Changes Of Gut Microbiota In The Occurrence And Development Of Atherosclerosis And Its Pathogenic Mechanism

Part I Changes of gut microbiota profile during the progression of atherosclerosis in miceObjective: Atherosclerosis（AS）is a complex disease,related to chronic inflammation and metabolism.The specific pathogenesis of AS has not been elucidated properly,which limited the basis of clinical treatment.The purpose of this study was to discover the changes of gut microbiota profile during the progression of AS,and to provide new ideas for the pathogenesis of the disease.Methods: Apo E-/-mice and wild-type（WT）mice fed on high-fat diet（HFD）for 12 weeks were set as AS group and AS control（AS CTL）group respectively.WT mice fed on normal diet（ND）for 12 weeks were set as control（CTL）group.16 S r DNA high-throughput sequencing was performed after intestinal microflora DNA was extracted from fresh feces of mice at 0,4,8 and 12 weeks.Serum total cholesterol（TC）,triglycerides cholesterol（TG）,high-density lipoprotein cholesterol（HDL-C）,and low-density lipoprotein cholesterol（LDL-C）were measured at 12 weeks.In the cluster analysis of operational taxonomic units（OTU）,Shannon index and Simpson index were used to compare the Alpha diversity of the gut microbiota and Uni Frac-PCo A analysis and NMDS analysis were used to compare the Beta diversity of the gut microbiota between the two groups.Results: At the beginning（0 week）,in Apo E-/-mice group,the relative abundance of Bacteroides was about（70.33±12.34）%,the relative abundance of Firmicutes was about（19.60±10.22）%,and the relative abundance of Proteobacteria was about（8.48±5.30）%.Whereas,in WT mice group,the relative abundance of Bacteroides in WT mice was about（56.98±10.85）%,the relative abundance of Firmicutes was about（26.18±10.47）%,and the relative abundance of Proteobacteria was about（14.35±9.31）%.The Alpha diversity of gut microbiota in Apo E-/-mice group was less than that in WT mice group（p<0.05）.PCo A analysis and NMDS analysis showed no significant difference in the Beta diversity between Apo E-/-mice group and WT mice group（p>0.05）.Compared with 0 week,the relative abundance of Bacteroides in all the AS group and AS CTL group decreased at 4 weeks,8 weeks,and 12 weeks,but the relative abundance of Firmicutes,Proteobacteria and Deferribacteraceae were increased.The Alpha diversity of gut microbiota in AS group increased significantly at 8 and 12 weeks compared with that at 0 week（p<0.05）.The Alpha diversity of gut microbiota in AS CTL group decreased at 4 weeks,8 weeks and 12 weeks（p<0.05）.PCo A analysis showed that the Beta diversity of gut microbiota significantly decreased in AS group and AS CTL group at 12 weeks（P<0.05）.NMDS analysis showed that the Beta diversity of gut microbiota in two groups changed at 4 weeks,8 weeks,and 12 weeks compared with that at 0 week.When the AS model was successfully constructed at 12 weeks,the data showed that relative abundance of Bacteroides decreased,while the relative abundance of Firmicutes and Proteobacteria increased more significantly in AS group than that in AS CTL group.No significant change in the Alpha diversity and Beta diversity was found between AS and AS CTL groups（p>0.05）.Simpson index showed no significant change in the Alpha diversity of gut microbiota in CTL group at 4,8,and 12 weeks of feeding（p>0.05）,while PCo A and NMDS analysis showed a significant decrease of the Beta diversity（p<0.05）.At 12 weeks,compared with AS CTL group,the Alpha diversity in CTL group was more noticeable（p<0.05）and the Beta diversity was less obvious（p<0.05）.Conclusion: The decreased relative abundance of Bacteroides and the increased relative abundance of Firmicutes and Proteobacteria in gut microbiota may be the relevant factors of occurrence and development in AS.But the Alpha diversity and Beta diversity of gut microbiota seemed to be no significant relevency to AS.Part 2 The effect of gut microbiota on the expression of plasma cytokines in atherosclerotic mice and its mechanismObjective: Atherosclerosis（AS）is a chronic inflammation and metabolism-related disease with complex pathogenesis.The role of inflammatory response in the development of AS has been widely recognized.However,the involvement of gut microbiota in inflammation and metabolism-related pathogenesis has not been elucidated yet.The purpose of this study was to discover a new mechanism for specific gut microbiota of AS mice models in regulating cytokines and inflammation.Methods: Apo E-/-mice and wild-type（WT）mice fed on high-fat diet（HFD）for 12 weeks were set as AS group and AS control（AS CTL）group respectively.Fresh feces of two groups were collected at 0,4,8,and 12 weeks,and subsequently fecal microbiota DNA was extracted for 16 S r DNA high-throughput sequencing.T-test was performed between the two groups to identify significant differences of gut microbiota at various taxonomic levels（Phylum,Family,Genus）or not.Plasma was collected at 12 weeks for measuring the levels of chemokine CXC motif ligand（CXCL）5 and CXCL11 in serum of two groups.High-performance liquid chromatography tandam mass spectrometry（HPLC-MS/MS）was used to determine the level of serum Trimethylamine N-oxide（TMAO）in mice.Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of endothelial cell injury-related proteins in serum of mice.The expressions of 96 cytokines in mice plasma were measured by AAM-CYT-G1000 chips.Foldchange was exerted to screen differentially expressed proteins（DEPs）of AS and AS CTL groups.Spearman correlation coefficient was used to analyze the correlations between DEPs and gut differential microbiota.Fisher exact test and cluster profiler data package of R/Bioconductor software were used for protein function annotation Gene Ontology（GO）enrichment analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis of DEPs.Vascular smooth muscle cells（VSCMs）in mice was stimulated by 100 u M/300 u M TMAOfor 12 hours,cell viability,apoptosis and adhered THP-1 monocytes were then determined.Western Blot was used to detect the protein expression of phosphorylated Janus kinase 2-signal transducer and activator of transcription 3（p-JAK2/p-STAT3）in VSCMs.2u M WP1066 was used as an inhibitor of STAT3.Results: At 12 weeks,the amount of Ruminiclostridium₉,Bacteroides,Akkermansia,Parabacteroides,teriumn_coprostanoligenes_group,unidentified_Ruminococcaceae and Intestinimonas in AS group were much more than that in AS CTL group（p<0.05）at genus classification level,while Rikenellaceae_RC9_gut_group,Odoribacter,Rikenella,Roseburia and Ruminococcaceae_UCG-009 were less than that in AS CTL group（p<0.05）.The results of LEf Se analysis showed that Ruminococcaceae,Bacteroidaceae and Bacteroides were the most influential bacterial species of AS mice.13 DEPs were detected in AS group,in which the top six species were interferon-gamma（IFN-gamma）,interleukin-6（IL-6）,interleukin-9（IL-9）,Chemokine CXC motif ligand 5（CXCL5/LIX）,monocyte chemo-attractant protein-1（MCP-1）and soluble tumor necrosis factor receptor-I（s TNF-RI）.Spearman correlation analysis revealed that 7DEPs were negatively correlated with the relative abundance of Akkermansia and Verrucomicrobia（p<0.05）,they were CXCL5,basic fibroblast growth factor（b FGF/FGF2）,E-selectin,glucocorticoid-induced TNFR-related protein（GITR）,interferon-induced T cell chemokines（ITAC/CXCL11）and tissue inhibitor of metalloproteinases-2（TIMP-2）.The relative abundance of Bacteroides and Bacteroidaceae was significantly negatively correlated with CXCL5 and CXCL11,and wereas a positive correlation with macrophage-derived chemokines（MDC/CCL22）（p<0.05）was observed.In addition,the relative abundance of Ruminococcaceae was negatively correlated with CXCL5 and CXCL11（p<0.05）,while the relative abundance of Rikenellaceae was positively correlated with b FGF and GITR（p<0.05）.GO analysis of DEPs revealed that the top four subtype found in biological processes were molecules of bacterial origin,lipopolysaccharide（LPS）,leukocyte migration,and JAK-STAT signaling pathway respectively.The top four subcategories found in molecular functions were cytokine activity,cytokine receptor binding,G-protein coupled receptor binding and growth factor activity.KEGG analysis showed that signaling pathway with the highest concentration of DEPs enrichment was related to cytokine-cytokine receptor interaction.Compared with AS CTL group,the serum TMAO levels,the expression levels of CXCL5 and CXCL11 significantly enhanced in AS mice,and the concentrations of serum endothelial cell injury-related factors also increased.Both 100 u M and 300 u M TMAO stimulation for 12 hours could obviously reduce the cell viability of VECs,while the cellular apoptosis and the adhered THP-1monocytes markedly increased.The protein expressions of p-JAK2 and p-STAT3 significantly were up-regulated.Inhibition of STAT3 expression could reverse the damage effects of TMAO on VECs.Conclusion: The changes of gut microbiota such as Akkermansia,Verrucomicrobia,Bacteroidaceae,Ruminococcaceae and Rikenellaceae were related to the expression levels of some serum cytokines including CXCL11,CXCL5,b FGF,MDC,and GITR.The activation of the JAK/STAT signaling pathway by TMAO,a metabolite of gut microbiota,may contribute to the pathogenesis of atherosclerosis by raising the expressive levels of inflammatory signaling molecules,which injured endothelial cells.