基于网络药理学与实验验证姜黄素改善心肌纤维化作用机制

Mechanisms of Curcumin in the treatment of myocardial fibrosis based on network pharmacology and animal experiment

目的网络药理学结合实验验证探究姜黄素(curcumin,Cur)改善心肌纤维化(myocardial fibrosis,MF)的作用及分子机制。方法运用网络药理学方法,从药物及疾病相关数据集筛选获取Cur和MF相关基因,借助STRING平台和Cytoscape软件构建蛋白互作网络,根据拓扑特征值筛选获得核心靶点,对核心靶点进行基因本体论(Gene Ontology,GO)与京都基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析并构建“姜黄素-靶点-通路”网络。运用AutoDock Vina软件对核心靶点进行分子对接验证。将C57BL/6J雄性小鼠随机分为对照组(Saline)、异丙肾上腺素组(isoproterenol,ISO)、ISO+姜黄素组(ISO+Cur)。Saline组小鼠每日颈部皮下注射等体积0.9%生理盐水,其余两组小鼠均采用皮下注射2.5 mg/kg·d ISO(0.9%生理盐水溶解)建立MF小鼠模型。ISO+Cur组小鼠同期给予Cur混悬液150 mg/kg·d灌胃3周。采用病理染色与RT-qPCR验证Cur对纤维化改善作用及其机制。结果共获得84个Cur治疗MF的潜在靶点,其中包括类固醇受体共激活因子(steroid receptor coactivator,SRC)、丝氨酸/苏氨酸蛋白激酶(protein kinase B1,AKT1)、热激蛋白90AA1(heat shock protein 90AA1,HSP90AA1)、雌激素受体1(estrogen receptor,ESR1)、表皮生长因子受体(epidermal growth factor receptor,EGFR)等16个核心基因。GO分析显示,上述靶点与激素介导的信号转导途径及激酶活性调控等生物学过程密切相关。KEGG结果提示,Cur改善MF与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、磷脂酰肌醇3激酶(phosphoinositide 3-kinase,PI3K)-蛋白激酶B(protein kinase B,Akt)等信号通路相关。分子对接结果显示,Cur与8个核心靶点之间具有良好的结合活性。HE与Masson染色显示,与ISO造模组比较,Cur干预后小鼠心肌间胶原纤维沉积减少,纤维化基因α-SMA、Col1α1及TGFβmRNA表达下调,抗氧化基因HO-1 mRNA表达上调,心室重构基因Nppa、Nppb mRNA转录水平明显下调(均P<0.05)。结论Cur可能通过多靶点、多途径抑制心脏炎症反应和氧化应激,从而改善MF,为临床使用及后续研究提供了实验依据。

Objective To explore the effect and molecular mechanism of curcumin in the treatment of myocardial fibrosis using an integrated network pharmacology and experimental verification.Methods The targets of curcumin and myocardial fibrosis(MF)were screened from disease and drug related databases,and the protein interaction network was constructed with the help of the STRING platform and Cytoscape software.The core targets were screened according to the topological eigenvalues.GO and KEGG enrichment analyses were performed and the"Curcumin-target-pathway"network was constructed.AutoDock Vina was applied to molecular docking.The C57BL/6J male mice were randomly divided into control(Saline),isoproterenol group(ISO),and isoproterenol+curcumin group(ISO+Cur).In the Saline group,an equal volume of 0.9%saline was injected subcutaneously into the neck daily,and in the remaining two groups,2.5 mg/kg·d of ISO(dissolved in 0.9%saline)was injected subcutaneously to establish a mouse model of myocardial fibrosis.Mice in the ISO+Cur group were given curcumin suspension 150 mg/kg·d by gavage for 3 weeks during the same period.Validation of the ameliorative effect of curcumin on fibrosis and its mechanism using pathological staining and RT-qPCR.Myocardial fibrosis was induced in mice by subcutaneous injection of isoprenaline(ISO)for three weeks,and pathological staining with RT-qPCR verified the ameliorative effect of curcumin on fibrosis and its mechanism.Results 84 potential targets of the curcumin for myocardial fibrosis were obtained,including 16 core genes such as SRC,AKT1,HSP90AA1,ESR1,EGFR,AR,RHOA and MAPK14.GO analysis showed that the above targets were closely related to biological processes such as hormone-mediated signal transduction pathway and kinase activity regulation.The results of KEGG suggested that curcumin amelioration of myocardial fibrosis was associated with signaling pathways such as MAPK and PI3K-AKT.The molecular docking results showed good binding activity between curcumin and eight core targets.HE and Masson staining showed that compared with the ISO model group,interstitial collagen fiber deposition in the mouse myocardium was reduced after curcumin intervention,fibrosis genesα-SMA,Col1α1 and TGFβmRNA expression was down-regulated,antioxidant gene HO-1 mRNA expression was up-regulated,and ventricular remodeling genes NPPA and NPPB mRNA transcript levels were markedly down-regulated(all P<0.05).Conclusion Curcumin can improve myocardial fibrosis by inhibiting cardiac inflammatory response and oxidative stress through multi-target and multi-pathway,providing an experimental basis for clinical use and subsequent research.