基于网络药理学、分子对接及实验验证探讨参附益心颗粒治疗心力衰竭的作用机制

Exploring the Mechanism of Action of Shenfu Yixin Granules in the Treatment of Heart Failure Based on Network Pharmacology,Molecular Docking and Experimental Verification

目的基于网络药理学、分子对接及实验验证探讨参附益心颗粒治疗心力衰竭的作用机制。方法(1)通过中药系统药理学数据库与分析平台(TCMSP)检索、筛选参附益心颗粒组方中药的有效活性成分;利用PubChem数据库、Swiss Target Prediction平台进行作用靶点预测;通过GeneCards、OMIM数据库检索、筛选心力衰竭疾病相关靶点;通过Venny 2.1.0平台对参附益心颗粒活性成分作用靶点与心力衰竭疾病相关靶点取交集(共同靶点),即为参附益心颗粒抗心力衰竭的潜在作用靶点。将潜在作用靶点导入STRING数据库,构建蛋白互作(PPI)网络,筛选参附益心颗粒治疗心力衰竭的核心靶点。通过Cytoscape 3.6.1软件构建“药物-活性成分-疾病-靶点”网络,筛选参附益心颗粒治疗心力衰竭的关键活性成分。利用DAVID数据库对潜在作用靶点进行GO功能及KEGG通路富集分析。利用AutoDock Vina软件对关键活性成分和核心靶点进行分子对接验证。(2)将SD大鼠随机分为假手术组、模型组、参附益心颗粒组(5.28 g·kg^(-1))及阳性药组(沙库巴曲缬沙坦组,20.8 mg·kg^(-1)),每组8只。采用结扎左冠状动脉前降支的方法复制急性心肌梗死后心力衰竭大鼠模型。灌胃给药,每日1次,连续4周。采用超声心动图检测大鼠心功能:左室射血分数(LVEF)、左室短轴缩短率(LVFS);HE、Masson染色法观察大鼠心脏组织病理变化;ELISA法检测血清脑钠素(BNP)、心钠肽(ANP)、醛固酮(ALD)水平;qPCR及Western Blot法检测心脏组织CAV1、F2、MAPK1 mRNA及蛋白表达水平。结果(1)共获得参附益心颗粒的活性成分210个,作用靶点1196个,心力衰竭疾病相关靶点801个;通过Venny 2.1.0平台取交集,共得到参附益心颗粒治疗心力衰竭的潜在作用靶点(共同靶点)97个。通过“药物-活性成分-疾病-靶点”网络分析得到槲皮素、木犀草素、花生四烯酸、山柰酚、丹参醛等关键活性成分。通过潜在作用靶点的PPI网络分析得到MAPK1、F2、CAV1、EDN1、GJA1等核心靶点。潜在作用靶点主要集中在基因表达的正向调节、心脏发育及对MAPK级联的正向调节等生物过程,涉及MAPK信号通路、HIF-1信号通路和PI3K/Akt等关键通路。MAPK1、CAV1、EDN1、F2与槲皮素、木犀草素、山柰酚、丹参醛以及MAPK1、F2与花生四烯酸均具有较好的结合活性。(2)与假手术组比较,模型组大鼠的LVEF、LVFS显著降低(P<0.01),心脏质量指数明显升高(P<0.05);心肌组织病理损伤明显,间质纤维化程度严重,心脏胶原容积分数显著升高(P<0.01);血清BNP、ANP、ALD水平显著升高(P<0.01);心肌组织中CAV1、F2、MAPK1 mRNA及蛋白表达水平显著升高(P<0.05,P<0.01)。与模型组比较,参附益心颗粒组、阳性药组大鼠的LVEF、LVFS均显著升高(P<0.01),但是心脏质量指数下降不明显(P>0.05);心肌组织病理损伤明显改善,间质纤维化程度明显减轻,心脏胶原容积分数显著降低(P<0.01);血清BNP、ANP、ALD水平显著降低(P<0.01);心肌组织中CAV1、F2、MAPK1 mRNA及蛋白表达水平显著下降(P<0.05,P<0.01)。结论参附益心颗粒可能是通过槲皮素、木犀草素、花生四烯酸、山柰酚、丹参醛等活性成分,作用于MAPK1、F2、CAV-1、EDN1等靶点,调控MAPK信号通路、PI3K/Akt信号通路等相关通路,从而改善心力衰竭大鼠的心功能及心肌纤维化。

Objective This study aims to examine the potential mechanism of Shenfu Yixin Granules on heart failure(HF)based on network pharmacology,molecular docking,and experimental verification.Methods(1)The active components of herbs in Shenfu Yixin Granules were screened and retrieved through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).PubChem database and Swiss Target Prediction platform were used to predict targets.GeneCards and OMIM databases were used to screen HF-related targets.The intersection of active ingredient targets of Shenfu Yixin Granules and HF-related targets was performed by using Venny 2.1.0 platform to obtain common targets,which were the potential targets for anti-HF effect of Shenfu Yixin Granules.The potential targets were imported into the STRING database to construct a protein-protein interaction(PPI)network and screen the core targets of Shenfu Yixin Granules for the treatment of HF.GO functional and KEGG pathway enrichment analysis of potential targets were carried out by using DAVID database.AutoDock Vina software was used for molecular docking validation of key active ingredients and core targets.(2)SD rats were randomly allocated into sham operation group,model group,Shenfu Yixin Granules(5.28 g·kg^(-1))group,and positive control group(sacubitril-valsartan,20.8 mg·kg^(-1)),with eight rats in each group.A rat model of HF after myocardial infarction was established by ligating the left anterior descending coronary artery.The rats were subsequently administered orally with the corresponding drugs once daily for a period of four weeks.Cardiac function including left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)in rats was assessed by echocardiography.Additionally,the histopathological alterations in rat heart tissue were examined using hematoxylin-eosin(HE)staining and Masson staining.The serum levels of brain natriuretic peptide(BNP),artial natriuretic peptide(ANP),and aldosterone(ALD)were determined by enzyme-linked immunosorbent assay(ELISA).Furthermore,real-time quantitative PCR and Western Blot were employed to detect mRNA and protein expressions of CAV1、F2 and MAPK1 in heart tissue.Results(1)A total of 210 active ingredients and 1196 targets of Shenfu Yixin Granules,as well as 801 HF-related targets were obtained.Venny 2.1.0 platform was used to acquire 97 potential targets(common targets)of Shenfu Yixin Granules for the treatment of HF.Key active ingredients,such as quercetin,luteolin,arachidonic acid,kaempferol,and tanshinaldehyde were screened by“drugs-active ingredients-disease-targets”network analysis.The core targets including MAPK1、F2、CAV1、EDN1 and GJA1 were identified through PPI network analysis.The potential targets are mainly concentrated in multiple biological processes,namely,the positive regulation of gene expression,cardiac development,and the positive regulation of MAPK cascade,and involve multi key pathways including MAPK signaling pathway,HIF-1 signaling pathway and PI3K/Akt signaling pathway etc.Good binding activities were observed between MAPK1,CAV1,EDN1,F2 and quercetin,luteolin,kaempferol,tanshinaldehyde,as well as MAPK1,F2 and arachidonic acid.(2)Compared with sham operation group,LVEF and LVFS of rats significantly reduced(P<0.01),heart mass index obviously increased(P<0.05)in the model group.Myocardial tissue appears obvious pathological damage,and the degree of interstitial fibrosis was serious.The collagen volume fraction of the heart significantly increased(P<0.01).The levels of serum BNP,ANP and ALD significantly increased(P<0.01).The mRNA and protein expressions of CAV1、F2 and MAPK1 in heart tissue significantly increased(P<0.05,P<0.01).Compared with the model group,LVEF and LVFS of rats obviously increased(P<0.01),but the decrease in heart mass index was not significant(P>0.05)in Shenfu Yixin Granules group and positive control group.The pathological damage in myocardial tissues was significantly improved,the degree of interstitial fibrosis was significantly reduced.The collagen volume fraction of the heart significantly decreased(P<0.01).The levels of serum BNP,ANP and ALD significantly decreased(P<0.01).The mRNA and protein expressions of CAV1、F2 and MAPK1 in heart tissue significantly decreased(P<0.05,P<0.01).Conclusion Shenfu Yixin Granules may improve heart function and myocardial fibrosis in heart failure rats through the interaction between the active ingredients(quercetin,luteolin,arachidonic acid,kaempferol,and tanshinaldehyde)and targets(MAPK1,F2,CAV-1,and EDN1),so as to regulate MAPK signaling pathway and PI3K/Akt signaling pathway.