Study On The Diagnosis,Severity Assessment And Trace Elements Of Coronary Atherosclerotic Heart Disease

Coronary heart disease(CHD)is an ischemic heart disease that includes chronic coronary artery disease and acute coronary syndrome.When coronary artery atherosclerosis causes lumen stenosis or occlusion,leading to myocardial ischemia,hypoxia,necrosis,the result is myocardial infarction.Coronary atherosclerosis(AS)is the cause of stable coronary heart disease(SCAD)and acute myocardial infarction(AMI).Atherosclerosis is a chronic inflammatory disease of arteries caused by the deposition of lipids under the intima of blood vessels.Atherosclerosis is progressive.According to the pathological classification of plaque by the American Heart Association,there are three stages in sequence:the first stage is the appearance of lipid spots and stripes,the second stage is the formation of atherosclerosis and thin-walled fibrous plaque,and the third stage is the occurrence of complex changes in arteries and veins.The clinical classification of coronary atherosclerosis is mainly based on symptoms,electrocardiogram,myocardial markers,etc.Atherosclerosis is caused by both environmental and genetic factors,and genetic factors account for 50%.Therefore,finding disease diagnostic markers is conducive to precise treatment.This study used the clinical data of over 10000 hospitalized patients in our hospital,as well as peripheral blood mononuclear cells(PBMC)and plasma.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blot were used to verify the expression of differential genes.Using proteomic methods(TMT),parallel response monitoring experiments(PRM),bioinformatics analysis,Mfuzz Analysis(Mfuzz),machine learning and statistical techniques such as multiple regression analysis,propensity matching,etc.,we studied the molecular markers for diagnosis of coronary atherosclerosis and the protein markers for judging severity,and analyzed the relationship between human nutrition,trace elements iron ion(Fe),zinc ion(Zn)and prognosis.The results showed that SOCS3 gene and RORA gene were highly expressed in the peripheral blood of patients with coronary atherosclerosis.Various protein markers define different stages of coronary atherosclerosis.Six of the proteins involved in the lipid metabolism pathways,namely ALB,SHBG,APOC2,APOC3,APOC4,and SAA4,have been further validated.Trace elements zinc and iron ions participate in the prognosis of patients with coronary atherosclerosis.The study supports the diagnosis of coronary atherosclerosis by iron ions,while appropriate supplementation of zinc ions may serve as a rehabilitation program for elderly menopausal women with coronary heart disease.The application value of this study is that through the differential expression of genes,the application of protein omics,and trace element analysis,we hope the study can predict the correlation of coronary atherosclerosis in patients at an early stage,increase the accuracy of judging coronary atherosclerosis at the middle stage,and increase the accuracy of treatment at the late stage.This study is divided into three parts.PartⅠ.Study on molecular markers for diagnosis of coronary atherosclerotic heart diseaseStudy on the correlation between peripheral blood SOCS3 and patients with acute myocardial infarctionObjective:Exploring whether the differential expression of the inhibitor of cytokine signaling 3(SOCS3)gene is associated with the development of AMI.Method:Peripheral blood samples from 113 patients with AMI and 92 patients with SCAD were collected,and RNA from the peripheral blood was extracted and reversely transcribed into cDNA.The expression level of SOCS3 mRNAin peripheral blood was measured by RT-qPCR,and the expression level of SOCS3 protein in mononuclear cells was measured by Western blotting.SPSS 24.0 software was used for statistical analysis,and P<0.05 was used to indicate statistical significance.Result:The mRNA expression level of SOCS3 gene in AMI patients was 1.33 times higher than that in SCAD patients,and the expression level of SOCS3 protein in peripheral blood mononuclear cells in AMI patients was 1.25 times higher(both P<0.05).Binary logistic regression analysis showed that the increased expression of SOCS3 gene was an independent risk factor for AMI.Study on the correlation between peripheral blood RORA and patients with acute myocardial infarctionObjective:To explore whether the differential expression of retinoic acid receptor-associated orphan receptor A(RORA)gene is related to the occurrence of AMI.Method:Peripheral blood samples from 93 patients with AMI and 74 patients with SCAD were collected,and RNA from the peripheral blood was extracted and reversely transcribed into cDNA.The expression level of RORA mRNA in peripheral blood was measured by RT-qPCR,and the level of RORA protein in mononuclear cells was measured by Western blotting.SPSS 24.0 software was used for statistical analysis,and P<0.05 was used to indicate statistical significance.Result:The mRNA expression level of RORA mRNA peripheral blood of patients with AMI was 1.57 times that of patients with SCAD.The expression level of protein RORA in peripheral blood mononuclear cells of patients with AMI was increased(both P<0.05).Binary logistic regression analysis showed that high expression of RORA was an independent risk factor for AMI,and it increased the risk of AMI by 2.990 times.PartⅡ.Proteomics in healthy people,patients with stable coronary heart disease and acute myocardial infarctionObjective:CHD is a complex disease caused by multiple interactions between genetic and environmental factors,which makes identifying candidate proteins and their associated risk markers a huge challenge.Coronary atherosclerotic heart disease,including SCAD and AMI.It is generally believed that plaque in patients with stable coronary heart disease is stable(possible),while plaque in patients with AMI is unstable(possible).Plaque stability or rupture is a pathological change,and we hope to use proteomic methods to find protein markers for diagnosis and assessment of coronary artery severity.Method:TMT was used to analyze 30 male patients aged 45 to 55 years who were diagnosed with coronary atherosclerosis.The inclusion criteria excluded patients diagnosed with hypertension and type 1 and type 2 diabetes.Mufuzz analysis is used to select target proteins,judge and select specific proteins involved in coronary atherosclerosis.PRM is used to validate selected target proteins while applying bioinformatics for analysis.Finally,the operating characteristic curve(ROC)is calculated through machine learning and random forest experiments.Statistics was analyzed using SPSS 24.0 software.Result:1147 proteins were identified in TMT mass spectrometry,of which 907 were quantifiable.In the PRM study,six proteins related to lipid metabolism pathways were validated,namely ALB,APOC2,APOC3,APOC4,SHBG,and SAA4.In healthy individuals,SCAD,and AMI patients,the expression gradients of APOC2,APOC3,APOC4,and SAA4 proteins increased,while the ALB and SHBG gradients decreased.PartⅢ.Correlation between nutrients and coronary atherosclerosisStudy on the relationship between the decrease of zinc ion concentration in peripheral blood and coronary atherosclerotic heart diseaseObjective:The imbalance of trace element concentration in blood caused by malnutrition may affect the development of CHD.This part explores the correlation between trace element zinc ion and coronary atherosclerosis.Method:To study the relationship between peripheral blood zinc ion concentration and CHD,the experimental group included 1253 CHD patients,and the control group included 2288 non coronary heart disease patients.In this study,multivariate logistic regression and hierarchical analysis were performed on the blood measurement results of 3541 patients,and baseline analysis and propensity matching were performed on the patient data set.SPSS 24.0 software was used for statistical analysis.Calculate the average and deviation of all indicators in each group,and use P<0.05 to indicate statistical significance.Result:Non-smoking,old age(especially postmenopausal women)and low blood zinc concentration were independent risk factors for CHD(P<0.05,zinc ion concentration is less than 13.82±2.91μmol/L).Study on the relationship between the decrease of peripheral blood iron ion concentration and coronary atherosclerotic heart diseaseObjective:The balance of trace elements in the body caused by nutritional factors can lead to the progression of coronary atherosclerosis.In this part,the study explored the correlation of trace element iron ion and coronary atherosclerosis.Method:The concentrations of sodium(Na),potassium(K),magnesium(Mg),calcium(Ca),zinc(Zn)and iron(Fe)in the peripheral blood samples of 4243 patients were measured,and the baseline analysis and trend matching were performed on the patient data set.The patients were divided into AMI(702 cases)group and SCAD(1253 cases)group.These two groups of patients were included in the coronary atherosclerosis group,a total of 1955 patients.The control group was composed of 2288 people.The concentrations of calcium,zinc,magnesium and iron in serum were measured by colorimetry.For comparison,15 external quality assessment(EQA)samples were selected from the Clinical Laboratory Center of the Ministry of Health of China.SPSS 24.0 software was used for statistical analysis.Calculate the mean value and deviation of all indicators in each group,and use P<0.05 to indicate statistical significance.Result:The iron ion concentration in the AMI group was significantly lower than that in the control group(P<0.05,area under the curve,AUC=0.724,AUC=0.702).Compared to the data from the SCAD group,the iron ion concentration in the AMI group was significantly lower(P<0.05,AUC=0.710,AUC=0.682).In addition,the iron ion concentration(median,IQR 13.73)in the coronary atherosclerosis group(AMI+SCAD)was significantly lower than that in the control group(IQR 15.56,P<0.05)Conclusion:The mRNA expression levels of SOCS3 and RORA genes in peripheral blood of patients with AMI,as well as the protein level in mononuclear cells,are higher than those of patients with SCAD,which are independent risk factors for the occurrence of AMI.In healthy individuals,SCAD,and AMI patients,the expression gradients of APOC2,APOC3,APOC4,and SAA4 proteins increased,while the ALB and SHBG gradients decreased.It provides accuracy for the judgment of coronary atherosclerosis in SCAD and AMI population.The serum zinc level of patients with CHD is lower,and its serum zinc level is significantly lower than that of the control group.It is further demonstrated that low serum zinc concentration is an independent risk factor for coronary heart disease.In addition,for non smoking elderly patients and women,especially postmenopausal women,decreased zinc ion concentration is an independent risk factor for coronary heart disease.Acute myocardial infarction and coronary atherosclerosis are associated with low levels of serum iron ion expression.Iron can be used as an additional biomarker and prognostic indicator of coronary atherosclerosis.