Epidemiological Investigation And Characteristic Comparison Of Phlegm-stasis Syndrome Of Coronary Heart Disease

ObjectivesIn Traditional Chinese Medicine(TCM)theory,phlegm syndrome and stasis syndrome have the same origin,same disease,and same treatment.Previous studies did not clearly reflect the difference between phlegm syndrome and stasis syndrome of coronary heart disease(CHD).It is necessary to conduct a comparative study on phlegm and blood stasis syndrome of CHD.In this study,we hypothesized that CHD phlegm and stasis syndrome had convergent phenotypic characteristics.Based on the principle of epidemiological investigation,we conducted a reasonable comparison of phlegm and stasis syndromes of CHD at macro and micro levels,and verified the differences based on elaboration model.MethodsPart 1:An Epidemiological Study on CHD Phlegm and Stasis SyndromeIn this study,cross-sectional data of CHD patients was collected in epidemiological investigation.According to the symptoms and signs of patients,the patients were divided into phlegm group,stasis group,phlegm-stasis group,and non-phlegm-stasis group.The primary symptoms,influencing factors and biochemical examination results were analyzed.Firstly,the comparison between phlegm group and stasis group preliminarily showed the difference characteristics.Then,multiple comparative analysis was carried out to verify the difference characteristics.Finally,based on the principle of elaboration model,relevant factors were controlled to verify the independent influence of different characteristics.Part 2:Proteomics and Metabolomics Analysis of CHD Phlegm and Stasis SyndromeProteomics and metabolomics analysis were carried out on biological specimens of CHD phlegm-stasis syndrome patients and healthy volunteers,respectively.The microscopic differences were shown in comparison between CHD group and healthy group,and comparisons between phlegm group,stasis group,and phlegm-stasis group.Finally,subgroup analysis was performed on patients according to different course of disease and degree of coronary stenosis to verify the stability of differential indicators.ResultsPart 1:An Epidemiological Study on CHD Phlegm and Stasis Syndrome1.A total of 198 CHD patients were included in this study.Among them,there were 74 patients in phlegm group,36 patients in stasis group,68 patients in phlegm-stasis group,and 20 patients in non-phlegm-stasis group.In comparisons of TCM symptoms and signs,fixed position in the chest pain,aggravating chest pain at night,fat tongue with teeth marks,dark purple tongue,etc were statistically significant different(P<0.05).Statistically significant differences were shown in cyanosis in face or fingers,fixed pain,squamous and dry skin,etc.In other words,the subjects of this study were not comparable in terms of demographic sociology and CHD condition.They were representative in terms of CHD phlegm and stasis syndrome.2.The comparisons of main symptoms,influencing factors and biochemical indexes between non-phlegm-stasis group,phlegm group,stasis group and phlegm-stasis group.There were statistical differences in character,location,degree,inducing factors,mitigating factors of chest pain and chest distress,age of diagnosis,body weight,BMI,diabetes,average daily sitting duration and cumulative years of exercise among the four groups(P<0.05).3.The main symptoms,influencing factors and biochemical indexes of CHD were compared between phlegm group and stasis group.A total of 11 factors were statistically significant differences(P<0.05),including character,location,degree,inducing factors,mitigating factors of chest pain and chest distress,body weight,BMI,average daily sitting duration.They were potential characteristics between CHD phlegm syndrome and stasis syndrome.4.Chest pain symptoms,location,degree and relieving factors were statistical differences between phlegm group and phlegm-stasis group.Among these four characteristics,degree of chest pain was the only statistical difference in comparison between stasis group and non-phlegm-stasis group.The inducing factors of chest pain,mitigating factors of chest distress and average daily sitting duration were statistical differences between stasis group and phlegm-stasis group.Among these three characteristics,no statistical difference was founnd in comparison between phlegm group and non-phlegm-stasis group.In the multiple comparative analysis,degree of chest pain was the only characteristic found in CHD stasis syndrome,which was the differential characteristic of phlegm syndrome and stasis syndrome.5.Stratified Chi-square test was applied to test the differential characteristics of CHD phlegm and stasis syndrome.The results showed that the degree of chest pain was still a significant difference in CHD phlegm and stasis syndrome when the factors including hyperlipidemia,course of disease and degree of coronary artery stenosis were controlled.Part 2:Proteomics and Metabolomics Analysis of CHD Phlegm and Stasis Syndrome1.Comparisons between healthy group and CHD group:there were 1247,1247 and 1245 different proteins in comparisons between health group(21 cases)and phlegm group(27 cases),stasis group(27 cases),phlegm-stasis group(27 cases).Different proteins enriched in immune inflammation response,complement and coagulation cascades,HIF-1 signaling pathway.There were 186,151 and 191 different metabolites identified in comparisons between health group(30 cases)and phlegm group(30 cases),stasis group(30 cases),phlegm-stasis group(35 cases).According to ROC curve analysis,PC(16:1E/18:2)(AUC=0.900),(2S,3R)-2-amino-1,3-Heptadecanediol(AUC=0.961),NPentacosanoylglycine(AUC=0.994)were significantly different metabolites.2.Comparison between the phlegm group and the phlegm-stasis group:a total of 1262 differential proteins were identified in proteomic analysis.Myelperoxidase(P05164)upregulated significantly,Ig heavy chain variable region(A0A2U8J975)down-regulated significantly.Different proteins enriched in cell adhesion molecules and defense response pathways.A total of 86 differential compounds were identified by metabonomics,including 33 up-regulated compounds and 53 down-regulated compounds.According to ROC curve analysis,N-Pentacosanoylglycine(AUC=1.000)was significantly different metabolites.3.Comparison between stasis group and phlegm-stasis group:a total of 1277 differential proteins were identified in proteomics analysis.Metalloproteinase inhibitor 1(B3KQF4)up-regulated significantly,extracellular protein of fibrin family(B7ZLY3)down-regulated significantly.Different proteins enriched in HIF-1 signaling pathway.Metabolomics identified 110 different compounds,including 38 up-regulated substances and 72 down-regulated substances.According to ROC curve analysis,marmesin(AUC=0.969)was significantly different metabolites.4.Comparison between phlegm group and stasis group:a total of 1265 different proteins were identified by proteomic analysis.α-1 antitrypsin(A0A024R6I7),myeloperoxidase(P05164)up-regulated significantly,and integrin β(L7UUZ7)downregulated significantly Differential proteins are enriched in blood coagulation and platelet activation pathways.A total of 29 differential compounds were identified in metabonomics,including 19 up-regulated substances and 10 down-regulated substances.ROC curve analysis showed that(2S,3R)-2-amino-1,3-Heptadecanediol(AUC=0.913)was significantly different metabolites.5.Complement C4-A(A0A140TA49)and porphyroglobulin(B4DE59)were specific proteins in phlegm group.Serine/threonine protein phosphatase(Q9BRF8)was only found in stasis group.There were 4 specific proteins in the phlegm-stasis group,including ubiquitin binding enzyme E2(F8VQQ8).6.Subgroup analysis:Subgroup analysis was performed on CHD patients with phlegm or stasis syndrome based on whether they had hyperlipidemia,the length of disease course and the degree of coronary artery stenosis.Proteomics and metabonomics subgroup analysis indicated that no protein or metabolic markers of phlegm-stasis syndrome of CHD were found in this study.Conclusions1.In this epidemiological investigation,degree of chest pain was the only difference between CHD phlegm syndrome and stasis syndrome.This result also proved that CHD phlegm and stasis syndrome had convergent phenotypic characteristics.2.This study found that a lot of substances and pathways related to CHD phlegm syndrome and stasis syndrome.But no protein and metabolite markers were found.This result showed that the microphenotype of CHD phlegm and stasis syndrome were similar.3.Summarizing the results of macro and micro research,this study found that differences between CHD phlegm and stasis syndrome were shown in quantification degree and manifestation.CHD phlegm and stasis syndrome were reflected in different emphasis directions with no essential difference.