网络药理学预测瓜蒌抗心肌缺血再灌注损伤作用机制及验证研究

Predicting and validating the mechanism of Trichosanthes mediated anti-myocardial ischemia-reperfusion injury by network pharmacology

基于网络药理学及大鼠(本实验所用动物经皖南医学院医学伦理委员会批准同意)心肌缺血再灌注损伤(myocardial ischemia-reperfusion injury,MIRI)模型验证的方法分析瓜蒌抗MIRI的作用机制。依据口服生物利用度(oral bioavailability,OB)≥30%、类药性(drug like,DL)≥0.18,通过TCMSP、TCM Database@Taiwan数据库筛选化合物。利用DRAR-CPI数据库获取化合物的PDB ID值(Z’-score <-0.5),并由UniProt数据库转换成靶点蛋白。通过CooLGeN数据库,以"myocardial ischemia reperfusion injury"为关键词,收集人类基因靶点蛋白。利用DAVID数据库进行与MIRI相关靶点蛋白的GOTERMBPDIRECT富集分析和Kyoto Encyclopedia of Genes and Genomes(KEGG)PATHWAY通路注释分析,使用Gephi0.9.2软件构建成分-靶点蛋白-信号通路网络。以复方丹参滴丸(85.05 mg·kg^-1)为阳性对照,瓜蒌滴丸(0.2、1.0和2.0 g·kg^-1)预处理MIRI大鼠,采用蛋白质免疫印迹(Western blot)法对丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路相关蛋白表达进行分析。网络药理学发现,瓜蒌中schottenol等12个化合物通过多靶点、多生物途径及多通路方式协同发挥抗MIRI作用,涉及ERK2(extracellular regulated protein kinase 2,ERK2)、JNK1 (c-jun-N-terminal kinase-1,JNK1)、p38MAPK等靶点蛋白及MAPK等信号通路。Western blot结果显示,瓜蒌滴丸预处理MIRI大鼠后,MAPK信号通路相关蛋白p-ERK1/2表达呈剂量依赖性上调,p-p38MAPK、p-JNK1表达呈剂量依赖性下调,与模型组比较,中、高剂量组(1.0、2.0 g·kg^-1)的ERK1/2、JNK1及p38MAPK蛋白磷酸化表达均有显著性差异(P<0.01),瓜蒌滴丸可通过调控MAPK信号通路的ERK1/2、JNK1、p38MAPK靶点蛋白及其磷酸化发挥抗大鼠MIRI作用。本文运用网络药理学阐释了瓜蒌抗MIRI的作用靶点和通路并进行了验证,为深入探讨瓜蒌抗MIRI的作用机制提供了依据。

Network pharmacology and rat ischemia-reperfusion injury(MIRI) model was used to analyze the mechanism of cardiac protection by Trichosanthes.The animal experiments were approved by the Medical Ethics Committee of Wannan Medical College.Compounds were screened by TCMSP database and TCM Database@Taiwan according to oral bioavailability(OB>30%) and drug like activity(DL>0.18).The PDBID value of the compound(Z’-score < 0.5) was obtained in DRAR-CPI database and converted into a target protein by UniProt database.Human genes of target proteins were identified using the term " myocardial ischemia reperfusion injury" as the keyword through the CoolGeN database.GOTERM_BP _ DIRECT enrichment analysis of target proteins related to MIRI and KEGG PATHWAY annotation analysis were performed using the DAVID database.The component-target protein-signal pathway network was constructed using Giphi0.9.2 software.The expression of mitogen-activated protein kinase(MAPK) signaling pathway-related proteins in MIRI rats pretreated with Trichosanthes(0.2,1.0 and 2.0 g · kg^-1) was analyzed by Western blot with compound Danshen(85.05 mg · kg^-1) as a positive control.Network pharmacology found that 12 compounds,including schottenol in Trichosanthes,synergistically inhibit MIRI through multiple targets or biological pathways,involving target proteins such as extracellular regulated protein kinase 2(ERK2),c-jun-N-terminal kinase-1(JNK1) and p38 MAPK in MAPK signaling pathways.Western blot results showed that phosphorylation of ERK1/2 was dose-dependently up-regulated in MIRI rats pretreated with Trichosanthes,while the level of p38 MAPK or JNK1 phosphorylation was down-regulated in a dose-dependent manner.Compared with the control group,phosphorylation of ERK1/2,JNK1 and p38 MAPK protein showed significant difference in medium and high dose groups(1.0 and 2.0 g·kg^-1)(P<0.01).Therefore,Trichosanthes could play an anti-MIRI role by regulating phosphorylation of ERK1/2,JNK1 and p38 MAPK proteins in rats.In conclusion,the targets and pathways of Trichosanthes on anti-MIRI were revealed by network pharmacology and verified in rat MIRI model,providing the scientific basis for further study on the mechanism of Trichosanthes for cardiac protection.