| Background:Acute cardiovascular events caused by atherosclerosis,including myocardial infarction and stroke,cause substantial disability or death worldwide.In China,the systematic survey of leading cause of death in the past 30 years shows that stroke and ischemic heart disease have risen to the first and second place among all risk factors.The increasing incidence of atherosclerosis and its acute complications have gradually become the greatest challenge in public and national health security.As one of the highly correlated independent risk factors for the formation of atherosclerosis,abnormal high glucose concentration in plasma has been shown to promote the formation of atherosclerotic lesions through related mechanisms,including the formation of advanced glycation end-products,increased oxidative stress,and the formation of chronic inflammation in the system.As a new research field,gut microbiota research has first attracted a wide attention in the prevention and treatment of chronic metabolic diseases.Not only because the genetic diversity of gut microbiota is directly related to the digestion,absorption and metabolism of nutrients in the digestive tract,but also because the antigens or specifically secondary metabolites produced by gut microbiota directly or indirectly stimulate enterocytes and enter the circulatory system to play a certain regulatory role in human immune system and metabolic capacity.A large number of studies have demonstrated that gut microbiota and its metabolites enter the circulatory system and participate in the pathways related to atherosclerosis development,such as the pro-plaque effect of trimethylamine oxide and the protection effect of short chain fatty acids in vascular endothelial.However,the detailed mechanism of altered microbiota associated with causative of atherosclerosis under the hyperglycemia is still unclear.Herein,we employed metagenomics,targeted metabolomics analyses of plasma and transcriptomics from subjects with both type 2 diabetes and non-diabetic subjects alike,coupled with integrative analysis,to reveal metabolites formed via meta-microbial pathways enhanced in subjects with atherosclerosis.Methods:Feces,plasma and complete blood cell samples were enrolled from the same individuals and divided into the healthy control(CON)group,atherosclerosis only(AS)group,type 2 diabetes only(DM)group and complication(COM)group.Fecal samples were sequenced by next-generation metagenomic sequencing,annotated at both taxonomy and gene function,and statistical differences among groups were analyzed.KEGG database was used to explore the differences of specific metabolite synthesis or modification gene pathways related to atherosclerosis formation and their key positions in various groups.Meanwhile,clinical data,targeted metabolome and metagenomics data were combined and analyzed.Three types of metabolites(TMAO,bile acids,short-chain fatty acids)in plasma samples were detected by targeted metabolomics using high resolution liquid chromatography-mass spectrometry,and statistical analysis was performed to determine whether all metabolites were significantly different among various groups.Complete blood cell samples were sequenced by mRNA transcriptome,and differentially expressed genes were identified in various groups.KEGG,GO and DO databases were used for annotation,and protein interaction network analysis was conducted for differentially expressed genes.Results:Metagenomic high-throughput sequencing of fecal samples showed that the diversity of bacteria in DM and COM groups decreased significantly.In the three disease groups(AS/DM/COM),the relative abundance of tow opportunistic pathogens:Escherichia and Shigella were significantly increased.In the CON group,several potential probiotic genera were enriched,including Clostridium,Faecalibacterium,Roseburia.The abundance of trimethylamine oxide(TMAO)metabolism-related genes(cutC/D)were significantly increased in AS and COM groups,while some microbial genes involved in butyric acid metabolism were enriched in CON group.Microbial genes in the secondary bile acid modification pathway were also significantly different between the three disease groups(AS/DM/COM)and the healthy control group.Moreover,A total of 61 metabolites were detected using targeted metabolome,and plasma concentrations of TMAO and its precursors,such as choline and carnitine increased in AS and COM groups.Several primary bile acid and secondary bile acid products were enriched in CON group.Butyric acid and propionic acid were enriched in CON group,while hexanoic acid was significantly increased in AS and DM group.A total of 1429 differentially expressed genes were found in the mRNA transcriptome sequencing.452(up-regulated and down-regulated)differential genes were found between the COM and CON groups,which were consistent with the results of Metagenomics,and were also related to the concentration differences of three types of plasma metabolites in different groups of targeted metabolites.Fifty-one differentially expressed genes were found in the condition of hyperglycemia,among which CLDN7 gene was found to be related to cell adhesion and pathogenic Escherichia coli infection.The synthesis,secretion and activation of parathyroid hormones and the EGR1 gene associated with AGE-RAGE signaling pathway in endocrine and metabolic diseases were significantly up-regulated.DGKK genes involved in triglyceride and glycerin phospholipid metabolism were significantly down-regulated.The TSHR gene related to lipid synthesis regulation and thyroid hormone recognition in adipocytes was significantly down-regulated.Conclusions:In this study,the multi-omics and multi-scales analysis of host and the abundance of gut microbiota in discovery cohort revealed that the significant differences in microbiota composition and functional gene abundance in hyperglycemia and normal cohorts,which caused changes in the concentration of relevant specific metabolites in the circulatory system,and ultimately affected the differential expression of blood cell genes in the host.On the one hand,under the hyperglycemia condition,a large number of advanced glycation end-products are formed in the circulatory system,which might bind to the surface receptors of immune cells and epithelial cells and trigger downstream cascades.Moreover,the enrichment of Shigella and Escherichia in the gut is significant under the hyperglycemia condition,while the potential probiotics such as Clostridium,Faecalibacterium and Roseburia are significantly reduced,which together lead to significant differences in the concentration of their related specific metabolites in plasma,thereby enhancing or reducing the activation of the related receptors.The activation of receptors affects downstream gene cascades.Finally,specific bacterial metabolites and host inflammatory responses mediated by MAPK and NF-κB pathways under hyperglycemia promoted the differences in the expression of EGR1,CLDN7 and other related genes in the circulatory system,which might promot the migration of immune cells and the formation of vascular endothelial dysfunction and lead to the formation and development of atherosclerosis.This study provides a new perspective for the mechanism of intestinal microbiota participating in the process of type 2 diabetes mediated-atherosclerosis,and theoretical foundation for the intervention,improvement or clinical treatment of intestinal microbiota disorder in chronic metabolic diseases in the future. |
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