| More and more studies show that the treatment mode of combining disease and syndrome of traditional Chinese medicine plays a significant role in the prevention and treatment of coronary heart disease and its complications.The domestic scholars found that the most common syndrome factors in patients with coronary heart disease angina were blood stasis and phlegm turbidity.Therefore,phlegm and blood stasis are common syndrome elements of coronary heart disease,and they are often combined with each other.According to the expert consensus on TCM diagnosis and treatment ofstable angina pectoris of coronary heart disease,Danlou tablet,a Chinese patent medicine,can be used for patients with phlegm and blood stasis syndrome.Modern research shows that Danlou tablet has beneficial clinical effects on patients with coronary heart disease of phlegm and blood stasis,which can regulate blood lipid metabolism system,improve atherosclerosis and restore myocardial blood supply.Objective:1.To construct the "Component-Target" network of Danlou tablet in the intervention of stable angina pectoris.2.Cluster analysis of the mechanism of Danlou tablet with different properties of "cold,cool and warm".3.Clinical research to obtain the differential genes of Danlou tablet before and after the intervention of stable angina pectoris,to conduct bioinformatics analysis and verify the pharmacological prediction network.Methods:1."Component-Target" network prediction of Danlou tablet in treating stable angina.Through the database and systematic literature search,the components of Danlou tablet were collected.TCMSP,TCMID database and literature were used to screen the pharmacological targets,and the "Component-Target" network was constructed by using Cytoscape software.STRING6 and other databases were used for protein-protein interaction mapping.The core nodes were identified with MCODE algorithm,and the PPI network function was annotated.2.Classification function of four properties in Danlou tablet.In the "Component-Target" database of Danlou tablet,the targets of active components in cold,cool and warm herbs were extracted respectively,and input into Webgestalt bio-analysis tool.The human genome was selected as the species,and the visual analysis of KEGG and GO(BP,CC,MF)projects was carried out by using ORA enrichment method.3.Differential gene detection of Danlou tablet in the treatment of stable angina with phlegm and blood stasis syndrome.In the experimental group and the control group,5 patients were selected,4 ml blood samples were collected before and after the intervention for high-throughput RNA sequencing.The differential gene expression profile of Danlou tablet in the treatment of stable angina with phlegm and blood stasis syndrome was found and unified into gene symbol format.Then,the genes were mapped to the predicted component targets to obtain the intersection,and carrying out biological annotation and pathway analysis.Results:1.A total of 190 active components of Danlou tablet were retrieved from TCMSP database.According to Chinese Pharmacopoeia and literature retrieval,57 components were added.After comparing the supplementary data with the data retrieved from TCMSP database,the research results obtained active components of Danlou tablet(15 ingredients of Gualou,13 ingredients of Xiebai,17 ingredients of Gegen,17 ingredients of Chuanxiong,80 ingredients of Danshen,36 ingredients of Chishao,13 ingredients of Zexie,26 ingredients of Huangqi,20 ingredients of Gusuibu and 17 ingredients of Yujin).Through TCMSP,TCMID database and literature search,the pharmacological targets of active components of Danlou tablet were screened out.After removing the repetitive values,a total of 369 component targets were obtained.OMIM,Drugbank,GeneCards,TTD and DisGeNET databases were used to search for genes related to stable angina.After data deduplication,there are 3385 disease targets.292 genes were obtained from the intersection of the component targets of Danlou tablet and the disease targets of stable angina.The "Component-Target" network was visualized by using Cytoscape software.According to the node degree value screening,the top five components were quercetin,daidzein,luteolin,kaempferol and puerarin.The degree values of the above components were more than 50,which were related to multiple disease targets.So they may be the main active components of Danlou tablet in the intervention of stable angina.Targets of the top five were PTGS1(Prostaglandin-Endoperoxide Synthase 1),PTGS2(Prostaglandin-Endoperoxide Synthase 2)and HSP90AA1(Heat Shock Protein 90 Alpha Family Class A Member 1),NCOA2(Nuclear Receptor Coactivator 2),ADRB2(Adrenoceptor Beta 2).The degree values of the above targets are more than 40,which were related to multiple herb components,and may be the core node in the pharmacological network of Danlou tablet in the intervention of stable angina.Based on 292 genes of the intersection of component targets and disease targets,we mapped the protein-protein interaction relationship of the above genes through the Metascape platform,and selected 7 representative MCODE modules for visual analysis.This study found that Danlou tablet may have many pharmacological effects,such as anti-virus infection,protection of spinal cord injury,regulation of steroid hormone metabolism,binding with GPCR ligand,mediating anti-inflammatory cytokines,transport regulation of growth factors,activation of neutrophils and so on.2.According to the Chinese pharmacopoeia in 2020 about the composition of Danlou tablet,the medicines of Danlou tablet were classified according to their properties.The severe cold,cold and slightly cold are unified into cold,and the warm and slightly warm are all classified into warm.This study found that there have cold herbs(Gualou,Zexie,Yujin,Danshen,Chishao),cool herb(Gegen),warm herbs(Xiebai,Chuanxiong,Gusuibu,Huangqi).Besides,there has no heat properties herb.Based on the results of the second part study,104 active components of cold herbs in Danlou tablet were extracted from the literature and TCMSP database,and 247 component targets of cold herbs were obtained;Among the 12 active components of cool herb,159 targets were found;Among the 58 active components of warm herbs,296 targets were found.The targets of active components were input into Webgestalt biochemical analysis tool respectively in cold herb,cool herb,warm herb.The species was selected as the human genome,and the enrichment of KEGG and GO pathway was carried out by ORA enrichment method,and then visualized analysis.The results showed that cold herbs and cool herb in Danlou tablet had the same function "fluid shear stress and atherosclerosis" on KEGG top 10 pathway,which may be related to the fact that both have the function of assisting blood circulation.Besides,cold herbs and warm herbs all act on IL-17 signaling pathway.The results of GO top 10 enrichment analysis showed that the biological processes of cold herbs and warm herbs all acted on the inflammatory and immune regulation,stress response and defense response,cell activation and secretion.Different from the other two groups,cool herb includes positive regulation of multicellular biological process,positive regulation of catalytic activity,positive regulation of cell migration,negative regulation of stimulation response,regulation of homeostasis process and intracellular signal transduction.In cell composition,all three groups were associated with neuronal projection,membrane raft and plasma membrane.Among them,cold and warm herbs are also involved in the dendritic compartment,which is the part of neurons that mainly receive signals.In molecular function,cold herbs and cold herb are related to neurotransmitter receptor activity,G protein coupled amine receptor activity,protein kinase binding and protein homodimeric activity.However,only warm drugs have nuclear receptor activity.3.10 subjects were included in the clinical study,and there was no significant difference between the two groups before the intervention.During the experiment,one patient in the control group drops out.After 4 weeks of drug intervention,a total of 5 samples in the experimental group and 4 samples in the control group were obtained.According to Efficacy Evaluation Criteria for Coronary Heart Disease Angina Pectoris with Traditional Chinese Medicine Treatment in 2018,research showed that there was no significant difference between the experimental group and the control group before intervention(P>0.05),but there had significant difference between the experimental group and the control group after intervention(P<0.05).After high-throughput sequencing,33 genes(confounding factors)in the experimental group and control group were removed,and 64 genes(52 up-regulated and 12 down-regulated)of the Danlou tablet were left.Volcano maps were drawn to see the overall distribution of different genes before and after treatment in two groups.Top of Go analysis showed that in the experimental group,the down-regulated items increased the "rhythm process" compared with the up-regulated items;In cell composition,,,synapse" is unique to down-regulation,while "extracellular matrix" is unique to up-regulation;Among the molecular functions,the up-regulated items increased "enzyme regulation activity",’molecular sensor activity","nucleic acid binding transcription factor activity" "protein binding transcription factor activity" and "receptor regulation activity" compared with the down-regulated items.KEGG top20 analysis showed that the experimental group increased "epithelial cells signaling in Helicobacter pylori infection","bacterial invasion of epithelial cells","regulation of actin cytoskeleton","hematopoietic cell lineage","tight junction","adhesion junction" and "EGFR tyrosine kinase inhibitor resistance" compared with the control group.Four genes(EGF,F3,MET,MGST1)were mapped by comparing the 64 genes obtained by high-throughput sequencing with the predicted composition targets database(369 targets)of Danlou tablet.Oral utilization of the components from above genes was all greater than 30,suggesting that high oral utilization may be the basis of drug efficacy.The results showed that only EGF and F3 were found also to be disease targets.In conclusion,high-throughput sequencing results verified some nodes in the "Component-Target" prediction network.Conclusion:1.Danlou tablet has a complex multi-target pharmacological mechanism in the intervention of stable angina pectoris.The core drug components may be related to flavonoids,and the main drug targets may be related to genes such as inflammation.2.In terms of drug commonality,KEGG top10 showed that cold and cool herbs in Danlou tablet act on the pathway "fluid shear stress and atherosclerosis",and its principle may be related to the effect that both have the function of assisting blood flow.Besides,both cold and warm herbs act on IL-17 signaling pathway.3.The results of high-throughput sequencing showed that quercetin may be one of the active components of Danlou tablet in the treatment of stable angina pectoris in coronary heart disease,and the main targets include EGF and F3,which is partially consistent with the results of "component target" prediction network. |
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