Clinical Research On Changes Of Gut Microecology And TMAO Level In Patients With ST-segment Elevation Myocardial Infarction Or Unstable Angina Pectoris

Objective Analyzing the changes of intestinal microecology in ST-segment elevation myocardial infarction（STEMI）patients,unstable angina pectoris（UAP）patients and controls and screen the relative predominant intestinal flora and marker species among the three groups.To analyze differences in the levels of Trimethylamine N-Oxide（TMAO）in secrum,a metabolite of intestinal flora among the three groups and screen the colonies related to TMAO levels closely.At the same time,to discuss the correlations between novel serological markers of novel cardiovascular diseases and STEMI,UAP,intestinal flora and TMAO.Methods The patients in the STEMI group（30 cases）and UAP group（30 cases）were hospitalized and diagnosed in the CCU ward of Tianjin Chest Hospital from June2017 to November 2017.The CONTROL group（25 cases）was from Tianjin Chest Hospital Health Checkup Center.The clinical basic conditions,past medical history and laboratory examination were recorded.Patients in the STEMI and UAP groups were enrolled for the first fresh feces within 72 hours and blood before angiography after hospital admission,and fresh feces and fasting blood samples were collected on the day of physical examination in CONTROL group.16S rDNA amplicon sequencing technique was used to detect the fecal flora of the subjects.Plasma TMAO levels were detected by liquid chromatography-mass spectrometry and enzyme linked immunosorbent assay（ELISA）were used to detect cardiovascular markers,including heart-type fatty acid binding protein（H-FABP）,myeloperoxidase（MPO）,Interleukin-6（IL-6）,proprotein convertase subtilisin kexin₉（PCSK9）,platelet-derived growth factor B（PDGF-B）,thromboxane B₂（TXB₂）.The QIIME1.9.1 platform was used for species sequencing,annotation,andαandβdiversity analysis of intestinal flora.The SPSS 19.0 statistical software was used for statistical analysis of clinical data and relative abundance of species.Results I.The changes of intestinal microecology1.There was no statistical difference in age,sex,body mass index and other indicators among the three groups（P>0.05）,but statistical differences existed in heart rate,systolic blood pressure,and some laboratory tests（P<0.05）.2.Combining with OTU analysis and species annotation results,at the level of phylum classification,intestinal flora with high relative abundance in three groups were of Firmicutes,Bacteroides,Proteobacteria,Actinomycetes,and Fusobacteria in order.At the level of genus classification,the relative abundances of Shigella,Acinetobacter,Lactobacillus,Akk.,and Aminococcus in STEMI and UAP patients were higher than those in healthy individuals.3.Theαdiversity analysis showed no significant difference in the abundance and diversity of intestinal flora among the three groups（P>0.05）.However,with species annotation results,the differences in species counts among three groups were statistically significant at the classification level of community,class,order,and family:CONTROL>STEMI>UAP（P<0.05）,but at the classification level of phylum and genus,species counts were not statistically significant（P>0.05）.4.Withβdiversity analysis,Anosim,MRPP,and Adonis index showed significant difference in intestinal flora structure between STEMI group and CONTROL group（P≤0.01）.PCoA analysis and PCA analysis further validated the results and the rationality of dividing into groups.TO the relative abundance of Firmicutes,Bacteroides and Actinomycetes,there were no significant difference among three groups（P>0.05）.The relative abundance of Proteobacteria among three groups was significantly different:[STEMI,UAP VS.CONTROL=5.39%（0.12）,3.24%（0.13）VS.3.12%（0.03）,P=0.018;STEMI>CONTRO,P=0.005)],but there was no difference between the UAP group and the other two groups（P>0.05）.The relative abundance of the three groups of Fusobacteria was significantly different（STEMI,UAP VS.CONTROL=0.04%（0.0006）,0.03%（0.0008）VS.0.01%（0.0002）,P=0.001）,and STEMI group and UAP group was significantly higher than the CONTROL group（P<0.05）,but the results were similar between the STEMI group and the UAP group（P>0.05）.At the same time,at the genus level,the relative abundances of Shigella,Acinetobacter,and Lactobacillus among three groups were also significantly different（P≤0.007）.5,LEfSe analysis showed that at genus level,Shigella,Clostridium,Streptococcus,Acinetobacter,Akk.,Cat.,Fae.and Aminococcus are dominant intestinal flora in STEMI group（LDA Score>3）.II.The relationship of intestinal microecology and trimethylamine N-Oxide1.There was significant difference in TMAO levels among three groups（STEMI,UAP VS.CONTROL=3.36±2.42,4.47±3.21 VS.1.92±1.01,P=0.008;UAP>STEMI>CONTROL）.2.Atgenuslevel,inSTEMIgroupTMAOwasrelatedto Ruminococcaceae_UCG.014,Ruminococcaceae_UCG.002,Akk.,Lac.,Alis.,Par.,Sub.,and Odo.（r=0.55,r=0.42,r=0.36,r=-0.40,r=0.45,r=0.49,r=0.40;P<0.05）correlation;in UAP group TMAO was related to Alis.,X.Eu.,Rom.,Lach.（r=0.47,r=0.48,r=-0.50,r=0.43;P<0.05）.III.Correlation of the biomarkers of cardiovascular disease and intestinal microflora and TMAO1.In STEMI and UAP groups the levels of H-FABP and MPO were significantly increased[H-FABP:STEMI group,UAP group vs.control group=99.14（171.79）,8.30（1.67）vs.7.82（0.91）,P<0.001;MPO:STEMI group,UAP group VS.CONTROL group=1.51（0.55）,0.79（0.66）vs.0.26（0.33）,P<0.001];however,although of the levels of There were differences statisticaliy in levels of IL-6,PDGF-B,PCSK9,TXB₂ among three groups（P>0.05）.2.At genus level,H-FABP was associated with Acinetobacter and Lactobacillus（r=0.44,P<0.01;r=-0.31,P<0.05）;MPO was associated with Roseburia and Prevotella₉（r=-0.46,P<0.01;r=-0.23,P<0.05）.3.The correlations between the biomarkers of cardiovascular disease（H-FABP,MPO,IL-6,PDGF-B,PCSK9,TXB₂）and TMAO have no significance statistically（P>0.05）.Conclusion 1.Bacterial diversity:At phylum level,intestinal flora with high relative abundance in three groups were of Firmicutes,Bacteroides,Proteobacteria,Actinomycetes,and Fusobacteria in order.The relative abundance of Proteobacteria and Fusobacteria in patients with STEMI and UAP was higher than that in healthy individuals.At genus level,the relative abundance of Shigella,Acinetobacter,Lactobacillus,Akk.,and Aminococcus in patients with STEMI and UAP was higher than that in healthy individuals.2.Dominant bacteria:At genus level,Shigella,Clostridium.,Streptococcus,Acinetobacter,Akk.,Cat.,Fae.and Aminococcus were the dominant bacteria in STEMI patients.3.Plasma TMAO levels were elevated in patients with STEMI and UAP.At genus level,plasma TMAO levels of patients with STEMI were related to Ruminococcaceae_UCG.014,Ruminococcaceae_UCG.002,Akk.,Lac.,Alis.Par.,Sub.and Odo.were;plasma TMAO levels of patients with STEMI were related to Alis.,X.Eu.,Rom.,and Lach.4.Akk.is the predominant intestinal flora,and its relative abundance was correlated with plasma TMAO levels in STEMI patients.5.Serum H-FABP and MPO levels were elevated in patients with STEMI and UAP.At genus level,the intestinal flora associated with H-FABP levels in patients with STEMI and UAP are Acinetobacter and Lactobacillus,and that associated with MPO levels are Roseburia and Prevotella₉.But serum biomarkers of cardiovascular diseases（H-FABP,MPO,IL-6,PDGF-B,PCSK9,TXB₂）were not associated with plasma TMAO levels.