Betulin Regulated The Progression Of Alcoholic Liver Disease Based On Lipin1/2-mediated P2X7R Signals

Objective:In this study,we explored the potential molecular mechanism of the regulation of betulin on the development of alcoholic liver disease based on alcohol-stimulated AML-12 cells and Lieber-DeCarli diet-induced mice in vitro and in vivo,providing an effective theoretical basis for the treatment of alcoholic liver disease.Methods:In vitro,,we measured the concentration of MTT to analyze the viability of AML-12 cells,which was treated with 0?100?M betulin or 0-400mM ethanol for 24h,and selected the effective concentration of betulin to establish the model of alcoholic liver disease.On the one hand,we performed oil red o staining and IFC staining to observe the effect of betulin on the lipid droplet accumulation of alcohol-stimulated AML-12 cells and the expression of fatly indicators;on the other hand,Western blotting was used to detect the protein expression of SREBP1,Lipin 1,and P2X7R etc.In vivo,male C57BL/6 mice were ad libitum fed with the Lieber-DeCarli liquid diets containing 5%ethanol for 28 days plus a single binge alcohol feeding to establish the model of alcoholic liver disease.In addition,the betulin group was administered betulin(20mg/kg or 50mg/kg)once a day by gavaging.Followed,serum of mice was taken to measure the levels of AST,ALT and TG.Then H&E staining,oil red o staining,IHC staining and IFC staining were used to observe the histopathological changes of liver in mice.Next,the total protein or total RNA of liver was extracted for Western blotting and RT-PCR experiments to determine the expression of SREBP1,PPARa,Lipin 1,P2X7R,IL-6 and other inflammatory indicators.Results:In vitro results showed that 0?100?M BT had no significant effect on the viability of AML-12 cells,betulin effectively reduced the expression of SREBP1,Lipin 1,P2X7R and NLRP3,increased Lipin2 expression,and significantly decreased the accumulation of lipid in alcohol-induced AML-12 cells.In vivo,betulin inhibited the increases in serum AST,ALT and TG levels in the mice with alcoholic liver disease,while suppressed the expression of SREBP1,Lipin 1,P2X7R and IL-6,enhanced Lipin2,PPARa expression by ethanol.Conclusion:Betulin has no obvious toxicity to hepatocytes,and effectively inhibit the alcohol-induced accumulation of lipid,which may ameliorate the development of alcoholic liver disease by regulating the Lipin 1/2-mediated P2X7R signaling pathway.