The RNA-seq Transcriptomics Analysis Of The Fatty Acid Degradation-related Genes Of Dihydromyricetin In The Prevention And Treatment Of Alcoholic Fatty Liver In Mice

[Objective] The dehydrobricetin is one of the main active components of hovenia raisin.Its chemical formula is 3,5,7,3 ',4 ',5 '-hexahydroxy 2,3-dihydroxyflavonol(Dihydromyricetin)DHM,also known as serpentine and dihydromyricetin,is a kind of flavonoid compound.Previous studies have confirmed that DHM has the pharmacological effect of significantly inhibiting hepatocyte steatosis and can be used for the treatment of alcoholic fatty liver disease,but the molecular mechanism of its prevention and treatment of alcoholic fatty liver disease has not been elucidated.Therefore,based on existing literature reports,we further studied the molecular mechanism of dihydromyricetin in the prevention and treatment of alcoholic fatty liver disease.[Methods] The experiment was divided into two sections.1.In vitro: AML12 cells were stimulated with 50 m M anhydrous ethanol to establish a cellular alcoholic fatty liver model.The extent of hepatocyte fat accumulation was observed by Nile Red cell fluorescence staining.The range of toxicity concentration of DHM on cells was determined by cck-8 cell viability assay.q RT-PCR was used to detect the expression of transcription factors related to fatty acid synthesis,such as,fatty acid synthesis related gene fatty acid synthase-acetyl-co A carboxylase(ACC),fatty acid translocation enzyme CD36 and alcohol metabolism enzyme cytochrome P450 2e1(CYP2e1),and to explore the molecular mechanism of DHM regulating alcohol to stimulate lipid metabolism in AML12 cells.The expression of CYP2e1 gene was detected to investigate the molecular mechanism of DHM to improve alcohol metabolism in AML12 cells.2.In vivo: Normal C57 mice were fed Lieber-decarli liquid feed containing or without 5% ethanol for 10 days,and the mice were induced to generate AFLD model.Meanwhile,DHM parallel administration group was set: the mortality of mice was observed.The degree of hepatic steatosis was determined by liver pathological sections,serum glutamate pyruvate transaminase(ALT),glutamic-oxaloacetic transaminase(AST)and triglycerides(TG).Through transcriptome RNA sequencing(RNA-seq)technology and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,the molecular mechanism of DHM regulating lipid metabolism in mouse liver was discussed according to gene functional classification and related signal pathways.[Results] 1.In vitro:(1)CCK8 showed that DHM at concentrations below 100?M had no cytotoxicity.(2)Nile Red fat staining showed that compared with the normal group,the intracellular fat accumulation increased in the model group treated with 50 m M anhydrous ethanol.Compared with the model group,the DHM group had significantly less fat accumulation.It indicated that DHM could inhibit fat accumulation in AML12 cells.(3)Compared with the normal group,the expression levels of ACC related to fatty acid synthesis were increased,PPAR related to fatty acid oxidation was decreased,CD36 related to fatty acid transport was increased,and CYP2e1 related to alcohol metabolism was increased in the model group;compared with the model group,the addition of DHM significantly inhibited the expression of ACC,CD36 and CYP2e1 genes induced by alcohol,and promoted the expression of PPAR,indicating that DHM could inhibit the fatty acid transport,lipid peroxidation and fat synthesis in alcohol-induced hepatocytes,and promote the metabolism of alcohol.2.In vivo:(1)H&E staining results showed that,compared with the normal group,the degree of steatosis of liver tissue cells in the model group was significantly increased.Compared with the model group,the liver tissue cell steatosis in the DHM group was reduced in a concentration-dependent manner,indicating that DHM could effectively treat liver fat deposition induced by alcohol in mice.(2)The biochemical test results showed that serum ALT,serum AST and serum TG were significantly increased in the model group compared with in the normal group.Compared with the model group,serum ALT,serum AST and serum TG in the DHM group significantly decreased.The results showed that DHM could inhibit liver injury and hepatocyte steatosis induced by alcohol.(3)RNA-seq transcriptional analysis of mouse liver tissue showed that DHM promoted the expression of downstream genes Ehhadh,CYP4a10,CYP4a14,CYP4a31 and CYP4a32 in the PPAR? signaling pathway,to promoted the oxidation and degradation of fatty acids in the liver,thus playing a pharmacological role in preventing alcoholic fatty liver disease.[Conclusion] DHM has the function of preventing and treating alcoholic fatty liver disease.Its molecular mechanism is to inhibit lipid deposition in liver cells caused by alcohol stimulation by promoting the expression of Ehhadh,CYP4a10,CYP4a14,CYP4a31 and CYP4a32 genes in the fatty acid oxidation PPAR? signaling pathway.