| Clinical trialObjectiveThe characteristics of gut microbiota in patients with unstable angina pectoris with heat toxin and blood stasis syndrome were observed through clinical research,and biomarkers with diagnostic and predictive value were explored through the theory of "stasis toxin".Methods1.A total of 60 hospitalized patients with unstable angina pectoris in the Cardiovascular Department of Guanganmen Hospital,China Academy of Chinese Medical Sciences from July 2022 to October 2022 were collected,including 30 patients with heat toxic blood stasis syndrome（UAP-THBS）and 30 patients with non-heat toxic blood stasis syndrome（UAPNTHBS）.The clinical data,score of Seattle angina pectoris scale（SAQ）and TCM syndrome were entered into excel table,and SPSS22.0 software was used to statistically analyze the differences in clinical characteristics between the two groups of patients;2.The serum TMAO levels of the two groups of patients were detected by liquid chromatography-mass spectrometry and the correlation between TMAO levels and various clinical indicators was analyzed;3.16S rRNA high-throughput sequencing technology was used to detect the fecal samples of the two groups of patients,and bioinformatics analysis was used to clarify the characteristics of gut microbiota.Results1.In all UAP-THBS patients:patients with hypertension and hyperlipidemia in the majority;The levels of glycosylated hemoglobin（HbA1c）,atherogenic index of plasma（AIP）、apolipoprotein B（ApoB）,high-sensitivity C-reactive protein（hsCRP）,fibrinogen（FIB）,mean systolic pressure,day-time mean systolic pressure,night-time mean systolic pressure in UAP-THBS patients were high,and the proportion of patients with drug resistance,electrocardiogram suggesting ST segment depression and T wave inversion in clopidogrel drug gene test results was significantly high（P<0.05）;the levels of high-density lipoprotein cholesterol（HDL-C）and apolipoprotein A1（ApoA1）,fastest heart rate,and E/A ratio were significantly reduced,and the difference was statistically significant（P<0.05）;2.The results of the analysis of related influencing factors in patients with UAP-THBS suggested that:（1）Admission systolic blood pressure,FIB,clopidogrel gene test,and T-wave inversion may be the influencing factors of disease progression in patients with UAP-THBS;The correlation coefficients r between serum TMAO level and ventricular septum thickness（IVST）,left ventricular end-diastolic thickness（LVPWd）,left ventricular ejection fraction（LVEF）,and lower limb artery plaque length were all close to 1,with significant correlation（P<0.05）.Among them,lower limb arterial plaque length had significant correlation with serum TMAO level（P<0.01）.In addition,serum TMAO level was negatively correlated with T-wave inversion;（2）Admission systolic blood pressure,T wave inversion,and FIB level have certain predictive value for evaluating patients with UAP-THBS;3.Results of 16S rRNA of gut microbiota in two groups of patients:（1）At the OTU level,OTU237 and OTU223 were significantly different between the two groups（P<0.05）.At the family level,the abundance of fLachnospiraceae was significantly different between the two groups（P<0.05）.At the genus level,the abundance of gSubdoligranulum and gRoseburia was significantly different between the two groups（P<0.05）;（2）The results of LEfSe analysis suggested that gBacteroides,fBacteroidaceae,sBacteroidesdorei,cActinobacteria and sunculturedbacteriumgPeptostreptococcus,gPeptostreptococcus,gEggerthella were the dominant species that distinguished UAP-THBS patients from UAP-NTHBS;（3）The results of correlation heat map showed that:1)At the phylum level,Bacteroidota was positively correlated with the level of HDL-C and negatively correlated with age（P<0.05）;Firmicutes were positively correlated with age and the level of APTT,and negatively correlated with the level of HDL-C（P<0.05）;2)At the family level,the level of TMAO was negatively correlated with unclassifiedpFirmicutes,Actinomycetaceae（P<0.05）;the level of Myo was negatively correlated with fLachnospiraceae（P<0.05）;the level of cTNI level was negatively correlated with Ruminococcaceae（P<0.05）;the level of HbA1c was positively correlated with Yersiniaceae and Eggerthellaceae（P<0.05）;the level of UA and AIP were negatively correlated with Akkermansiaceae（P<0.05）;3)At the genus level,the level of APTT were positively correlated with Faecalibacterium,Agathobacter,Fusicatenibacter,LachnospiraceaeND3007group,Roseburia,Blautia,Tyzzerella,unclassifiedfLachnospiraceae and LachnospiraceaeUCG-001（P<0.05）;the level of TT4 were positively correlated with Roseburia and negatively correlated with Bifidobacterium（P<0.05）;the level of NT-proBNP and PT were positively correlated with Klebsiella（P<0.05）;the level of FT3 was positively correlated with Olsenella,but negatively correlated with Ruminococcus and Klebsiella（P<0.05）;the level of TMAO was positively correlated with Acidaminococcus and negatively correlated with Eubacteriumhalliigroup（P<0.05）;（4）The results of random forest model and ROC curve prediction analysis suggest that:1)gRoseburia,gFusicatenibacter,fLachnospiraceae,gSubdoligranulum and sRuminococcaceaebacteriumGD6 as biomarkers have certain diagnostic and predictive value for distinguishing UAP-THBS from UAP-NTHBS;2)At the OTU level,the comprehensive score of the two groups of patients have high accuracy for diagnosing UAPTHBS（AUC:0.92）;（5）The results of network analysis suggest that:Patescibacteria,Proteobacteria,Actinobacteriota,Fusobacteriota,Firmicutes,Lachnoclostridium,Bifidobacterium,Subdoligranulum,Blautia,and Intestinibacter may be closely related to the formation of UAP-THBS;（6）The results of 16S PICRUSt2 suggest that:1)COG function prediction:unknown function,amino acid transport and metabolism,carbohydrate transport and metabolism,nucleotide transport and metabolism may be closely related to UAP-THBS;2)KEGG function prediction results suggest that metabolism-related pathways（mainly including energy metabolism,amino acid transport and metabolism,carbohydrate transport and metabolism,nucleotide transport and metabolism）and the upregulation of ko01120 and ko01100 function are closely related to UAP-THBS（P<0.05）;ConclusionsHeat toxin and blood stasis are the key factors leading to the onset of UAP-THBS patients,changes in the abundance of gRoseburia,gFusicatenibacter,fLachnospiraceae,gSubdoligranulum and sRuminococcaceaebacteriumGD6 may promote the progression of UAP-THBS by upregulating the expression of amino acid metabolism,microbial metabolism,energy metabolism,glucose and lipid metabolism,endocrine and metabolic diseases and other related gene functions.Basic researchObjectiveThrough in vivo study,the mechanism of the anti-atherosclerotic efficacy of the compatibility of traditional Chinese medicine components for detoxifying and activating blood circulation（polydatin combined with total flavonoids of hawthorn）was discussed from the perspective of intestinal flora and its metabolite TMAO through the theory of "stasis toxin".Methods1.Fifty-five SPF male ApoE-/-mice aged 6-8 weeks were selected and fed with high fat diet（containing 1%choline）for 13 weeks,confirming the success of AS model.The control group selected 10 homologous C57BL/6J mice and fed with ordinary diet for 24 weeks.After successful modeling,ApoE-/-mice were randomly divided into detoxifying and bloodactivating traditional Chinese medicine components low（PHL）,medium（PHM）,high dose groups（PHH）,simvastatin group,and model group,once a day,and continuously administered for 11 weeks;The general situation and basic data of mice were recorded.After the experiment,serum,aorta,liver and stool samples were taken and delivered for inspection after the experiment;2.Detect the pathomorphological indexes of the aorta of each group of mice,including aortic oil red staining,aortic valve oil red "O" staining,HE staining;3.Detect the levels of serum lipids and inflammatory factors in each group of mice,and use immunohistochemistry and Western blot to detect the levels of NF-κB and TNF-α in the plaque;4.The serum TMA and TMAO levels were detected by liquid chromatoc-mass spectrometry（LC-MS）,and the protein and mRNA levels of FMO3 were detected by Western blot and qPCR;5.16SrRNA high-throughput sequencing and metagenomics were used to detect the changes of gut microbiota and functional genes in each group of mice.Results1.Pathomorphology:The results of aortic oil red staining,aortic valve oil red "O" staining,and HE staining of various mice suggest that compared with the Model group,the aortic plaque area of the PHL,PHM,PHH,and Simvastatin groups was significantly reduced after the intervention（P<0.05）;2.Serum inflammatory factors and lipid levels:（1）Compared with Model group,the detoxifying and blood-activating traditional Chinese medicine components decreased the levels of inflammatory factors IL-1β,IL-2,IL-6,IL17A,TNF-α and lipid levels TC,TG,LDL-C,VLDL-C in each group in a dose-dependent manner,and increased the HDL-C level,the difference was statistically significantly（P<0.05）;（2）The level of inflammatory factors in the aortic plaque:Immunohistochemical staining results and Western blot results suggested that detoxifying and blood-activating traditional Chinese medicine components could reduce the expressions of inflammatory factors NFκB and TNF-α in the aortic plaques of mice to a certain extent,compared with the model group（P<0.05）;3.Gut microbial TMAO metabolic pathway:（1）Compared with the Control group,the serum levels of TMA and TMAO in the Model group were significantly increased;compared with the Simvastatin group,the serum levels of TMAO in the PHH group were significantly reduced,and the difference was statistically significant（P<0.05）;（2）The results of qPCR and Western blot showed that compared with the Model group,the expression levels of FMO3 protein in the PHH group and the Simvastatin group were significantly reduced,and the difference was statistically significant（P<0.5）;4.Results of 16S diversity analysis of:（1）The results of the difference analysis between species groups showed that:1)At the phylum level,the abundance of Bacteroidota in the Control group was significantly higher than that in the other groups（P<0.05）;compared with the Control group,the abundance of Actinobacteriota in the PHH group and the Simvastatin group was significantly increased（P<0.01）;compared with the PHL group,the abundance of Desulfobacterota in the Control group,the PHM group and the Simvastatin group was reduced（P<0.05）;compared with the Control group,the abundance of Patescibacteria in the PHM group was significantly increased（P<0.05）;2)At the family level,the species composition of the samples in each group was significantly different The bacteria were Muribaculaceae、Lactobacillaceae、Atopobiaceae,Bifidobacteriaceae,Oscillospiraceae,Prevotellaceae.Compared with the Control group,the abundance of Muribaculaceae in each group was lower than that in the Control group（P<0.05）;the abundance of Prevotellaceae in the PHH group was lower,and the difference was statistically significant（P<0.05）;the abundance of Lactobacillaceae and Bifidobacteriaceae were higher in the simvastatin groups,while the abundance of Oscillospiraceae was lower,compared with the Control group,the difference was statistically significant（P<0.05）;the abundance of Prevotellaceae was lower than the other groups（P<0.05）;3)At the genus level,the bacteria with significant differences in species composition of each group were norankfMuribaculaceae,Lactobacillus,CoriobacteriaceaeUCG-002,Bifidobacterium,Dubosiella,and Allobaculum.Compared with the control group,the PHH group had the lowest norankfMuribaculaceae abundance,and a higher abundance of Allobaculum,the difference was statistically significant（P<0.01）;Compared with the Model group and the PHM group,the PHH group had a higher abundance of Coriobacteriaceae UCG-00,and the difference was statistically significant（P<0.01）;（2）The results of heatmap showed that:1)At the phylum level,the abundance of Desulfobacterota was positively correlated with serum lipid levels TC,TG,LDL-C,and inflammatory factors hs-CRP,IL-2,IL-1β,VLDL-C,IL-17A（P<0.05）;the abundance of Firmicutes was positively correlated with the level of serum TMAO,TG,LDL-C,hs-CRP,IL-2,IL-1β（P<0.05）;2)At the family level,the abundance of Firmicutes was positively correlated with serum TMAO,serum lipid levels TG,LDL-C,inflammatory factors hs-CRP,IL-2,IL-1β（P<0.05）.The abundance of Clostridiaceae was positively correlated with serum lipid levels TG,TC,VLDL-C,and inflammatory factors IL-17A,IL-6,TNF-α,hsCRP,IL-1β,IL-2（P<0.05）;3)At the genus level,the abundance of Turicibacter,RikenellaceaeRC9gutgroup,and Lachnoclostridium was positively correlated with serum TMA,TMAO,serum lipid levels TG,TC,LDL-C,VLDL-C,and inflammatory factors IL-17A,TNF-α,hs-CRP,IL-2,IL-6,and IL-1β levels（P<0.05）;the abundance of Roseburia was positively correlated with serum lipid levels TG and LDL-C（P<0.05）;the abundance of Desulfovibrio and Alobaculum was positively correlated with serum lipid levels TG,TC,LDL-C,VLDL-C,and inflammatory factors IL-17A,TNF-α,hs-CRP,IL2,IL-6,IL-1β（P<0.05）;（3）The results of species correlation network analysis showed that at the genus level,the top ten species in abundance belong to a larger proportion of genera in the phylum Firmicutes;5.Results of metagenomic analysis of gut microbiota:（1）COG function annotation analysis:1)Compared with the Model group,the number of COGs in each group of the drug intervention increased,and the PHM group and the Simvastatin group had the largest number of specific COGs,and the number of specific COGs in the two groups was similar;2)ENOG410YF7P,COG0463,COG0745,ENOG410XNMH,COG 1961 were highly enriched in each group.In addition,the abundance of ENOG410YF7P in PHM group and the simvastatin groups was significantly higher than that in other groups;3)The Heatmap map suggested that the abundance of COG and Function in the Control group was higher than that in the other groups,while that in the Model group was relatively low;4)There were significant differences in COG 1132,COG0582,COG1136,COG3436,ENOG410XNNV among the groups（P<0.05）;5)LEfSe difference discrimination showed that the abundance of COG related to carbohydrate and amino acid transport and metabolism in the Control group was higher,while the abundance in the Model group was significantly reduced;（2）KEGG metabolic pathway annotation analysis:1)Compared with the Model group,the number of bacterial genes in each intervention group increased,and the number of bacterial genes unique to the PHM group and the Simvastatin group was the largest;2)The Heatmap showed that compared with the Control group,the abundance of KO gene enriched in PHL and PHH groups increased significantly after drug intervention,and the KO gene function of each group was mainly enriched in metabolic related pathways at level1,level2,and level3 levels;3)The KO functional genes in each group were mainly enriched in K21572（starch-binding outer membrane protein）;at the Module level,they were mainly enriched in M00001（glycolysis）,M00048（purine total synthesis pathway）,and M00173（reduced citric acid cycle）;in terms of lipid metabolism,the functional gene enrichment abundance in the Model group was the highest.After drug intervention,the functional genes related to lipid metabolism in the PHL and PHH groups were significantly down-regulated（P<0.05）;in Pathway level3,the abundance of functional genes such as amino acid biosynthesis,carbon metabolism,microbial metabolism,ABC transporter,and purine metabolism was higher;4)The results of LefSe differential discriminant analysis showed that compared with the Model group,the types and abundance of functional genes in the samples of each group after intervention were increased;（3）CAZy（carbohydrate active enzymes）functional annotation analysis:1)At the Family level,the Control group had the highest CAZy species;at the Class level,the CAZy species common to all groups were the same;2)There were significant differences in GH,GT,CE,AA,PL and CBM among the groups（P<0.05）.In GH and PL,the abundance of CAZy in the Model group was lower.After drug intervention,the abundance of CAZy in each intervention group increased to a certain extent;the abundance of CE-related active enzymes in the PHH group increased significantly compared with other groups;3)The results of LefSe difference discriminant analysis showed that In terms of Class,the functional enrichment of active enzymes in the Control group was mainly concentrated in CBM and PL;the functional enrichment of active enzymes in the PHH group was mainly concentrated in CE;the PHL group was mainly enriched in GT and AA;the Simvastatin group was mainly enriched in GH,and the differences between the groups were statistically significant（P<0.05）;in terms of Family,compared with the Model group,the functional types and abundance of CAZy in the samples of each group of drug intervention were significantly increased;（4）The results of the analysis of the differences in key species and ko metabolic pathways between groups suggest that:1)Comparison of group differences in key species（genus level）:the abundance of Clostridium and Parabacteroides in Model group was higher,and the abundance of Lachnoclostridium,Bacteroides,and Alistipes decreased after drug intervention;2)The results of inter-group comparison of key ko metabolic pathways showed that the functional metabolic pathways of each group were mainly enriched in ko01100 and ko01110;in addition,the abundance of each group on ko00121 was significantly higher than that of ko00120;（5）The results of metabolic pathway differences between groups suggest that primary and secondary bile acid（BAs）biosynthesis,glycolysis/gluconeogenesis biosynthesis may be the main pathways;（6）The correlation analysis between different species and inflammation,serum lipids,and TMAO in COG function suggests that:COG1136 and COG0577 were positively correlated with the level of serum TMA and TMAO（P<0.05）;COG 1132 and COG2826 were positively correlated with the level of serum TMA（P<0.05）;COG0515 was negatively correlated with serum TMAO and TMA,inflammatory factors IL-2,IL-6,IL-17A,IL-1β,TNF-α,hs-CRP,and serum lipid level HDL-C（P<0.05）;（7）The correlation analysis of different species with inflammation,serum lipids,and TMAO in the KEGG metabolic pathway showed that:Inflammatory factors IL-2,TNF-α,IL-6,IL-17A,IL-1β,hs-CRP,and serum lipid levels TG,TC,LDL-C,VLDL-C are negatively correlated with K21572（P<0.05）;K01990,K02003,and K01992 were positively correlated with the level of serum TMA and TMAO（P<0.05）;ABC transporters were positively correlated with the levels of TMA,TG,TC,LDL-C,VLDL-C,IL-2,TNFα,IL-6,IL-17A,IL-1β,and hs-CRP（P<0.05）;（8）The correlation analysis of different species with inflammation,serum lipids,and TMAO in CAZy function showed that:1)At the Class level,serum lipids and inflammation-related indicators were mostly positively correlated with AA（P<0.05）;the levels of TMA and TMAO were positively correlated with AA and negatively correlated with PL（P<0.05）;at the Family level,GH1 and GH109 were positively correlated with all inflammatory factors and serum lipid-related indicators（P<0.05）;Serum TMAO levels were positively correlated with GH109 and negatively correlated with GH4310,while serum TMA levels were positively correlated with CE7,GH109,GH25,and negatively correlated with GH23,GH24,and GH78（P<0.05）;（9）The results of species and function contribution analysis suggest that PActinobacteria,P Bacteroidetes,P Chlamydiae,PFirmicutes,PProteobacteria,GunclassifiedfCoriobacteriaceae,GunclassifiedoBacteroidales,GunclassifiedfMuribaculaceae,GunclassifiedfLachnospiraceae,,and GT2Glycostransf2 play an important role in the functional gene abundance of gut microbiota in each group;（10）Gene ontology（GO）function enrichment:The functional genes of each group of samples were mainly concentrated in metabolism-related processes on BP（Biological Process）level;on CC（Cellular Component）level,they were mainly concentrated in the intrinsic components of the membrane and intracellular parts;on MF（Molecular Function）level,they were mainly concentrated in nucleic acid binding,nucleoside phosphate binding,and nucleotide binding.ConclusionsBased on the theory of blood stasis and toxin,the detoxifying and blood-activating traditional Chinese medicine components plays an important role in the prevention and treatment of atherosclerosis by changing the composition of gut microbiota in atherosclerotic mice,reducing the activity of liver FMO3,inhibiting the transformation of TMA to TMAO to a certain extent,and reducing the level of TMAO,thereby reducing the plaque area of atherosclerotic mice,reducing inflammatory factors and blood lipid levels. |
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