基于网络药理学和实验验证的丹参-红花药对治疗心肌缺血的作用机制探讨

Study on mechanism of Salvia miltiorrhiza-Carthamus tinctorius in treatment of myocardial ischemia based on network pharmacology and experimental verification

目的通过网络药理学和动物实验探究丹参-红花药对治疗心肌缺血的潜在作用和机制。方法检索中药系统药理学数据库与分析平台(TCMSP)和文献手动补充确定丹参-红花的活性成分及治疗心肌缺血靶点。以“心肌缺血”及“急性心肌缺血”为关键词,在CTD、DisGeNET、GeneCards和OMIM数据库中检索疾病的潜在靶点。利用Venny 2.1.0将筛选得到的有效化学成分靶点与疾病靶点进行交集,确定丹参-红花药对治疗心肌缺血的作用靶点。将交集靶点信息上传至STRING v11.0,构建活性成分-靶点网络及蛋白质相互作用网络(PPI)。通过DAVID数据库对丹参-红花治疗心肌缺血的靶点进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路的富集分析。40只SD雄性大鼠,根据体质量随机分成4组,每组10只,即对照组、模型组、复方丹参滴丸(73 mg·kg^(−1)·d^(−1))组、丹参-红花(生药量16 g·kg^(−1)·d^(−1))组。对照组和模型组大鼠每天ig等量0.9%氯化钠溶液,给药组ig相应剂量的药物,每天1次,共7 d。于第6天,除对照组外,其余各组给药1 h后sc异丙肾上腺素(ISO,85 mg·kg^(−1)),连续2 d造模。HE染色观察各组大鼠心肌组织病理变化;蛋白免疫印迹法检测各组大鼠心肌组织磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、磷酸化转录激活蛋白3(p-STAT3)、缺氧诱导因子1α(HIF-1α)、血管内皮生长因子A(VEGFA)、血小板衍生生长因子A(PDGFA)和碱性成纤维细胞生长因子(bFGF)蛋白表达水平。结果网络药理学筛选得到丹参-红花药对治疗心肌缺血的活性成分91个,靶点246个。PPI网络结果显示主要靶点包括STAT3、MAPK1、JUN、RELA、MAPK3、AKT1、SRC、APP、TNF、PIK3CA、CXCL8、KNG1、IL6、VEGFA、MAPK14、HSP90AA1、MAPK8、EGFR、FOS和IL2等。主要靶点相关的通路涉及血管生成(如HIF-1信号通路和VEGF信号通路)、炎症反应(如NF-κB信号通路和TNF信号通路)和细胞凋亡(如细胞凋亡信号通路和p53信号通路)等。动物实验HE染色结果表明,对照组大鼠心肌细胞正常;模型组大鼠心肌纤维肿胀且有断裂,心肌细胞增宽,局部有明显水肿、渗出、炎性细胞浸润;复方丹参滴丸组与模型组大鼠比较,心肌纤维排列略微紊乱且较窄,病理改变程度有所减轻。丹参-红花组大鼠的心肌纤维排列整齐,细胞形态结构完整,心肌纤维肿胀轻,无毛细血管扩张,接近正常心肌形态。蛋白质免疫印迹检测结果表明,与对照组比较,模型组大鼠心肌组织中HIF1α、VEGFA、PDGFA和bFGF的蛋白表达水平显著升高(P<0.05、0.01),p-mTOR和p-STAT3的蛋白表达水平显著下降(P<0.05、0.01)。与模型组比较,丹参-红花显著上调了p-mTOR、p-STAT3、HIF-1α、VEGFA、PDGFA和bFGF的蛋白表达水平(P<0.05、0.01)。结论丹参-红花可能通过上调与血管生成密切相关的蛋白水平来促进急性心肌缺血模型大鼠缺血心肌组织的血管生成,从而改善其缺血性损伤。

Objective To explore the potential effect and mechanism of Salvia miltiorrhiza-Carthamus tinctorius on myocardial ischemia through network pharmacology and animal experiments.Methods The pharmacological database and analysis platform(TCMSP)of the traditional Chinese medicine system and the literature were searched and manually supplemented to determine the active components of S.miltiorrhiza-C.tinctorius and the therapeutic target of myocardial ischemia.With"myocardial ischemia"and"acute myocardial ischemia"as the keywords,search the potential targets of diseases in CTD,DisGeNET,GeneCards and OMIM databases.Venny 2.1.0 was used to intersect the screened effective chemical component target with the disease target,and determine the target of S.miltiorrhiza-C.tinctorius for the treatment of myocardial ischemia.Upload the intersection target information to STRING v11.0 to build the active component-target network and protein-protein interaction(PPI)network.The DAVID database was used to enrich and analyze the Gene Ontology(GO)function and the Kyoto gene and genome encyclopedia(KEGG)pathway of the target of S.miltiorrhiza-C.tinctorius in the treatment of myocardial ischemia.Forty SD male rats were randomly divided into four groups according to their body mass,10 in each group,namely,control group,model group,Compound Salvia Miltiorrhiza Dropping Pills(73 mg·kg^(−1)·d^(−1))group,and S.miltiorrhiza-C.tinctorius(16 g·kg^(−1)·d^(−1))group.The rats in the control group and model group were given the same amount of 0.9%sodium chloride solution once a day for seven days.On the 6th day,except for the control group,the rats in other groups were administered with isoproterenol(ISO,85 mg·kg^(−1))by sc for two days.HE staining was used to observe the pathological changes of myocardial tissue of rats in each group.Detection of p-mTOR,p-STAT3,HIF-1α,VEGFA,PDGFA and bFGF protein expression level in myocardial tissue of rats in each group by Western blotting.Results 91 active components and 246 targets of S.miltiorrhiza-C.tinctorius were screened by network pharmacology.PPI network results show that the main targets include STAT3,MAPK1,JUN,RELA,MAPK3,AKT1,SRC,APP,TNF,PIK3CA,CXCL8,KNG1,IL6,VEGFA,MAPK14,HSP90AA1,MAPK8,EGFR,FOS and IL2.The main target-related pathways involve angiogenesis(such as HIF-1 signal pathway and VEGF signal pathway),inflammatory response(such as NF-κB signal pathway and TNF signal pathway)and apoptosis(such as apoptosis signal pathway and p53 signal pathway).The results of HE staining in animal experiments showed that the myocardial cells in the control group were normal.In the model group,the myocardial fibers were swollen and the area was broken,the myocardial cells were widened,and there were obvious edema,exudation and inflammatory cell infiltration in the local area.Compared with the model group,the arrangement of myocardial fibers in the Compound Danshen Dropping Pills group was slightly disordered and narrow,and the degree of pathological changes was alleviated.The myocardial fibers of the rats in the S.miltiorrhiza-C.tinctorius group were arranged in order,the cell morphology and structure were complete,the myocardial fibers swelled slightly,and there was no telangiectasia,which was close to the normal myocardial morphology.The result of Western blotting showed that compared with the control group,the protein expression levels of HIF-1α,VEGFA,PDGFA and bFGF in the myocardial tissue of rats in the model group were significantly increased(P<0.05,0.01),while the protein expression levels of p-mTOR and p-STAT3 were significantly decreased(P<0.05,0.01).Compared with the model group,S.miltiorrhiza-C.tinctorius significantly up-regulated the protein expression level of p-mTOR,p-STAT3 and HIF-1α,VEGFA,PDGFA and bFGF(P<0.05,0.01).Conclusion S.miltiorrhiza-C.tinctorius may promote the angiogenesis of ischemic myocardium in rats with acute myocardial ischemia by up-regulating the protein level related to angiogenesis,thus improving the ischemic injury.