Omics Analysis Of Heterogeneity In Metabolic Associated Fatty Liver Disease

Objective:Taking the heterogeneity of metabolic associated fatty liver disease(MAFLD)as the research content,we explored from the perspective of dietary intake to explore changes in liver functional gene expression and intestinal flora behind changes in liver metabolic homeostasis.Changes in species composition and functional metabolic pathways.Under the same phenotype of fatty liver disease(FLD),whether there are many different patterns of changes in genes and flora,and which pathogenic mechanisms are responsible for these different patterns FLD.Fully understanding the heterogeneity behind this can allow us to better understand the etiology and pathogenesis of MAFLD,and in clinical practice,it can target different heterogeneous patient subtypes to more accurately interfere with liver molecular targets and intestinal flora functional metabolism,thus achieving the goal of personalized precision medicine.Methods:High fat diet(HFD),2%methionine-added diet(high-methionine diet),and folate deficient diet(FD diet)were used to construct HFD mice and hyperhomocysteinemia(Hyperhomocysteinaemiain,HHcy)mice,FD mice,all three models of dietary intervention induced mouse models of liver steatosis.Then,liver tissues and feces of FLD mice induced by three different intervention methods and normal chow mice were taken.After RNA/DNA extraction and library construction,liver transcriptome sequencing(RNA-seq)and 16S rDNA amplicon sequencing were performed on the machine.Finally,the sequencing data were processed and analyzed and comprehensive bioinformatics analysis was performed.Results:Frozen sections of mouse liver+oil red O staining:Compared with the normal group,mice in the three diet intervention groups showed significant fat deposition.The liver of HFD mice is mainly bullous steatosis,while the HHcy and FD mice are mainly bullous steatosis.Bioinformatics Analysis of Transcriptome Sequencing:① The results of principal component analysis(PCA)and cluster heatmap analysis(heatmap)suggest that the liver transcripts of the four groups of N,HFD,HHcy,and FD are not similar to each other as a whole.② GO-BP results of differential genes showed that oxidation reduction and fatty acid metabolic process were significantly enriched in the three intervened groups,but the degree of enrichment in each group was significant difference.After analyzing the enrichment results of the KEGG pathway,it was found that the metabolism of xenobiotics by cytochrome P450 pathway and fatty acid metabolism pathway were in a significant position in the three groups.The remaining enriched pathways are different.③ We put forward the genes of the oxidation reduction biological processes and the CYP450 family members in the Metabolism of xenobiotics by cytochrome P450 pathway involved in each group to draw a heatmap,showing that there are different expression profiles on the enzymes of the oxidation reaction.Such as genes CYP2E1(which can cause liver damage by promoting oxidation leading to NASH)is higher in the FD and HHcy groups than in the HFD group④ The genes contained in the BP and Pathway involved in the metabolism of lipids in each group were separately extracted,and the core genes were selected by protein protein interaction(PPI)analysis.The results show that the key genes involved in the biological processes and pathways of lipid metabolism in the three intervention groups are not exactly the same in type.Using the Hub genes of each group to draw a heatmap,it can be seen that not only are the types of Hub genes different between the groups,but even if they have the same Hub gene,their expression levels in the groups are not exactly the same.It can be concluded that FLD caused by different etiology has different core pathways and molecular mechanisms in the level of lipid metabolism in liver cells.For example,squalene epoxidase(SQLE)is a key oncogene that leads to fatty liver-associated hepatocellular carcinoma,and this gene is the core gene of the HHcy group.Biomarker analysis of 16SrDNA amplicon sequencing:① The results of PCA analysis indicate that the intestinal flora of each intervention group and the normal group are different from each other at the genus level.②The histogram of relative abundance at the phylum level clearly shows that there is a significant difference in the specie composition between different groups.③ Alpha and Beta diversity analysis indicated that there were differences in intestinal flora diversity in each intervention group.④ The classification branch and distribution bar graphs obtained by LEfSe analysis show the specific bacteria with significant changes in abundance in each group,and each group is different from each other.The main intestinal flora of the normal group of mice was Bacteroidia,the most significant change in the intestinal flora of HFD mice was Actinobacteria,HHcy mice were Firmicutes,and FD mice were Unassigned(Classification unknown).⑤ The analysis results of PICRUSt showed that the intestinal flora of different intervention groups had differences in functional metabolic pathways.HFD mice’s intestinal flora have upregulated in the anabolic pathway of propionate(a short-chain fatty acid salt that increases the risk of diabetes and obesity)compared to the normal group;HHcy mice’s intestinal flora have upregulated anabolic pathways in selenium compounds(that increase intracellular ROS levels and induce apoptosis);FD mice’s intestinal flora have downregulated anabolic pathway of phenylpropanin(which can resist obesity and hyperlipidemia).Conclusion:From the perspective of dietary intake,we explored changes in the liver transcriptome and intestinal flora behind MAFLD heterogeneity,and found that different liver gene transcription profiles and patterns of species composition and functional metabolic pathway of intestinal flora.While discussing the existence of the differences,we also analyzed the specific possible functional genes,bacteria and metabolites that cause MAFLD heterogeneity.It provides a basis for further verification and research on the mechanism of action by subsequent wet experiments,functional genomics,culturomics,and metabolomics.