Study On The Mechanism Of Action Of Danlou Tablets In The Treatment Of Stable Angina Pectoris With Phlegm And Blood Stasis Syndrome Based On Bioinformatic

Purpose: 1.Based on bioinformatics,explore and analyze the potential molecular biological mechanism of coronary heart disease and explore its potential biomarkers and therapeutic targets.2.To reveal the material basis and key target of the excellent effect of TCM in treating stable angina pectoris with phlegm and blood stasis syndrome,and to clarify the mechanism of Danlou tablets and its flavoring to exert the effect.3.Improve the design of clinical research on the combination of evidence-based medicine and TCM differentiation and treatment,fully reflect the characteristics and advantages of TCM combination of disease and syndrome differentiation and treatment,and provides a methodological reference for clinical research of Traditional Chinese Medicine..At the same time,optimize the existing diagnosis and treatment plan,improve the clinical efficacy,and form high-quality clinical evidence for the clinical plan of treating stable angina pectoris with phlegm and blood stasis.Material and method: 1.Research on the pathogenesis of coronary heart disease based on bioinformatics analysis(1)The human atherosclerosis related transcriptome expression profile gene set(GSE100927)was retrieved from the GEO database,and the differentially expressed genes related to AS were screened using the Limma package of R language.(2)Use the WGCNA package of R language to construct a weighted gene co-expression network,determine the relationship between gene modules and sample grouping phenotypic data,find out the modules that are significantly related to grouping phenotypic data as target modules,and screen out relevant genes.(3)Intersect the differentially expressed genes and the genes screened by WGCNA analysis to obtain the common key gene set.(4)Metascape database was used to analyze the GO function and KEGG pathway enrichment of the intersection genes screened from the differential genes and the WGNCA co-expression part.(5)Through GO and KEGG functional annotation of differential genes,biological processes related to pathological changes of atherosclerosis were analyzed from a biological perspective,so as to screen biomarkers closely related to atherosclerosis.2.Study on the Intervention of Danlou Tablets on Plasma Protein Markers in Stable Angina Pectoris with Phlegm and Blood Stasis Syndrome(1)Case inclusion and intervention measures: 304 patients with stable angina pectoris with phlegm-stasis syndrome were included and randomly divided into the experimental group(152 cases in the TCM treatment group)and the control group(152 cases in the conventional western medicine treatment group).The cases were from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine,Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine,Yunnan Provincial Hospital of Traditional Chinese Medicine Outpatients of 12 sub-centers of the Affiliated Hospital of Southwest Medical University,Longhua Hospital of Shanghai University of Traditional Chinese Medicine,the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine,Hubei Provincial Hospital of Traditional Chinese Medicine,Gansu Provincial Hospital of Traditional Chinese Medicine,the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,and the First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine.The control group received routine western medicine treatment;The test group received Danlou tablet and its modified combination with conventional western medicine treatment.Danlou tablet was 5 tablets each time,and the corresponding concomitant granules were 1 bag/time for each variety.After being dissolved in 100 ml of boiling water at 90-100 ℃,it was taken warm,three times a day,and the intervention lasted for 6 months(24 weeks).During the trial period,all test cases shall not use other TCM treatment methods(such as traditional Chinese medicine preparations,acupuncture and moxibustion treatment,etc.)other than those specified for the treatment of the disease.(2)Preparation of clinical biological samples: 4m L of peripheral venous blood were collected at 0,1(4 weeks),3(12 weeks)and 6(24 weeks)after the subjects were enrolled in the group,and the plasma was centrifuged and extracted,and then stored at-80 ℃ refrigerator for long-term low-temperature storage for testing.(3)The levels of IL-6,VCAM-1,MMP9,MPO and Ang-Ⅰ in plasma of subjects with stable angina pectoris syndrome of phlegm and blood stasis were detected by enzyme-linked immunosorbent assay(ELISA).(4)Efficacy index evaluation: The plasma levels of IL-6,VCAM-1,MMP9,MPO and Ang-Ⅰ in the test group and control group were statistically analyzed.3.A Study on the m RNA Expression of Protein Markers in Stable Angina Pectoris with Phlegm and Blood Stasis Syndrome Intervention of Danlou Tablets(1)Case inclusion and intervention measures: 26 patients with stable angina with phlegm-stasis syndrome were included and randomly divided into the experimental group(12 cases in the TCM treatment group)and the control group(14 cases in the conventional western medicine treatment group).The cases were from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine and Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine.The control group received routine western medicine treatment;The test group received Danlou tablet and its modified combination with conventional western medicine treatment.Danlou tablet was 5 tablets each time,and the corresponding concomitant granules were 1 bag/time for each variety.After being dissolved in 100 ml of boiling water at 90-100 ℃,it was taken warm,three times a day,and the intervention lasted for 6 months(24 weeks).During the trial period,all test cases shall not use other TCM treatment methods(such as traditional Chinese medicine preparations,acupuncture and moxibustion treatment,etc.)other than those specified for the treatment of the disease.(2)Preparation of clinical biological samples: 4m L of patients’ peripheral venous blood was collected at 0,1(4 weeks),3(12 weeks),and 6(24 weeks)after the subjects were enrolled in the group,and the peripheral blood leukocytes were separated and extracted and stored in a freezer at-80 ℃ for detection by real-time fluorescent PCR.(3)Quantitative real-time PCR was used to detect the expression of Ang-Ⅰ,IL-6,MPO,MMP-9 and VCAM-1 in peripheral blood leukocytes of patients with stable angina pectoris with phlegm-stasis syndrome.(4)Efficacy index evaluation: The expression of Ang-Ⅰ,IL-6,MPO,MMP-9 and VCAM-1 in peripheral blood leukocytes of patients in the test group and the control group was statistically analyzed.Results:1.Research results on the pathogenesis of coronary heart disease based on bioinformatics analysis(1)In this study,441 differential genes highly related to AS lesions were screened through differential expression analysis.Among them,MMP9 is an up-regulated gene highly related to atherosclerotic pathological changes.(2)Through weighted gene co-expression network analysis,turquoise(correlation coefficient=0.73,P<0.001),which was significantly related to the grouping phenotype,was screened out,and 976 genes with synergistic effect in the module were obtained.(3)The genes screened by WGCNA analysis were intersected with 441 differentially expressed genes,and 282 differentially co-expressed genes were obtained.(4)The GO and KEGG functional annotation of the obtained differentially expressed genes found that these differentially expressed genes are mainly involved in the regulation of leukocyte activation,negative regulation of immune system process,regulation of cytokine production,positive regulation of immune response,leukocyte differentiation,myeloid leukocyte activation,leukocyte migration,phagocytosis,regulation of defense response,myeloid leukocyte differentiation,cytokine-mediated signal pathway Regulation of ERK1 and ERK2 cascade,production of interleukin-6,interferon γ Biological processes such as production of molecular mediators involved in inflammatory reaction,toll-like receptor signal pathway,macrophage activation,antigen-stimulated inflammatory reaction,positive regulation of hydrolase activity,leukocyte-mediated cytotoxicity,etc.(5)Based on the chronic inflammatory process of atherosclerosis and relevant literature,this study considered that MMPs,adhesion molecules,IL-6,myeloperoxidase and other inflammatory biomarkers played an important role in the process of inflammatory lesions of atherosclerotic plaque.MMP-9,IL-6,VCAM-1,MPO,Ang-1 were studied with subsequent experiments.2.Research results on the intervention of Danlou tablets on plasma protein markers of stable angina pectoris with phlegm stasis syndrome(1)Sub-center and case distributionSince 4 subjects fell out during the trial,300 patients were finally included in the study for statistical analysis,including 151 cases in the trial group and 149 cases in the control group.The cases were from 12 sub-centers in six major regions of the country,and the number of cases in each sub-center accounted for about 8%.The number of cases in each sub-center trial group was equal to that in the control group,with a wide range of cases,relatively balanced distribution and certain representativeness.(2)Demographic dataIn terms of demographic data of 300 patients,there was no statistically significant difference between groups(P>0.05),and the baseline was balanced and comparable.(3)Efficacy index evaluation1)Efficacy analysis of full analysis setAfter treatment,the plasma levels of IL-6,VCAM-1,MMP-9,Ang-1 and MPO in the two groups were lower than those before treatment(P.Therefore,it can be considered that the curative effect of the two groups is different,and the curative effect of the traditional Chinese medicine treatment group is more significant,indicating that the modified Danlou tablet can significantly reduce the plasma levels of IL-6,VCAM-1,MMP-9,Ang-1 and MPO in patients,and the curative effect is more obvious than that of the conventional western medicine treatment group.2)Analysis of therapeutic effect in accordance with the plan setAfter treatment,the plasma levels of IL-6,VCAM-1,MMP-9,Ang-1 and MPO in the two groups were lower than those before treatment(P.Therefore,it can be considered that the curative effect of the two groups is different,and the curative effect of the traditional Chinese medicine treatment group is more significant,indicating that the modified Danlou tablet can significantly reduce the plasma levels of IL-6,VCAM-1,MMP-9,Ang-1 and MPO in patients,and the curative effect is more obvious than that of the conventional western medicine treatment group.(4)Safety analysisThere was no significant difference in the occurrence of all adverse events,the severity of adverse events,the number of cases and the incidence of serious adverse events between the traditional Chinese medicine treatment group and the conventional western medicine treatment group(P>0.05).There was no statistically significant difference between the two groups in the blood routine,liver function,kidney function,urine routine and stool routine safety assessment items at each visit point(P>0.05),and there was no significant change from baseline after treatment(visit 4),and there was no deterioration,suggesting that Danlou tablets had good safety.3.Research results on the m RNA expression of protein biomarkers in stable angina pectoris with phlegm stasis syndrome treated with Danlou tablets(1)Demographic dataIn terms of demographic data of 26 patients,there was no statistically significant difference between the groups(P>0.05),and the baseline was balanced and comparable.(2)Efficacy index evaluationThe modified Danlou tablet can down-regulate the expression of IL-6,VCAM-1,MMP9,MPO,Ang-Ⅰ m RNA in patients with stable angina pectoris with phlegm and blood stasis syndrome,and has statistical significance(P<0.01).Conclusions:1.From the perspective of biological information,it has revealed that inflammatory reaction is the key molecular biological mechanism of coronary heart disease.It is believed that IL-6,VCAM-1,MMP9,MPO,Ang-I may be biomarkers and potential targets of coronary heart disease.2.From the perspective of proteins,it is revealed that IL-6,VCAM-1,MMP9,MPO,Ang-I are the material basis for Danlou Tablets and its flavoring to play an excellent role in TCM differentiation and treatment.From the perspective of m RNA,it is clear that IL-6,VCAM-1,MMP9,MPO,Ang-I is the key target of Danlou Tablets and its flavoring to play a role,and it is inferred that it may be through regulating NF-κ B down-regulates the expression of downstream related inflammatory factors such as IL-6,VCAM-1 and MMP9;It inhibits Ang-Tie2,VEGF/VEGFR signaling pathway by downregulating Ang-Ⅰ and MMP-9,so as to inhibit inflammatory reaction,adjust endothelial function,stabilize plaque,delay the pathological process of atherosclerosis,and play a therapeutic role.3.This study selects the multi-center practical randomized controlled trial method,adopts the design of superior effect,fully reflects the characteristics and advantages of the combination of diseases and syndromes of traditional Chinese medicine and treatment based on syndrome differentiation,and provides a methodological reference for clinical research of traditional Chinese medicine.At the same time,it provides high-quality evidence-based evidence for the differentiation and treatment of stable angina pectoris with phlegm and blood stasis syndrome in traditional Chinese medicine.On the basis of conventional treatment of western medicine,combined with the differentiation and treatment plan of traditional Chinese medicine,it can improve the clinical efficacy and provide the basis for the promotion and application of Danlou tablet and its modified TCM differentiation and treatment plan in clinical practice.