Rev-erbα Regulates Steatosis Of Alcoholic Fatty Liver Through Autophagy

Alcoholic liver disease(ALD)is a kind of liver systemic disease caused by long-term excessive drinking.ALD is a complex pathological process,which includes alcoholic fatty liver,alcoholic hepatitis,alcoholic liver fibrosis and alcoholic cirrhosis,eventually develops into liver cancer.Alcoholic fatty liver disease(AFLD)is considered to be the initial stage of pathological development of ALD.It is mainly a liver disease characterized by fat accumulation,mild inflammatory infiltration and slight liver cell damage.If the fatty degeneration is not controlled,it can further lead to liver cell death and damage of liver cell function and lead to further deterioration of the disease.Therefore,it is very important for the treatment and prevention of AFLD to study the regulatory mechanism of alcohol induced lipid metabolism disorder.Studies have showed that Rev-erbα is involved in a variety of metabolic diseases,and it has a certain regulatory effect on lipid metabolism.However,the role of Rev-erbα in AFLD has not been studied.The purpose of this study is to explore whether Rev-erbα can participate in the development of AFLD by regulating lipid metabolism,and to explore the possible molecular mechanism of Rev-erbα affecting lipid metabolism.The main research contents are as follows:1.The expression of Rev-erbα in the liver of EtOH-fed mice and EtOH induced L02 cellsIn vivo,we adopted the chronic ethanol plus a single ethanol binge feeding on mice described by the NIAAA model protocol to create AFLD animal model.The results of H&E,Oil Red O staining,immunohistochemistry,RT-PCR and Western blot showed that there were more fat vacuoles and lipid droplets in EtOH-fed mice.The levels of triglyceride(TG),total cholesterol(T-CHO)and alanine aminotransferase(ALT)in the serum of EtOH-fed mice were higher,and the expression of Pparα was decreased while Srebplc was increasedIn vitro,we used 150 mM ethanol to stimulate L02 cells,the results of Oil Red O staining,TG content detection,RT-PCR and Western blot showed that after ethanol stimulation,lipid droplet deposition and TG level in the cells was increased,and Pparαand Srebp1c was abnormal expressed in EtOH-stimulated L02 cellsComparing the expression of Rev-erbα with Rev-erbβ in EtOH fed mice liver and ethanol induced L02 cells,we found that the expression of Rev-erbα mRNA in CD-fed mice liver and L02 cells in control group was significantly higher than that in Rev-erbβThe expression of Rev-erbα mRNA in the liver of mice and cells of the model group increased significantly,but the expression of Rev-erbβ has no significantly change.The results of immunohistochemistry,immunofluorescence,RT-PCR and Western blot showed that the expression of Rev-erbα in AFLD and ethanol induced L02 cells increased significantly,and the expression of Rev-erba only increased in the nucleus but not in the cytoplasm.It is suggested that the expression of Rev-erbα in the liver of AFLD and L02 cells induced by ethanol is significantly increased2.Effects of Rev-erbα on lipid metabolism in the liver of EtOH-fed mice and EtOH induced L02 cellsIn vivo,the mice fed with alcoholic liquid feed was injected with Rev-erbαantagonist SR8278(10 μM,three days)in tail vein.H&E,Oil Red O staining,immunohistochemistry and Western blot results showed that the fat vacuoles in the liver tissue of the mice were significantly reduced and the lipid accumulation was relievedIn vitro,Rev-erbα ShRNA was used to knockdown the Rev-erbα expression in L02 cells,Then,we treated the cells with ethanol(150 mM)and analyzed the effect of Rev-erbα silencing on lipid metabolism.TG content analysis,Oil Red O staining,RT-PCR and Western blot showed that the lipid accumulation in the cells was alleviated.The same results was found in EtOH+SR8278-stimulated L02 cells compared to EtOH-stimulated L02 cells3.The mechanism of Rev-erbα regulating steatosis3.1.The activity of autophagy in the liver of EtOH-fed mice and EtOH induced L02 cellsThe results of transmission electron microscopy and immunohistochemistry showed that autophagy formation decreased in EtOH fed group.In vitro,Western blot and lyso tracker green DNA-26 lysosomal probes showed that autophagy was also significantly reduced in ethanol induced L02 cells3.2.Effects of Rev-erbα on autophagy activity in the liver of EtOH-fed mice and EtOH induced L02 cellsIn order to further study the effect of Rev-erbα on autophagy activity in EtOH-fed mice and ethanol induced L02 cells,SR8278 was injected into tail vein of EtOH-fed mice.The results of transmission electron microscopy and immunohistochemistry showed that the formation of autophagy in the liver was increased after administration of SR8278.In vitro,Western blot and Lyso tracker green DNA-26 lysosomal probes showed that when Rev-erbα was silenced or treated with Rev-erbα antagonist,the formation of autophagic bodies increased,and the acidity of lysosomes returned to normal.It is suggested that Rev-erbα may affect lipid metabolism by regulating autophagy activity 3.3.Effect of Bmal1 on Rev-erbα regulating autophagy activityIn order to further explore the mechanism of Rev-erbα regulating autophagy activity,it was found that Bmall expression was increased after silencing Rev-erbαwith Rev-erbα ShRNA,while Bmall expression was decreased in EtOH-fed mice liver tissue and ethanol induced L02 cells.Further,reducing Bmall on the basis of silence Rev-erbα,the expression of Lc3 was decreased,the expression of P62 was increased and the acidity of lysosome was also decreased.Western blot showed that after Bmall was down expressed,the expression of Pparα was also down regulated,but there was no significant difference in Srebp1 expression.The results suggest that Rev-erba may negatively regulate autophagy by targeting Bmal1 and then affect liver steatosis.