The Study On The Changes Of Microbiota And Metabolites In Cecum Of Mice With Nonalcoholic Fatty Liver Disease Based On Multi-omics Technologies

Non-alcoholic fatty liver disease(NAFLD)is currently the most common chronic liver disease in the world.Its pathogenesis research has gradually deepened and received widespread attention with the extensive application of omics technology,and it has been clarified that the intestine-liver axis plays a vital role in the occurrence of NAFLD.Researchers have confirmed that gut microbiota and metabolites in patients with NAFLD undergo significant changes,and the relationship between dysbacteriosis and changed metabolites has become a research hotspot.Therefore,in order to elucidate the mechanism of high-fat diet-induced cecum microbiota in mice that affects the formation of NAFLD,and to build a cecum microbiota-metabolites-NAFLD network,this study carried out the following experiments:1.Preparation of a High Fat Diet-induced Mouse Model of Nonalcoholic Fatty Liver.Twenty C57 BL / 6 male rats of 6-week-old were selected and fed into the SPF barrier system,and were randomly divided into a common feed group(CK group)and a high-fat feed group(OB group),with 10 in each group.The results showed that:(1)The weight and adipose tissue of mice in the OB group were significantly higher than those in the CK group(P<0.01).(2)Serum test results showed that liver injury indexes ALT and AST in the OB group were significantly higher than those in the CK group(0.01<P<0.05),LDL and TG were significantly higher than those in the CK group(P<0.01),and TC was significantly higher CK group(0.01<P<0.05),but HDL was significantly lower than that of CK group(0.01<P<0.05).(3)Liver pathological slices showed normal morphology of hepatocytes in the CK group and no lipid droplets in the cytoplasm;hepatocytes in the OB group showed varying degrees of steatosis.With reference to the National Institutes of Health standards and the Kleiner scoring system,the NAS score was 3.4.All above results proved that the non-alcoholic fatty liver disease model was successfully established.2.To study the changes of cecum microbiota in nonalcoholic fatty liver model mice based on 16 S rDNA technology.The cecum of all mice in Test 1 was collected for 16 S rDNA analysis.Venn distribution showed the OB group had more OTUs than CK group.The relative abundance plots showed that the composition of two groups is different at the phylum and genus levels.Alpha diversity analysis showed no significant difference in the abundance and average of the microbiota between the OB and CK groups.In the Beta diversity analysis,PCo A showed significant differences between the two groups;T-test tests revealed that in the OB group,Firmicutes increased significantly at the phylum level and genus level,while Bacteroides decreased significantly.At the same time,the ratio of Firmicutes to Bacteroides was also significantly increased.Eight biomarkers were screened out by LEf Se analysis.The bacterial species with significantly increased abundance in the OB group were Helicobacter,Blautia,Unidentified-Lachnospiraceae,Romboutsia,Faecalibaculum and Ileibacterium;while the abundances of Allobaculum and Enterhabdus were significantly reduced.3.Study on the changes of metabolites caused by nonalcoholic fatty liver and the correlation analysis with significantlydifferentialgenera.All mice in the test 1 were collected for cecum for non-targeted metabolomics analysis based on UHPLC-Q-TOF MS.Multivariate statistical analysis PCA,PLS-DA,OPLS-DA combined with FC analysis and t test,using VIP> 1 and P <0.05 as the screening conditions to obtain 167 significantly differential metabolites,including amino acids and their derivatives,lipids,bile acids,nucleotides and their derivatives.The KEGG enrichment map screened metabolic pathways such as ABC transporters,protein digestion and absorption,arginine biosynthesis,metabolism of glycine,serine,and threonine,and biosynthesis of unsaturated fatty acids.Correlation analysis between 167 significantly differential metabolites screened by metabolomics and 8 significantly differential genera screened by 16 S rDNA technology,found that Blautia,Romboutsia,Helicobacter,Unidentified-Lachnospiraceae were positively correlated with anti-inflammatory amino acids(aspartate,glutamine,arginine and so on),AAAs,BCAAs,creatine,tyramine and SFAs;negatively correlated with PUFAs;Blautia、Romboutsia、Helicobacter、Unidentified-Lachnospiraceae、Faecalibaculum 、 Ileibacterium were negatively correlated with asparagine and histamine.Romboutsia and Ileibacterium were positively correlated with chenodeoxycholate,taurodeoxycholic acid,and taurodeoxycholic acid.Furthermore,Blautia,Romboutsia,Helicobacter,Unidentified-Lachnospiraceae are also negatively correlated with uracil,inosine,mannose,melibiose,Vitamin C,Vitamin B6 and so on.Conclusions: In the non-alcoholic fatty liver disease model of C57 BL / 6 mice induced by high-fat diet,16 S rDNA technology was used to determine that the cecal microbiota of mice showed significant differences at the phylum and genus levels;167 significantly different metabolites were identified by non-targeted metabolomics,including amino acids,lipids,bile acids,nucleotides,etc.through association analysis,it is clear that there is a markedly relationship between cecal microbiota and the changes of metabolites.