A Study Of Plasma Exosome Protein Spectrum In Coronary Heart Disease With Phlegm Turbidity Syndrome

BackgroundThe global incidence of coronary heart disease is on the rise,and it is the leading cause of death in the world.About 7.4 million people die of coronary heart disease every year.Approximately,10 million people in the United States suffer from angina,with more than 500,000 cases diagnosed each year.According to the 2020 Report on Cardiovascular Health and Diseases in China,there are about 330 million cases of cardiovascular diseases in China,including 11.39 million cases of coronary heart disease.Cardiovascular disease is a serious threat to national health and brings a serious economic burden to the country and society.Dyslipidemia is an important risk factor for cardiovascular diseases,but the awareness rate,treatment rate and control rate of dyslipidemia in Chinese adults are all at a low level.Traditional Chinese medicine believes that dyslipidemia is related to addiction to fat,sweet,cold,tobacco and alcohol,dysfunction of the spleen and stomach,and production of phlegm and dampness.Phlegm-turbid syndrome(PTS)is an important syndrome of coronary heart disease.Exosomes carry a large amount of biological information,participate in the transfer of substances and information between cells,and can directly act on target organs and tissues.Exosomes participate in biological processes such as immunity and inflammatory response,and play an important role in the diagnosis and treatment of cardiovascular diseases.Therefore,it is of great significance to further study the expression levels of plasma exosome proteins in patients with coronary heart disease with PTS.ObjectiveThe purpose of this study was to explore the expression of plasma exosomal proteins in coronary heart disease and healthy people,coronary heart disease with PTS and coronary heart disease with non-phlegm-turbid syndrome(NPTS).Then the study verified some differential expression proteins by using targeted protein labeling technology,in order to provide more biological evidence for clinical diagnosis and treatment of coronary heart disease patients with PTS.MethodsExperiment one:This study collected 120 patients with coronary heart disease who visited the Cardiovascular and General Departments of Guang ’anmen Hospital,China Academy of Traditional Chinese Medical Sciences from July 2021 to November 2021.In addition,30 healthy subjects who visited the Preventive health care department Department of Guang’anmen Hospital,China Academy of Traditional Chinese Medical Sciences at the same time and age were included as the control group.Fasting venous blood was drawn from all subjects,and chemical analysis was used to determine Cholesterol(CHO),Triglyceride(TG),High density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C),very low density lipoprotein(VLDL),apolipoprotein A1(Apo Al),apolipoprotein B(Apo B)levels.Randomly selected 20 patients with coronary heart disease and 10 healthy subjects.Exosomes from fasting plasma were extracted by qEV size exclusion method,and identified by transmission electron microscopy,Nanoparticle Tracking analysis(NTA)and Western Blot.Label free unlabeled protein spectrometry was used to detect the protein expression levels of exosomes in each group,and bioinformatics techniques were used to analyze the differentially expressed proteins in each group.In addition,20 patients with coronary heart disease and 10 healthy subjects were randomly selected.MRM targeted protein labeling technique was used to verify some differential proteins.Experiment two:This study collected 80 patients with coronary heart disease with PTS and 40 patients with coronary heart disease with NPTS who were treated in cardiovascular and general wards from July 2021 to November 2021.Fasting venous blood was drawn from all subjects,and chemical analysis was used to determine CHO,TG,HDL-C,and LDL-C,VLDL,Apo A1,Apo B levels.Randomly selected 10 patients with coronary heart disease with PTS and 10 patients with coronary heart disease with NPTS.Exosomes from fasting plasma were extracted by qEV size exclusion method,and identified by transmission electron microscopy,NTA and Western Blot.Label free unlabeled protein spectrometry was used to detect the protein expression levels of exosomes in each group,and bioinformatics techniques were used to analyze the differentially expressed proteins in each group.In addition,10 patients with coronary heart disease with PTS and 10 patients with coronary heart disease with NPTS were randomly selected.MRM targeted protein labeling technique was used to verify some differential proteins.SPSS 25.0 statistical software was used for data analysis,and P<0.05 was used as the basis for judging statistically significant differences.Measurement data conforming to normal distribution or approximately normal distribution are expressed as X±S,and t-test is used for comparison between groups.Measurement data that does not conform to normal distribution are expressed as quartiles,and rank sum test is used for comparison between groups.Expressed as frequencies and percentages,comparisons between groups were performed using chi-square test and Fisher’s exact test.ResultsExperiment one:1.Baseline Results:There was no statistical difference between the coronary heart disease group and the healthy control group in gender,age,alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine(Cr)(P>0.05).2.Body mass index(BMI),Glycated hemoglobin(HbA1c),and blood lipids:Compared with healthy controls,the levels of BMI,HbA1c,TG,and VLDL in patients with coronary heart disease were significantly higher(BMI:25.69±3.65 kg/m2 VS 20.98±2.10 kg/m2;HbA1c:6.71±1.35%VS 4.73±0.37%;TG:1.73±0.96 mmol/L VS 0.88±0.30 mmol/L;VLDL:0.78±0.44 mmol/L VS 0.43±0.16 mmol/L;P<0.001);HDL-C was significantly reduced(1.06±0.24 mmol/L VS 1.32±0.30 mmol/L,P<0.001);There were no significant differences in the levels of CHO,LDL-C,Apo Al,and Apo B(P>0.05).3.Results of plasma exosome protein profile in patients with coronary heart disease3.1 Identification results of exosomes in patients with coronary heart disease:①Clear circular or quasi-circular vesicle structures can be seen under the field of electron microscope,similar to the "cup mouth saucer",and the size is about 100nm;②NTA detected that the particle size of the sample is 125.2nm,which meets the detection standard of exosomes;③Using WB to detect the surface marker proteins of exosomes extracted,3 positive proteins:CD9,CD81,HSP70;1 negative protein:Calnexin;the above results all meet the detection standards of exosomes,which can prove that the extracted substances are exosomes.3.2 Unlabeled quantitative proteomics results in patients with coronary heart disease:Compared with the healthy control group,92 proteins including ANXA6 were up-regulated in the coronary heart disease group(P<0.05);101 proteins including GSTM5 were downregulated(P<0.05).3.3 Results of functional analysis of differentially expressed proteins in patients with coronary heart disease;Compared with the healthy control group,GO enrichment analysis found that the differentially expressed proteins in the coronary heart disease group were mainly involved in biological processes such as "immune effector process","complement activation",and "vesicle-mediated transport";Molecular functions such as"particle receptor binding" and"lipid binding";affect cellular components such as "extracellular space","extracellular exosome",and "extracellular vesicle".Compared with the healthy control group,KEGG enrichment analysis showed that the differentially expressed proteins in the coronary heart disease group were mainly involved in "Complement and coagulation cascades","Cholesterol metabolism","Focal adhesion" and other signaling pathways.3.4 Validation results of some differentially expressed proteins in patients with coronary heart disease:29 differentially expressed proteins were selected for MRM-targeted protein validation,and it was finally found that ANXA6,C4BPB,PRG4,F8,CFB,Apo E,C9,CLU and unlabeled quantitative proteomics The expression trend of the results was consistent and statistically significant(P<0.05).3.5 ROC curve results of differentially expressed proteins in patients with coronary heart disease:The area under the ROC curve of differentially expressed proteins ANXA6,C4BPB,PRG4,F8,CFB,Apo E,and C9 between the coronary heart disease group and the healthy control group were all Above 0.79(ANXA6:0.82;C4BPB:0.90;PRG4:0.83;F8:0.81;CFB:0.80;Apo E:0.85;C9:0.86;P<0.01),the area under the ROC curve of the CLU protein was as high as 0.93(P<0.001).Experiment two:1.Baseline Results:There was no significant difference in gender,age,ALT,AST,Cr between the coronary heart disease with PTS group and the coronary heart disease with NPTS group(P>0.05).2.BMI,HbA1c,blood lipids and use of lipid-lowering drugs:Compared with coronary heart disease with NPTS,BMI,prevalence of hyperlipidemia,TG level,and VLDL level in patients with coronary heart disease with PTS were increased(P<0.05);HbAlc,prevalence of diabetes,CHO,HDL-C,LDL-C,Apo A1,Apo B and the use of lipid-lowering drugs were not significantly different between the two groups(P>0.05).3.Study results of plasma exosome protein profile in patients with coronary heart disease with PTS3.1 The identification results of exosomes:①Clear circular or quasi-circular vesicle structure can be seen under the field of electron microscope,similar to the "cup mouth saucer",and the size is about 100nm;②NTA detected the particle size of the sample The size is 116.0 nm,which meets the detection standard of exosomes;③The surface marker proteins of exosomes extracted by WB detection,3 positive proteins:CD9,CD81,HSP70;1 negative protein:Calnexin;the above results are in line with the exosomes.The detection standard of exosomes can prove that the extracted substances are exosomes.3.2 Unlabeled quantitative proteomics results in patients with coronary heart disease with PTS:Compared with coronary heart disease with NPTS,a total of 28 proteins including ANXA6 were found to be up-regulated in the coronary heart disease with PTS(P<0.05);14 proteins including C4BPB were down-regulated(P<0.05).3.3 Bioinformatics analysis results of differentially expressed proteins in patients with coronary heart disease with PTS:Compared with the coronary heart disease with NPTS group,GO enrichment analysis found that the differentially expressed proteins in the PTS group of coronary heart disease were mainly involved in "leukocyte mediated immunity","Biological processes such as neutrophil degranulation;exert molecular functions such as "phospholipid binding","lipase inhibitor activity","lipoprotein particle receptor binding","cholesterol binding","lipid binding";change "extracellular exosome","extracellular vesicle",etc.cellular components.Compared with the NPTS of coronary heart disease,KEGG enrichment analysis showed that the differentially expressed proteins in the PTS group of coronary heart disease were mainly involved in "Endocytosis","Complement and coagulation cascades","Cholesterol metabolism" and other signaling pathways.3.4 Validation results of some differentially expressed proteins in patients with coronary heart disease with PTS:8 differentially expressed proteins were selected for MRM targeted protein validation,and it was finally found that ANXA6,C4BPB and PLEK had the same expression trend with unlabeled quantitative proteomics and statistical significance(P<0.05).Conclusion1.Compared with the healthy control group,the expression levels of 92 proteins including ANXA6 in plasma exosomes of patients with coronary heart disease were up-regulated,and the expression levels of 101 proteins including GSTM5 were down-regulated.The differentially expressed proteins between the two groups were mainly related to immune effects,cholesterol metabolism,complement and Coagulation cascade.2.Compared with the NPTS of coronary heart disease,a total of 28 proteins including ANXA6 were up-regulated in plasma exosomes of patients with coronary heart disease with PTS,and a total of 14 proteins including C4BPB were down-regulated.The differentially expressed proteins between the two groups were mainly related to lipid binding,complement and coagulation cascade,and cholesterol metabolism.