Berberine Ameliorates Abnormal Lipid Metabolism Via The AMPK/SIRT1 Pathway In Alcoholic Fatty Liver Disease

Background: Alcoholic fatty liver disease(AFLD)is an early stage of alcohol-related liver disease characterized by abnormal lipid metabolism in hepatocytes.To date,there is no effective strategy to prevent or treat alcohol-related liver disease other than abstinence from alcohol.Berberine(BBR)is a major bioactive component extracted from Chinese herbal medicines such as Huang Lian and Scutellaria baicalensis.Numerous studies have shown that it has a wide range of pharmacological effects on various diseases,including vasodilation,blood pressure regulation,inflammation inhibition,immunomodulation,and cancer treatment,in both animal models and clinical practice.Berberine can also have a protective effect in the liver by reducing inflammatory responses and oxidative stress In vivo and In vitro having a protective effect on liver function.In addition,berberine exerts lipid-regulating effects by regulating apoptosis,autophagy and improving insulin resistance.However,the potential function of BBR in AFLD remains unclear.Objective: This study was designed to investigate the role of berberine in the regulation of lipid metabolism and molecular mechanisms in alcoholic fatty liver disease through a mouse model of alcohol-related liver disease and an In vitro cellular model.Materials and Methods.pharmacodynamics study:1.In vivo experiment: to investigate the hepatoprotective effect of BBR and the effect on alcohol-induced hepatic steatosis in mice.Male C57BL/6J mice were divided into six groups(8 mice per group)according to the randomization principle: control liquid diet group,control liquid diet + 200 mg/kg BBR,alcohol liquid diet group,alcohol liquid diet+200mg/kg silybin(Sil)group,alcohol liquid diet + 50 mg/kg BBR group,alcohol liquid diet + 100 mg/kg BBR group and alcohol liquid diet + 200 mg/kg BBR group.The alcoholic fatty liver disease model was established by chronic feeding of sixteen-day ethanol diet according to the Gobin model.From the first day of model establishment,mice in different groups were given different doses of berberine and fed a liquid diet.During the acclimatization feeding period,all mice were fed a control liquid diet for three days,and during the next thirteen days of the modeling period,the control liquid diet group,the control liquid diet + 200 mg/kg BBR group was fed a control liquid diet,while the other groups were fed an ethanolic liquid diet.On day sixteen,the mice fed the ethanol liquid diet were administered a single dose of 5 g/kg of ethanol at a concentration of 33%by gavage.Nine hours later,the mice were treated under anesthesia and blood samples and liver tissue were collected for further analysis.The extent of liver damage,lipid accumulation and changes in genes related to lipid metabolism in mice with alcoholic diet-induced alcoholic fatty liver disease were measured by histopathology(H&E staining,Oil Red O staining),serology(TG,TC content measurement),liver mass/body mass ratio,and by using ELISA,protein immunoblotting and real-time fluorescence quantitative PCR methods.2.In vitro experiments: To investigate the effect of BBR on alcohol-induced lipid metabolism in AML-12 cells In vitro.100 m M alcohol stimulated AML-12 cells were established as an In vitro model of lipid metabolism disorder.The effect of different concentrations of BBR administration on cell viability of ethanol-stimulated AML-12 cells was examined.The groupings in the experiments to explore the In vitro pharmacodynamic effects were as follows: normal control,model group(100 m M alcohol),model + BBR(2.5 μM),model + BBR(5 μM),model + BBR(10 μM).And the improvement of cellular lipid metabolism by different concentrations of BBR administration was verified by protein immunoblotting,oil red O staining and real-time fluorescence quantitative PCR experiments.Mechanism study:1.In vitro experiment: to investigate the effect of BBR on the expression level of SIRT1 in the SIR2 family in alcohol-stimulated AML-12 cells.Set up grouping: normal control group,model group(100 m M alcohol),model + BBR(10 μM),normal + BBR(10 μM).Real-time fluorescence quantitative PCR assay was performed to detect the effect of BBR on the expression level of SIR2 family in alcohol-stimulated AML-12 cells.Protein immunoblotting assay was performed to detect the changes of SIRT1 expression in alcohol-stimulated AML-12 cells after BBR administration.In vivo experiment: to investigate the effect of BBR on the expression level of SIRT1 in alcohol diet-stimulated mice.Set up groups: control liquid chow group,control liquid chow + 200 mg/kg BBR,alcohol liquid chow group,alcohol liquid chow + 50 mg/kg BBR group,alcohol liquid chow + 100 mg/kg BBR group and alcohol liquid chow + 200 mg/kg BBR group.ELISA experiments and protein immunoblotting assays were performed to detect the effects of BBR administration on alcohol diet-stimulated the changes of SIRT1 expression level in mice stimulated by alcohol diet after BBR administration.2.In vitro experiment: to investigate the effect of SIRT1 on lipid metabolism in alcoholstimulated AML-12 cells.Set up grouping: model group(100m M alcohol)+ empty vector group,model group(100m M alcohol)+ empty vector + BBR(10μM)group,model group(100m M alcohol)+ si-SIRT1 group,model group(100m M alcohol)+ si-SIRT1 + BBR(10μM)group.The changes of genes related to lipid metabolism were detected by protein immunoblotting and real-time fluorescence quantitative PCR methods.And the changes of AMPK,p-AMPK protein levels were detected by protein immunoblotting.3.In vitro experiment: to investigate how SIRT1 affects lipid metabolism in alcoholstimulated AML-12 cells via AMPK.Set up grouping: model group(100m M alcohol)+empty vector group,model group(100m M alcohol)+ empty vector + BBR(10μM)group,model group(100m M alcohol)+ Dorsomorphin(AMPK phosphorylation inhibitor)group,model group(100m M alcohol)+ Dorsomorphin + BBR(10μM)group.Changes in SIRT1 expression were detected by protein immunoblotting and real-time fluorescence quantitative PCR methods.Intermolecular interaction simulation experiments were performed using Discover Studio software.4.In vivo experiment: to investigate the effect of BBR on AMPK phosphorylation in the liver of alcohol diet fed mice.Set up groups: control liquid diet group,control liquid diet+ 200mg/kg BBR,alcohol liquid diet group,alcohol liquid diet + 200mg/kg BBR group.The changes of AMPK,p-AMPK protein levels were detected by protein immunoblotting.In vitro experiment: to investigate the effect of BBR on alcohol-induced AMPK phosphorylation in AML-12 cells.Normal control group,model group(100 m M alcohol),model + BBR(10μM).Changes in AMPK,p-AMPK protein levels were detected by protein immunoblotting.5.In vitro experiment: to investigate the effect of AMPK on alcohol-induced lipid metabolism in AML-12 cells.Set up grouping: model group(100m M alcohol)+ empty vector group,model group(100m M alcohol)+ empty vector + BBR(10μM)group,model group(100m M alcohol)+ si-AMPK group,model group(100m M alcohol)+ si-AMPK+ BBR(10μM)group.Lipid droplet accumulation in AML-12 cells was detected by oil red O staining,and the expression levels of genes related to lipid metabolism were detected by immunoprotein blotting and real-time fluorescence quantitative PCR.Results:1.BBR is protective against alcoholic fatty liver disease.Histopathology,serology,ELISA assay,protein immunoblotting and real-time fluorescence quantitative PCR experiments showed increased expression levels of lipid metabolism-related genes in the alcoholic liquid-fed group.Alcohol diet-fed mice showed reduced symptoms of liver injury and decreased expression levels of genes related to lipid metabolism after administration of berberine.2.BBR attenuated alcohol-induced disorders of lipid metabolism in AML-12 cells In vitro.Protein immunoblotting and real-time fluorescence quantitative PCR experiments showed that the expression levels of lipid metabolism-related genes were decreased in alcohol-induced AML-12 cells after administration of berberine.3.BBR promotes the expression and activity of SIRT1 In vivo and In vitro.Protein immunoblotting and real-time fluorescence quantitative PCR experiments showed that SIRT1 mRNA levels in AML-12 cells were significantly decreased after alcohol stimulation;however,its expression returned to normal after BBR administration.ELISA and real-time fluorescence quantitative PCR experiments showed that compared with the alcohol liquid feed feeding group,alcohol liquid feed + alcohol liquid feed + 200 mg/kg SIRT1 concentration was significantly increased in the BBR group.4.The silencing of SIRT1 reduced the lipid regulation of alcohol-induced AML-12 cells by BBR.The results of protein immunoblotting and real-time fluorescence quantitative PCR experiments showed that the silencing of SIRT1 increased the levels of genes related to lipid metabolism.The results of protein immunoblotting experiments showed that the silencing of SIRT1 did not affect AMPK,p-AMPK protein levels.5.BBR upregulated the expression of SIRT1 by activating the AMPK/SIRT1 pathway.The results of protein immunoblotting and real-time fluorescence quantitative PCR experiments showed that the expression of SIRT1 was suppressed after inhibition of AMPK phosphorylation.The intermolecular interaction simulation experiments suggested that BBR could bind to AMPK highly.6.BBR ameliorated the alcohol-induced reduction of AMPK phosphorylation In vitro and In vivo.The results of protein immunoblotting experiments showed that BBR administration restored the alcohol-induced reduction in ex vivo p-AMPK protein levels.7.BBR attenuated EtOH-stimulated hepatic steatosis in AML-12 cells via the AMPK/SIRT1 pathway,and the results of oil red O staining assay showed that alcoholinduced lipid droplet accumulation was reduced in AML-12 cells after BBR administration,and the results of immunoprotein blotting and real-time fluorescence quantitative PCR experiments showed that the expression of genes related to lipid metabolism was also reduced.Conclusion: Berberine may act as a regulator of lipid metabolism via the AMPK/SIRT1 pathway in alcoholic fatty liver disease.Berberine may be a potential therapeutic agent for alcoholic fatty liver disease.