Diallyl Disulfide Alleviates Alcoholic Liver Disease By Regulating The "Enteric-liver Axis" And Maintaining The Fatty Acid Catabolism In Hepatocytes

AimsThe incidence of alcoholic liver disease(ALD)keeps on increasing,but effective prophylactic and therapeutic medicines are still lacking.Diallyl disulfide(DADS)is an organosulfur compound derived from garlic,which has been demonstrated to alleviate alcohol-induced oxidative stress and lipid accumulation in the liver,but the molecular mechanisms remain to be clarified.This study was designed to investigate the protective effects of DADS against ALD in mice and the molecular mechanisms by focusing on the"gut-liver" axis and nuclear factor erythroid E2-related factor 2(NRF2).MethodsIn this study,ethanol-induced murine AML12 hepatocyte steatosis model,ethanol-plus LPS-induced AML12/J774A.1 macrophage co-culture model,and chronic alcohol plus binge feeding model were used to study the hepatoprotective effect of DADS against ALD and explore the underlying mechanisms.The levels of lactate dehydrogenase,alanine aminotransferase,aspartate aminotransferase,and triglyceride in cell culture medium and serum of mice were determined using commercial kits.The levels of TNF-α and LPS levels were measured by ELISA kits.The protein levels and mRNA levels of key components involved in ethanol metabolism,NF-κB signal,NLRP3 inflammasome pathway,and fat metabolism-related pathways were determined using western blotting and qPCR technique,respectively.Oil red O staining and H&E staining were performed to detect pathological changes in the liver and intestine of mice.The immunohistochemical technique was used to detect the expression of F4/80 in macrophages and Ly6G in neutrophils.Apoptosis was measured using the TUNEL kit.The homeostasis of small intestinal flora was detected by 16s rRNA sequencing.In addition,NRF2-/-AML12 cells,established by the Crispr-Cas9 technique,were used to explore the roles of NRF2 in the protection of DADS against ethanol-induced fat accumulation.ResultsResults of the in vivo studies:(1)DADS significantly inhibited the increase of serum ALT and AST activities,the elevation of hepatic MDA level,the decrease of hepatic GSH level,the decline of ADH1 and ALDH2 level,the elevation of CYP2E1 level,the upregulation of protein expression of NRF2 and the downstream targets.(2)DADS significantly suppressed ethanol-induced damage to the small intestine,the increased abundance of Escherichia coli Shigella and reduced abundance of Ackermanella,the elevation of serum LPS level,the activation of NF-κB and NLRP3 inflammasome activation,and the M1-type polarization of macrophages.(3)DADS significantly attenuated the alcohol-induced increase of hepatic TG levels,upregulation of hepatic SREBP1 protein expression,downregulation of hepatic PPARα,and inactivation of the AMPK pathway.Results of in vitro studies:(1)DADS could suppress the downregulation of AMPK signaling and PPARα expression,and the upregulation of SREBP1 expression.(2)Knockout of NRF2 abrogated the protection of DADS against ethanol-induced disturbance of lipid metabolism.(3)DADS could suppress the elevation of ALT,LDH,and TNF-α levels in the culture medium,and inhibit the upregulation of key components involved in NF-κB and NLRP3 inflammasome pathway in ethanol/LPS-stimulated AML12/J744.1A co-culture model.ConclusionsDADS could mitigate ethanol-induced liver inflammation in mice by activating ethanol/acetaldehyde dehydrogenase activity,inhibiting liver oxidative stress,regulating the gut-liver axis,and inhibiting liver NLRP3 inflammasome activation.DADS could also reduce ethanol-induced liver fat accumulation in an NRF2-dependent manner.These results suggest that DADS could be used as a dietary supplement in preventing the development of ALD in heavy drinkers.