR inhibitor RAPA was treated in steatosis-cells after RNA interference experiment.Results1.Experiment In vitro1.1 The SREBP1 expression was significantly increased,whereas the expression of CAV1 and autophagy-related proteins(Beclin1 and LC3-II/I)were significantly decreased and the level of p62 were significantly increased in HFD mice compared to control mice.The protein results in L02 cells were consistent with those in vivo.The immunofluorescence cytochemistry assay showed that CAV1 was almost negative for steatosis-cells.The above results suggest that CAV1 and autophagy might be involved in the development of NAFLD.1.2 HE and Oil Red O staining revealed extensive lipid deposition manifested as microvesicular steatosis and red lipid droplets in the HFD group.Besides,serum levels of ALT,AST and TG were markedly decreased in the CSD group compared to the HFD group.Electron microscopy showed that the number of fat vacuoles and damaged mitochondria were decreased,whereas autophagosomes were increased in the CSD group compared with the HFD group.Western blotting showed that CSD treatment decreased SREBP1 level in the HFD group.Furthermore,CSD treatment increased the expressions of CAV1,LC3-II/I and Beclin1,whereas decreased the accumulation of p62.Altogether,these results indicated that the upregulation of CAV1 could reduce lipid accumulation and promote autophagy in HFD mice.2.Experiment in vitro2.1 Up-regulation of CAV1 appeared to alleviate lipid accumulation with reduced lipid droplets,whereas the down-regulation of CAV1 aggravated lipid accumulation.Likewise,Western blot indicated that the level of SREBP1 was decreased in the GV146-CAV1 group compared with the GV146-Control group,whereas SREBP1 were increased in the CAV1-si RNA group compared with the Control-si RNA group The results of autophagy-related proteins showed that caveolin1 overexpression plasmid can significantly increase the reduced autophagy level in the model group,and CAV-si RNA silencing further inhibited autophagy in the model group.And CQ reversed the reduced lipid accumulation and increased autophagy level regulated by CAV1 overexpression in steatosis cells.These results indicated that the autophagic induction of CAV1 might be involved in the reduction of lipid accumulation in L02 cells.2.2 CSD treatment significantly reduced the expression of phosphorylated Akt and phosphorylated m TOR in HFD mice.Besides,transfection with GV146-CAV1 significantly reduced the upregulation of phosphorylated Akt and phosphorylated m TOR in A/O treated cells.On the contrary,after transfection with CAV1-si RNA,phosphorylated Akt and phosphorylated m TOR were further increased in A/O treated cells.And RAPA reversed the aggravated lipid accumulation and decreased autophagy level regulated by CAV1-si RAN in steatosis cells..Together,these results indicated that the inhibition of the m TOR pathway may contribute to the effects of CAV1 on lipid accumulation and autophagy.Conclusion1.The protein level of CAV1 was lower in HFD(high-fat diet)fed mice liver and in A/O(alcohol and oleic acid mixture)treated L02 cells.2.Mechanistically,the inhibition of Akt/m TOR pathway was involved in the protective role of CAV1 in autophagy induction and lipid metabolism in NAFLD...