Mechanism Of Xuefu Zhuyu Decoction In Treating Coronary Heart Disease Based On Network Pharmacology And Transcriptome Sequencing

Objective:To screen the therapeutic targets of Xuefu Zhuyu Decoction for intervention of coronary heart disease by network pharmacological methods,looking for pivot genes,network modules and co-expressed genes in the therapeutic target interaction network,predicting significant biological processes and pathways.To screen differentially expressed genes of Xuefu Zhuyu Decoction intervention of coronary heart disease by transcriptome and sequencing and bioinformatics verification.To further explain the possible mechanism of Xuefu Zhuyu Decoction in treating coronary heart disease.Method:The active ingredients and important targets of Xuefu Zhuyu Decoction were initially determined with the help of SymMap database,GeneCards and CHD@ZJU databases were integrated to screen for coronary heart disease-related targets;Cytoscape software was used to construct a Chinese medicine component-target network and protein interaction network.By using CytoHubba,MCODE tools and Coexpedia database we analyzed the network,and hub genes,network modules,and co-expressed genes were screen out.Through Metascape database analysis the Xuefu Zhuyu Decoction’s therapeutic targets towards coronary heart disease,the significant biological processes and enrichment pathways were extracted out.The combination of transcriptomics and next-generation sequencing was used to select Xuefu Zhuyu Decoction to interfere with the clinical research of coronary heart disease in before-treatment and after-treatment.Four peripheral blood samples were collected,after leukocyte isolation,total RNA extraction,cDNA library construction,genomic sequence alignment,differential quantification,and screening for differentially expressed genes through bioinformatics methods.Compared prediction results of the network pharmacology with the results of the clinical analysis to the differentially expressed genes,therapeutic mechanism of Xuefu Zhuyu Decoction on coronary heart disease was reasonably inferred.Results:(1)The study found that a total of 1839 compounds in Xuefu Zhuyu Decoction were obtained by searching the database and a total of 800 active ingredients in under the conditions of oral bioavailability>30%,of which Semen Pruni Persicae has 33 active ingredients,70 active ingredients in Safflower,26 active ingredients in Rehmannia glutinosa,70 active ingredients in Radix Paeoniae Rubra,74 active ingredients in Angelica sinensis,116 active ingredients in Ligusticum wallichii,and 11 active ingredients in Fructus Aurantii,27 active ingredients in Platycodon grandifloras,182 active ingredients in Radix Bupleuri,49 active ingredients in Achyranthes bidentata,and 142 active ingredients in Licorice(2)The study obtained 2124 drug targets in Xuefu Zhuyu Decoction.Among them,Semen Pruni Persicae has 124 potential targets,Safflower has 307 potential targets,Rehmannia glutinosa has 84 potential targets,and Radix Paeoniae Rubra has 202 potential targets,Angelica sinensis has 84 potential targets,Ligusticum wallichii has 175 potential targets,Fructus Aurantii has 78 potential targets,Platycodon grandifloras has 147 potential targets,Radix Bupleuri has 365 potential targets,Achyranthes bidentata has 271 potential targets and Licorice has 286 potential targets.The important targets were screened on the condition of P value<0.05,and the results were as follows:72 important targets in Semen Pruni Persicae,224 important targets in Safflower,36 important targets of Rehmannia glutinosa,and 126 important targets of Radix Paeoniae Rubra.There are 28 targets in Angelica sinensis,88 important targets in Ligusticum chuanxiong,59 important targets in Fructus Aurantii,79 important targets in Platycodon grandiflora,218 important targets of Radix Bupleuri,146 important targets in Achyranthes bidentate and 167 important targets of Licorice.(3)In this study 6785 coronary heart disease targets were obtained,of which 1,436 were coronary heart disease targets of homo sapiens,and 251 were the intersection targets of Xuefu Zhuyu Decoction and coronary heart homosapiens targets.(4)In this study,15 core targets in the Chinese Medicine Ingredients-Drug targets network of Xuefu Zhuyu Decoction through network topological analysis.Protein-protein interaction network analysis of the therapeutic targets,we got 32 targets with a very-high degree of network distance centrality,which are INS,IL6,GAPDH,TNF,VEGFA,TP53,MAPK3,PTGS2,JUN,EGFR,MAPK8,EGF,STAT3,MAPK1,SRC,IL1B,MYC,CXCL8,MMP9,CCL2,FN1,IL10,NOS3,FOS,ESR1,CCND1,PPARG,ACTB,FGF2,PTEN and MMP2.(5)In this study the enrichment analysis results of therapeutic targets showed that biological processes mainly involved such as signal transduction,intercellular communication,energy pathways,metabolism,and immune response(P<0.01):molecular functions mainly involved transcription factor activity,catalytic activity,cytokine activity(P<0.01);Cell components part mainly involved extracellular,cytoplasmic,extracellular space,and extracellular area(P<0.05).Eight core signaling pathways are manifested including PI3K-Akt signaling pathway,HIF-1 signaling pathway,TNF signaling pathway,FoxO signaling pathway,NF-κB signaling pathway,ErbB signaling pathway,Jak-STAT signaling pathway and AMPK signal pathway(P<0.01).Thirteen modules with similar biological functions were obtained from the network module analysis,and the largest module gene group was composed of 12 genes and 78 edges.The Top 10 hub genes were identified based on the MCC method,including VEGFA,GAPDH,IL6,TNF,PTGS2,MMP9,MARK3,INS,FN1 and TP53.Gene co-expression analysis revealed that 14 co-expressed genes with the same expression profile as coronary heart disease;they are PLG,CYP1A2,PON1,CYP2E1,CYP2C8,CTH,CRP,XDH,TGM2,GSTM2,GSTM1,C5AR1,RAF1 and BCL2L1.(6)By transcriptomic sequencing the differentially expressed genes of Xuefu Zhuyu Decoction intervention in coronary heart disease were determined.The results are as follows:2 groups after treatment included 2832 differentially expressed genes before and after treatment,of which 1747 genes were up-regulated and 1085 genes were down-regulated;There were 2656 differentially expressed genes in the two groups before treatment,of which 1045 genes were up-regulated and 1611 genes were down-regulated.There were 2410 differential genes in the group before treatment and 1 group after treatment,1315 genes were up-regulated,and 1095 genes were down-regulated genes.There were 2737 differentially expressed genes in group 1 compared with group 1 after treatment,of which 1579 were up-regulated and 1158 were down-regulated genes.(7)Functional enrichment analysis of differentially expressed genes in treatment-before and treatment after Xuefu Zhuyu Decoction intervention with western medicine basic therapy showedd that 10 significant biological processes involved they are inflammatory response,immune response,positive regulation of endothelial cell proliferation,positively regulates histone H3-K4 methylation,positively regulate histone H3-K9 methylation,positively regulate T helper cell differentiation,response to mechanical stimulation,regulation of dendritic spine morphogenesis,calcium-mediated signals,cell response to extracellular stimuli(P<0.05).There are four significant signaling pathways involved,PI3K-Akt signaling pathway,Wnt signaling pathway,Fc epsilon RI signaling pathway,and B cell receptor signaling pathway(P<0.01).Differentially expressed genes after western medicine basic treatment and Xuefu Zhuyu Decoction intervention after western medicine basic treatment involved inflammatory response,drug metabolism,peptidyl-glutamic acid carboxylation,positive feedback of inflammatory response,neutrophil chemotaxis,nitric oxide biosynthesis process,lipopolysaccharide signal transduction,positive regulation of NF-κB entry into the nucleus,natural killer cell chemotaxis and positive regulation of histone H3-K9 methylation biological processes significantly(P<0.016).Six core signaling pathways were manifested in the KEGG pathway enrichment,they are MARK signaling pathway,Nod-like receptor signaling pathway,NF-κB signaling pathway,p53 signaling pathway,Toll-like receptor signaling pathway,TNF signaling pathway(P<0.01).By comparison,the significant biological process both appeared in two main group which Xuefu Zhuyu Decoction interfered are the inflammatory response.By comparing therapeutic targets obtained from the network pharmacology section and transcriptome sequencing,the differential expressed genes in before and after the intervention of Xuefu Zhuyu Decoction plus Western medicine,22 common targets were found which are CXCL10,MPO,MYC,PLAT,PTGS2,CCL2,CCL5,HNF1A,THBD,TNF,TRPC3,VEGFA,CD163,BIRC5,C5AR1,CDKN1A,CH13L1,FOS,JUN,PLAU,SLC6A4 and HK2.Comprehensive analysis have shown that the mechanism of Xuefu Zhuyu Decoction intervention with coronary heart disease is possibly related to inhibition of inflammatory response,anti-platelet aggregation,protecting vascular endothelium,regulation of apoptosis and promotion of immune response and immunological stress.The mechanism of Xuefu Zhuyu Decoction treating on coronary heart disease is significantly related to NF-κB signaling pathway,complement and coagulation cascades,cytokine-cytokine receptor interaction,and TNF signaling pathway.Conclusion1)The mechanism of Xuefu Zhuyu Decoction intervention in coronary heart disease involves multiple targets,multiple processes and multiple pathways.2)Combining network pharmacology and the results of bioinformatics differential expression analysis though transcriptomics sequencing technology,both results jointly revealed:the mechanism of Xuefu Zhuyu Decoction intervention in coronary heart disease have close relationship with mechanism of inhibition of inflammatory response,anti-platelet aggregation,protecting vascular endothelium,regulating cell proliferation,differentiation,apoptosis process and regulating immunological stress.3)The mechanism of Xuefu Zhuyu Decoction intervention of coronary heart disease is significantly related to NF-κB signaling pathway,complement and coagulation cascades,cytokine-cytokine receptor interaction,and TNF signaling pathway.