Study On The Biological Mechanism Of Phlegm And Blood Stasis Syndrome In Stable Angina Pectoris Of Coronary Heart Disease Based On Multi-omics

The biological basis of phlegm and blood stasis syndrome of coronary heart disease(CHD PBSS)is one of the hot spots in syndrome research.Some progress has been made in genome,proteome and metabonomics.However,there are some problems in the research,such as relatively isolated research level,failure to establish the interconnection between different omics information,and unable to systematically explain its complex biological basis.In order to solve the above problems,this study combined high-throughput sequencing,high-resolution mass spectrometry and other multi omics technology with syndrome research,screened genes,proteins and metabolites related to phlegm and blood stasis syndrome of coronary heart disease,used multi algorithm bioinformatics analysis,integrated multi omics bioinformatics,and observed the influence of blood activating and phlegm resolving drugs on key factors through "syndrome detection by drugs",so as to provide reference It provides a new way to explain the biological basis of the syndrome of phlegm and blood stasis in coronary heart disease.ObjectiveTo explore the biological basis of CHD NPBSS at three levels of genome,proteome and metabonome;to discover the gene,protein and metabolite biomarkers of phlegm and blood stasis syndrome of stable angina pectoris of coronary heart disease;to verify the biological mechanism of phlegm and blood stasis syndrome of stable angina pectoris of coronary heart disease.Methods1.Transcrip tome study of stable angina pectoris with phlegm and blood stasis syndrome.The high throughput sequencing technology was used to detect the mRNA in peripheral blood nucleated cells of patients with PBSS,CHD of non phlegm and blood stasis syndrome(CHD NPBSS),non-CHD of phlegm and blood stasis syndrome(NCHD PBSS)and healthy people.The differentially expressed genes were screened by biomedical statistics,test and grouping comparative analysis.The differentially expressed genes were analyzed by bioinformatics methods with the help of DAVID and STRING platform of the gene function,signal pathway and protein-protein interaction.According to the biological basis,fold change and significance of differentially expressed genes,the target gene were screened.The target gene was verified by qPCR and analyzed by ROC curve to determine their possibility as biomarkers.2.Study on plasma proteomics of stable angina pectoris with phlegm and blood stasis syndrome.The DIA proteomics technology was used to detect the plasma proteins of patients with CHD PBSS,CHD NPBSS,NCHD PBSS and healthy people.The differentially expressed protein were screened by biomedical statistics,test and grouping comparative analysis.The differentially expressed genes were analyzed by bioinformatics methods with the help of DAVID databse and STRING platform of the gene function,signal pathway and protein-protein interaction.According to the biological basis,fold change and significance of differentially expressed genes,the target protein were screened.The target protein was verified by ELISA and analyzed by ROC curve to determine their possibility as biomarkers3.Study on the serum metabonomics of stable angina pectoris with phlegm and blood stasis syndrome.The LC-MS non targeted metabonomics technology was used to detect the serum metabolites of patients with CHD PBSS,CHD NPBSS,NCHD PBSS and healthy people.The differential metabolites were screened by multivariate statistics and univariate analysis.The signal pathway and diagnostic efficacy of the differential metabolites were analyzed by Metaboanalysis platform.According to the biological basis,diagnostic efficacy,VIP,fold change and significance the biomarkers at the metabonomic level were screened4.Clinical intervention of traditional Chinese medicine for activating blood circulation and resolving phlegm on target genes of stable angina pectoris with phlegm and blood stasis syndromeAccording to the randomized controlled trial design,30 patients with CHD PBSS were included according to the diagnostic criteria of Western medicine and traditional Chinese medicine.There were 15 patients in the experimental group(Danlou tablet)and 15 patients in the control group(Danlou tablet simulant).The treatment course was 4 weeks.PCR was used to detect the expression of target gene before and after treatment.The observation indexes included the difference of syndrome score before and after treatment,the effectiveness of TCM syndrome and the change of target gene expression before and after treatment between the two groups.The curative effect was evaluated according to the objective standard.Nonparametric test was used to compare the difference of syndrome score before and after treatment between the two groups,chi square test was used to compare the effectiveness of TCM syndrome between the two groups,and t test and U test were used to compare the change of target gene expression between the two groups.The relative expression and the overall distribution of the expression changes were compared between two groups to determine the suitable biomarker for CHD PBSS.Results1.Ninety-eight differentially expressed mRNA of CHD PBSS were screened by high-thoughput sequencing,including 56 up-regulated genes and 42 down regulated genes.Through bioinformatics analysis of the biological process and signal pathway of differential genes,it is found that at the mRNA level,the occurrence of CHD PBSS may be closely related to immunity and inflammation.The main biological processes include inflammatory response,adaptive immune response,immune response,etc.And the main molecular functions include chemokine activity,etc.The main signaling pathways include cytokine cytokine receptor interaction,antigen processing and presentation,chemokine signaling pathway,TNF signaling pathway and so on.Six target genes were screened and verified by qPCR.The results showed that the expression of CXCL8 was 56%lower than that of the control group,P=0.000,CCL2 expression decreased 25%compared with the control group,the difference between the two groups was not significant,P=0.168,and the expression of csfl increased 23%compared with the control group,The difference between the two groups was not significant(P=0.103).ROC curve analysis shows that AUC of CXCL8 is 0.844,AUC of CCL2 is 0.699,AUC of CSF1 is 0.626.Through comprehensive analysis,CXCL8 can be used as a biomarker of CHD PBSS.CCL2 and CSF1 can be used as candidate biomarkers.2.Through dia proteomics identification technology,104 differential proteins of CHD PBSS were screened out,including 92 non functional annotated proteins,mostly immunoglobulin fragments,22 functional annotated proteins,of which 21 proteins were up-regulated and 1 protein was down regulated.Through bioinformatics analysis of the biological processes and signal pathways of differentially expressed proteins,we found that at the protein level,CHD PBSS may be closely related to immune and coagulation.The main biological processes involved include opsonization,innate immune response,blood coagulation,etc.The main molecular functions include structural molecule activity,cell adhesion molecule binding,etc.The main signaling pathways include complement and coagulation cascades and platelet activation.Two target proteins were screened and verified by ELISA.The results showed that the expression of SERPIND1 was significant between two groups,P=0.003.ROC curve analysis showed that the AUC of SERPIND1 is 0.768.Through comprehensive analysis,SERPIND1 can be used as a candidate biomarker.3.Through LC-MS non targeted metabonomics technology and multivariate statistical methods,20 differential metabolites of CHD PBSS were screened.Through bioinformatics analysis of the signal pathways involved in the differentially expressed metabolites,we found that at the level of metabolites,the occurrence of CHD PBSS may be related to amino acid metabolism,glycerophospholipids metabolism and steroid hormones biosynthesis and so on ROC curve analysis showed that the AUC of 8 metabolites are more than 0.85.There are 5 alpha pregnan 20 alpha ol 3 one,dopamine,2 arabidonylglycerol,allantoic acid.acetylhomoserine,5 hydroxy L tryptophan,ribothymidine,1,2,3 propanetricarboxylic acid.Through comprehensive screening,5alpha Pregnan 20alpha ol 3 one、Dopamine、Allantoic acid,5 Hydroxy L tryptophan,LysoPC(20:4(8Z,11Z,14Z,17Z)),3,4 Dihydroxybenzeneacetic acid、Homogentisic acid could be used as biomarkers of CHD PBSS.4.Through the randomized controlled trial,the difference of syndrome score between the two groups was compared,the experimental group reduced 27(15,29)points vs.the control group reduced 11(3,15)points,P=0.004;compared the effectiveness of the two groups,12 people in the experimental group were effective,accounting for 80.00%,while 5 people in the control group were effective,accounting for 33.33%,P=0.010.qPCR result showed that compared with the control group,the expression of CXCL8 increased by 22%,CCL2 increased by 57%,CSF1 decreased by 20%.The P value of t test was 0.201,0.201,0.153 and 0.908,respectively,and of U test was 0.165,0.110,0.237 and 0.820.Compared with the healthy control group,CXCL8,CCL2 and SERPIND1 were down regulated,while CSF1 was up regulated.After treatment,compared with the placebo group,the expression of CXCL8 and CCL2 increased while CSF1 and SERPIND1 decreased.The distribution of the target gene samples also showed the same trend with the expression level.It suggests that the above four genes can be used as biomarkers of CHD PBSSConclusion1.There are differential expression profiles of transcriptome in CHD PBSS,and their functions and pathways are mostly related to immune and inflammatory response2.There are differential expression profiles of proteomic CHD PBSS,and their functions and pathways are closely related to immune response and coagulation3.There are differential expression profiles of metabonomics in stable angina pectoris of coronary heart disease with phlegm and blood stasis syndrome and the involved pathways are mostly related to amino acid metabolism,glycerophospholipids metabolism and steroid hormone biosynthesis.4.Integrative analysis of multi-omics found that immune response is one of the biological basis of the occurrence and development of CHD PBSS.5.CXCL8,CCL2,CSF1 and SERPIND1 can be used as biomarkers of CHD PBSS.