基于网络药理学与实验验证探讨通脉颗粒治疗缺血性脑卒中的作用机制

Functional Mechanism of Tongmai Granule in Treating Ischemic Stroke Based on Network Pharmacology and Experimental Validation

目的采用网络药理学探讨通脉颗粒治疗缺血性脑卒中(ischemic stroke,IS)的作用机制,并开展体内实验进行验证。方法通过TCMSP数据库和TCMIP数据库检索丹参、川芎、葛根3味中药的化学成分及其对应的作用靶点,在GeneCards数据库、Drugbank数据库、TTD数据库、OMIM数据库收集IS的相关靶点;将化学成分靶点和IS靶点绘制韦恩图取交集,借助STRING数据库和Cytoscape软件构建药物-潜在活性成分-作用靶点网络图;利用DAVID数据库对关键靶点进行富集分析。采用大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)法复制IS大鼠模型,造模成功后用通脉颗粒低、中、高剂量药物干预1周。采用Longa评分、免疫荧光染色等方法进行动物体内药效验证和机制探讨。结果经网络药理学分析,共获得通脉颗粒87个潜在活性成分,作用于IS的关键靶点为白细胞介素(interleukin,IL)-6、丝氨酸/苏氨酸蛋白激酶AKT(serine/threonine-protein kinase AKT,AKT)1、肿瘤坏死因子(tumor necrosis factor,TNF)、β-肌动蛋白(beta-actin,ACTB)、血管内皮生长因子(vascular endothelial growth factor,VEGF)A,可能的作用机制与丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号通路、磷脂酰肌醇-3-激酶-丝氨酸/苏氨酸蛋白激酶AKT(phosphatidylinositide 3-kinases-serine/threonine-protein kinase AKT,PI3K-Akt)信号通路、TNF信号通路、IL-17信号通路等有关。动物实验验证结果表明,与模型组比较,通脉颗粒可显著降低IS大鼠神经功能缺损评分(P<0.05),能明显缩小脑组织梗死灶、有效改善脑组织病理学表现,改变小胶质细胞活化状态,显著降低IL-6、TNF-α的表达水平(P<0.05),显著升高IL-4和IL-10的表达水平(P<0.05)。结论通脉颗粒可抑制炎症因子的表达,改善神经功能损伤,其作用机制可能与调控小胶质细胞介导的炎症反应有关。

Objective The mechanism of Tongmai Granules in the treatment of ischemic stroke was investigated by network pharmacology and verified in vivo.Methods The chemical constituents and their corresponding targets of Salviae miltiorrhizae radix et rhizome,Chuanxiong rhizome and Puerariae lobatae radix were searched through TCMSP database and TCMIP database.The related targets of ischemic stroke(IS)were collected in GeneCards database,Drugbank database,TTD database and OMIM database.The intersection of chemical composition targets and IS targets is drawn by Venn diagram.The network diagram of drug-potential active ingredient-targets was constructed by STRING database and Cytoscape.DAVID database was used to perform the enrichment analysis of key targets.An IS rat model was established by MCAO method.The IS rats were administered with low,medium and high dose of Tongmai Granules for 1 week.The efficacy and mechanism of Tongmai Granules were verified by Longa score and immunofluorescence staining.Results Through network pharmacological analysis,87 potential active components of Tongmai granules were obtained.The key targets were IL-6,AKT1,TNF,ACTB and VEGFA.The possible mechanism was related to MAPK signaling pathway,PI3K-Akt signaling pathway,TNF signaling pathway,IL-17 signaling pathway and other signaling pathways.The results of animal experiments showed that compared with the model group,Tongmai Granules could significantly reduce the neurological deficit score of IS rats(P<0.05),significantly shrink the infarct of brain tissue,effectively improve the pathological manifestations of brain tissue,change the activation state of microglia,significantly reduce the expression levels of IL-6 and TNF-α(P<0.05),and significantly increase the expression levels of IL-4 and IL-10(P<0.05).Conclusion Tongmai Granules can inhibit the expression of inflammatory factors and improve nerve function injury,the mechanism of which may be related to the regulation of microglia-mediated inflammatory response.