The Effects And Molecular Mechanisms Of High Fat Diet On Alcoholic Hepatotoxicity

ObjectivesExcessive drinking can lead to alcoholic liver disease(ALD).Epidemiological studies have shown that obesity is a risk factor for the progression of ALD.The molecular mechanism of ALD pathogenesis is still not fully understood,but But liver inflammation is a key feature of ALD.Nuclear factor kappa B(NF-κB)is the main factor that regulates the inflammatory process.The nuclear translocation of activated NF-κB binds to the promoter of the inflammatory gene,thereby activating the transcription of Interleukin 1β(IL-1β).The maturation of IL-1β requires the participation of the NLR family Pyrin domain containing protein 3(NLRP3).Both high fat diet and alcohol can activate NF-κB and NLRP3 inflammasome in the liver,leading to the activation of Caspase-1 and the production of IL-1β.This study explored the effect of high-fat diet on alcoholic hepatotoxicity and its molecular mechanism based on inflammation related pathways.Methods1.The animal model was constructed by combining high fat diet with alcohol.40 Male C57BL/6N mice were randomly divided into 4 groups:control group,alcohol group,high-fat diet group,the exposure group of high-fat diet combined with alcohol.Mice were fed a high fat diet for 2 months.Other mice were also fed a control diet for 2 months.Mice were given ethanol(5 g/kg body weight as 53%solution in water)or isocaloric dextrin-maltose by oral gavage from the 5th week to 8th week and then euthanized at 9 h following the last treatment.Liver inflammation of mice was evaluated by determination of serum aminotransferase activity,triglyceride content,and liver histopathological examination.And the mRNA level of related molecules was determined by RT-qPCR technology.The expression of NF-κB and NLRP3 signaling pathway proteins and alcohol metabolism proteins was determined by Western blotting.2.The mouse normal liver cell(AML 12)model and mouse monocyte macrophage(J774A.1)model were constructed by combining free fatty acid with alcohol.Cells were divided into four groups:control group,alcohol(100 μM)group,free fatty acid(1 μM)group,the exposure group of free fatty acid combined with alcohol(1μM free fatty acid+100 μM alcohol).The content of Tumor necrosis factor α(TNF-α)in cell culture medium was determined by ELISA.Cell steatosis was detected by oil red O staining.The mRNA levels of related molecules were determined by RT-qPCR.And protein expressions of NF-κB and NLRP3 signaling pathway were determined by Western blotting.Results1.Compared with control group,high fat diet significantly increased body weight and fat gain ratio of mice(P<0.05).Compared with the alcohol group,the liver of the exposure group of high fat diet combined with alcohol had significant steatosis,and the serum aminotransferase activity,serum and liver tissue triglyceride content were significantly increased(P<0.05),and the liver coefficient was significantly reduced(P<0.05).Compared with the alcohol group,the mRNA expression level of anti-inflammatory-related genes was decreased in the exposure group of high fat diet combined with alcohol,and the expression of Cd206 and Il10 was decreased with statistical significance(P<0.05);the mRNA expression of pro-inflammatory genes was increased,and the expression of Ccl2,Il6 and Il1b was increased with statistical significance(P<0.05);the NF-κB and NLRP3 related protein expression increased significantly,and the expression of NF-κB,IKBα,COX-2,pro-Caspase-1,cleaved-Caspase-1 was increased with statistical significance(P<0.05);The expression of alcohol metabolism-related proteins increase significantly(P<0.05);the positive staining of F4/80 immunohistochemical staining was increased.2.The results of the AML 12 cell model showed that compared with the alcohol group,the exposure of free fatty acid combined with alcohol could cause significant cell steatosis,but there was no significant change in the expression of NF-κB and NLRP3 related proteins(P<0.05).3.The results of J774A.1 cell model of free fatty acid alcohol joint exposure showed that compared with the alcohol group,the exposure of free fatty acid combined with alcohol could cause significant cell steatosis,the TNF-α in cell culture medium and the mRNA of inflammation(Tnfa,Ccl2 and Il1b)was significantly increased(P>0.05),and the expression of NF-κB and NLRP3 related proteins was increased,the expression of IKBα,P-IKBα,iNOS,cleaved-Caspase-1,pro-Caspase-1,NLRP3 was significantly increased(P>0.05).Conclusions1.The high fat diet increased alcohol-induced liver inflammation in mice,which may be related to the activation of NF-κB and NLRP3 signaling pathways in the liver.2.After exposure of free fatty acid combined with alcohol,there was no significant change in the expression of NF-κB and NLRP3 signaling pathway proteins in AML12 cells,suggesting that the hepatotoxicity of alcohol increased by high fat diet may not be related to the NF-κB and NLRP3 signaling pathways of hepatocytes.3.After exposure of free fatty acid combined with alcohol,J774A.1 cells increased the secretion of inflammatory factor and activated the expression of the NF-κB and NLRP3 pathways,suggesting that the hepatotoxicity of alcohol increased by high fat diet may be related to the NF-κB and NLRP3 signaling pathways of macrophage.