A Study On The Pathogenesis Of Alcoholic Liver Disease In Mice Based On Multi-omics

Objective:The global burden of alcoholic liver disease is becoming more and more serious.The existing research theories cannot accurately explain the pathogenesis of alcoholic liver disease.Therefore,there is no specific clinical treatment for alcoholic liver disease.Enhancing the understanding of the pathogenesis of alcoholic liver disease is helpful to the discovery of its potential therapeutic targets.Therefore,this study aims to investigate the changes in liver metabolism in mice with alcoholic liver disease and to understand the effect of alcohol on liver metabolism in mice.Combining the changes of upstream regulatory genes and downstream metabolites to explore the possible toxic mechanism of alcohol on the liver,and provide new ideas for the research on the pathogenesis of alcoholic liver disease.Methods:1.The mouse model of alcoholic liver disease was established by NIAAA method,including two stages.In the first stage,30 mice adapted to liquid diet for 5 days,and then they were randomly divided into EtOH-fed group and paired-fed group,15 mice in each group;in the second stage,10 days of modeling period,during which isocaloric feeding was used.They were gavaged on the 16th day and sampled 9 hours later.2.Collecting 6 cases of EtOH-fed group and 6 cases of Paired-fed group mouse liver tissues,based on non-target liquid chromatography mass spectrometry technology for qualitative and quantitative analysis of metabolic small molecules.The metabolites with VIP>1,FC>1 and P<0.05 were defined as differential metabolites by univariate and multivariate statistical analysis.The pathways mapped by differential metabolites were further comprehensively analyzed to find the pathway with the highest correlation with differential metabolites.3.liver tissues of 3 mice in EtOH-fed group and 3 mice in paired-fed group were collected.The differentially expressed genes in liver tissues of mice with alcoholic liver disease were screened by transcriptomics based on RNA-sequencing technology,and the metabolic pathways with significantly enriched differentially expressed genes were analyzed.4.The mouse liver tissue RNA was extracted with the kit,and then reverse transcribed into cDNA,and real-time reverse transcription PCR experiment was used to verify the RNA sequencing results.Results:1.The serum ALT,AST,TG and TC levels of mice in the EtOH-fed group were significantly higher than those in the Paired-fed group;HE staining of liver tissue in the EtOH-fed group indicated that alcohol feeding caused steatosis in the liver of the EtOH-fed group.2.Comparison of liver tissue metabolites between EtOH-fed group and Paired-fed group showed that 110 differential metabolites were screened in positive ion mode,of which 100 were up-regulated differential metabolites and 10 were down-regulated differential metabolites.In the negative ion mode,63 differential metabolites were screened.There were 55 differential metabolites up-regulated and 8 differential metabolites down-regulated.In the positive and negative mode,14/15 metabolic pathways changed significantly.3.n the process of transcriptome analysis,a total of 2017 differentially expressed genes were screened,and 1281 and 736 genes were up-regulated and down regulated respectively.The top 20 pathways that are significantly enriched include:chemical carcinogenesis,retinol metabolism,drug metabolism-cytochrome P450,exogenous substance metabolism-cytochrome P450,bile secretion,alanine,aspartic acid and glutamine Acid metabolism,glycine,serine and threonine metabolism,peroxisomes,ABC transporters,malaria,glutathione metabolism,histidine metabolism,arginine and proline metabolism,platinum drug resistance,Steroid hormone biosynthesis,steroid biosynthesis,amino acid biosynthesis,primary bile acid biosynthesis,glyoxylic acid and dicarboxylic acid metabolism,tryptophan metabolism.4.Alanine,aspartic acid and glutamate metabolism,arginine and proline metabolism,tryptophan metabolism and glutathione metabolism showed significant changes in both metabolism and transcription.Conclusions:1.Combined analysis of metabonomics and transcriptomics found that four metabolic pathways were involved in the progress of ALD,namely alanine,aspartate and glutamate metabolism,arginine and proline metabolism,tryptophan metabolism and glutathione metabolism.2.Increased metabolism of tryptophan via the kynurenine pathway may be involved in the progression of alcoholic liver disease.