MMP9介导的活血化瘀药对丹参-川芎反向调节血管新生作用物质基础揭示

Revealing the material basis of MMP9-mediated activating blood and removing blood stasis drugs on Danshen-Ligusticum chuanxiong anti-vascular effect

目的:揭示活血化瘀药对丹参-川芎反向调节血管新生作用药效物质基础及作用机制。方法:在中药系统药理学数据库(TCMSP)中搜索出冠心宁注射液中丹参和川芎两种中药的有效成分,并在Swiss Target Prediction数据库中检索药物作用靶点;在疾病-基因数据库(DisGeNET)中检索促进血管新生靶点和抑制血管新生靶点;使用蛋白质相互作用数据库(STRING)数据库和Cytoscape软件分析网络核心靶点;使用DAVID数据库对药物-疾病靶点交集进行基因本体(gene ontology,GO)功能富集分析与京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析;使用SYBYL-X 2.0软件进行分子对接分析;RT-qPCR实验验证木犀草素对MMP9基因表达的影响;体外小管形成实验分析木犀草素对内皮细胞新血管生成的作用。结果:经分析得出丹参有10个有效成分,50个血管新生作用靶点;川芎有2个有效成分,4个血管新生作用靶点。取药靶交集得出9个作用靶点,包括AR、PARP1、MMP9、MMP2、MMP12、AKR1B10、ABCC1、CDK6、STAT3。经过丹参/川芎活性成分-靶点网络图分析,经分析得出木犀草素是关键化学成分。经蛋白相互作用分析,结果显示MMP9是核心靶点。KEGG通路主要富集在5条信号通路,最主要的通路是癌症相关通路。GO生物学过程主要有22条,主要涉及胶原分解代谢过程、蛋白质细胞外基质、金属内肽酶活性等,经小管形成实验分析初步得出木犀草素抑制血管新生并下调MMP9基因的表达。结论:丹参-川芎药对的关键化学成分木犀草素通过调节MMP9靶点抑制血管新生作用,为活血化瘀中药在已知治疗缺血性疾的基础上开拓了反向调节血管新生作用,进而为丹参-川芎药对在临床癌症病人的使用提供新的视角和理论依据。

Objective:To reveal the material basis and mechanism of angiogenesis effect of Danshen-chuanxiong herb-part⁃ners.Methods:The effective components of Danshen and Chuanxiong were searched in the TCM System Pharmacology Database(TCMSP),and the drug targets were searched in the Swiss Target Prediction database;the Disease-Gene Database(DisGeN⁃ET)was used to search for angiogenesis-promoting and inhibiting angiogenesis targets;the protein interaction database(STRING)database and Cytoscape software were used to analyze the network core targets;the DAVID database to enrich the drug-disease target intersection for GO and KEGG;SYBYL-X 2.0 software was used to analysis the molecular docking;RT-qP⁃CR experiments verified the effect of luteolin on MMP9 gene expression;in vitro tube formation experiments analyzed the effect of luteolin on endothelial cell neovascularization.Results:After analysis,it was found that Danshen had 10 active ingredients and 50 angiogenesis targets;Chuanxiong had 2 active ingredients and 4 angiogenesis targets.The intersection of drug-taking targets yielded 9 targets,including AR,PARP1,MMP9,MMP2,MMP12,AKR1B10,ABCC1,CDK6,and STAT3.After analyz⁃ing the active ingredient-target network graph of Danshen/Chuanxiong,it was concluded that luteolin was the key chemical ingredi⁃ent.After protein interaction(PPI)analysis,the results showed that MMP9 was the core target.The KEGG pathway was mainly enriched in 5 signaling pathways;the most important pathway was cancer-related pathways.There were 22 main biological pro⁃cesses of GO,which mainly involved collagen catabolism,protein extracellular matrix,metal endopeptidase activity,etc.The pre⁃liminary analysis of tubule formation experiment showed that luteolin inhibited angiogenesis and down-regulated the expression of MMP9 gene.Conclusion:Luteolin,the key chemical component of the Danshen-Ligusticum Chuanxiong drug pair,inhibits angio⁃genesis by regulating the MMP9 target,and develops the reverse regulation of angiogenesis for the traditional Chinese medicine for promoting blood circulation and removing blood stasis on the basis of the known treatment of ischemic diseases,which provides a new perspective and theoretical basis for the use of Danshen and Chuanxiong medicine in clinical cancer patients and reveals the material basis and mechanism of angiogenesis effect of Danshen-chuanxiong herb-partners.