| Background: The prevalence of Type 2 diabetes mellitus(T2DM)rate was increased year by year,and there is a tendency of the younger patients,T2 DM has become a serious global health problem.Some studies have shown that the gut microbiota of T2 DM patients have specific changes,and the correlation studies between gut microbiota and the occurrence,development,treatment and prognosis of T2 DM has been paid more and more attention.Objective: This study intends to observe the specific changes of gut microbiota before and after metformin treatment of T2 DM by detecting the changes of related gut microbiota in healthy people,T2 DM patients,and the corresponding population who receive metformin treatment,so as to provide research direction for exploring the regulatory mechanism of gut microbiota participating in the occurrence,development and treatment of T2 DM.Methods: Patients from the department of endocrinology,affiliated hospital of Chengdu university of traditional Chinese medicine,Recruited 30 initially diagnosed with T2 DM was integrated as the primary diagnosis group.After one month treatment with metformin,the patients in the initial diagnostic group were included in the treatment group.Randomly choose 30 healthy subjects who include as the normal control group.We collected Serum and whole blood from the control group.The primary diagnosis group and the treatment group,measured their Kidney function,blood lipid and diabetes,and then performed Anova analysis.We next collected fecal samples from three groups,and extracted fecal DNA for 16 Sr DNA analysis.After that,we detected species composition and abundance by using real-time(SMRT)Sequencing based on Pac Bio Sequencing platform,which including Circular Consensus Sequencing(CCS)sequence and the cluster of Operational Taxonomic Unit(OTU);and further analyzed the correlation between the gut microbiota abundance,the general data and hematological indicators among three groups.The metabolic pathways and enrichment of functional proteins were predicted by Kyoto Encyclopedia of Genes and Genomes(KEGG)and Clusters of Orthologous Groups of proteins(COG),so as to speculate the relevant mechanisms and explore the species composition differences of gut microbiota among three groups.Results: 1.Analysis of general data and hematological indicators: the differences in age,weight and BMI was statistically significant between the primary diagnosis group and the control group but except for height.There was no significant differences in renal function,total cholesterol and Low density lipoprotein among the three groups(P>0.05).The difference of fasting insulin between the newly diagnosed group and the normal control group was statistically significant(P<0.05).Compared with the normal control group,the differences of triglycerides and High density lipoprotein(HDL)between the treatment group and the treatment group were statistically significant(P<0.05),however,there was no statistically significant(P>0.05)differences of triglycerides and HDL after metformin treatment.2.Analysis of gut microbiota species composition and relative abundance: in this study,the gut microbiota composition analysis indicated that the composition ratio of intestinal microbiota from phylum classification is unique among the three groups.The Firmicutes accounted for 54%.Bacteroidetes and Chlorobi accounted for 21%,Proteobacteria accounted for 18%,Cyanobacteria accounted for 4%,while unknown attribution of bacteria accounted for 3%.Compared to the control group,the relative abundance of Escherichia and Megamonas was statistically significant(P<0.05),while compared with the T2 DM patients group,Megamonas was increased after treatment with metformin,and the difference was statistically significant(P < 0.05).Further analysis from species classification was performed among the three groups of gut microbiota showed that Bacteroides.Coagulant,Coprostanoligenes.Bacillus and Eubacterium were specificity increased in the primary diagnosis group(P < 0.05);Megamonas uniformed and Streptococcus salivation were significantly increased in the treatment group(P < 0.05).3.Correlation analysis of gut microbiota with general data and hematological indicators: relative abundance of Eubacterium and Bacteroides is positively correlated with HDL and LDL;while the relative abundance of Faecalibacterium and Ruminococcus was positively correlated with height,weight,age,BMI,urea,creatinine,HDL and LDL,and negatively correlated with total cholesterol.The comparative abundance of Prevotella,Akkermansia,Escherichia and Megamonas was positively correlated with body weight,age,BMI,triglycerides and urea.4.Gut microbiota participating in the pathogenesis mechanism of T2 DM.We used bioinformatics analysis databases such as KEGG and COG predicted the molecular mechanism of gut microbiota in which regulates the development of T2 DM.The difference of cytoskeleton protein enrichment in the three groups was statistically significant(P<0.05).Compare to the normal control group,the differences in enrichment of fatty acid biosynthetic pathway,phosphatidylinositol signaling system and peroxisome proliferator-activated receptor(PPAR)signaling pathway were statistically significant(P<0.05)in the primary diagnosis group.While the enrichment of phosphatidylinositol signaling system between the treatment group and the control group was statistically significant(P<0.001).Dioxin analysis of protein function and signaling pathways and literature retrieval predicted that the cytoskeleton protein and radiopacity signaling pathways play a regulatory role through the PI3K/AKT signaling pathway.Conclusions: 1.Patients with T2 DM are prone to coexist with abnormal blood lipid indexes such as triglyceride and high-density lipoprotein.The results of fasting blood glucose and hemoglobin hemoglobin A1 b were significantly improved after metformin treatment,but the fasting insulin was not significantly reduced.2.Through the analysis from the top 10 microbiota which with the highest relative abundance of genera,we found the relative abundance of Escherichia and Megamonas was significantly different among three groups.Then we further analyzed the differences of all the bacteria from species classification in the three groups,we obtained the exact bacteria characteristics in the primary diagnosis group,in which Bacteroide.Coagulant and Eubacterium.Coprostanoligenes was significantly increased.Besides this,Megamonas uniformed and Streptococcus salivation were significantly increased in the treatment group.3.The relative abundance of Escherichia and Megamonas was positively correlated with body weight,age,BMI,triglyceride and urea.The relative abundance of Escherichia was positively correlated with LDL,but negatively correlated with HDL and the total cholesterol.The relative abundance of Megamonas was negatively correlated with the total cholesterol,HDL and LDL.4.Through analyzing the sequencing results of gut microbiota among three group,and comparing with the bioinformatics analysis databases such as KEGG and COG,we found that cytoskeleton proteins and the adipocytokine signaling pathway respectively play an authoritative role as functional proteins and metabolic pathway in the occurrence,development and treatment of T2 DM.Cytoskeleton protein and adipose cytokine signaling pathway may mediate T2 DM through PI3K/AKT signaling pathway. |
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