脉络舒通丸治疗缺血性卒中的网络药理学分析和实验验证

Effect of Mailuo Shutong Pills in treatment of ischemic stroke based on network pharmacological analysis and experimental verification

网络药理学结合体内实验探究脉络舒通丸治疗缺血性卒中的作用机制,为临床用药提供理论依据。利用中药系统药理分析平台和文献挖掘获得脉络舒通丸的主要活性成分,利用SwissTargetPrediction和DisGeNET数据库获得脉络舒通丸化学成分治疗缺血性卒中的潜在靶点,通过STRING数据库进行蛋白互作分析,并对其进行基因本体论(GO)功能分析和京都基因与基因组百科全书(KEGG)通路富集分析。采用AutoDock Vina软件对关键靶点和成分进行分子对接验证。建立小鼠缺血性卒中模型,分为假手术组、模型组和脉络舒通丸组,2,3,5-氯化三苯基四氮唑染色分析脑梗死体积占比,Western blot法检测组织中磷脂酰肌醇-3-激酶(PI3K)、蛋白激酶B(AKT)、核因子κB(NF-κB)及其磷酸化蛋白的表达水平。结果共筛选出脉络舒通丸222个主要活性成分,包括β-谷甾醇、槲皮素、甘草查尔酮B、甘乌内酯等,作用于701个靶点;与1 079个缺血性卒中靶点取交集,得到192个共同靶点。核心靶点包括AKT1、磷脂酰肌醇-3-激酶催化亚基α(PIK3CA)、磷酸肌醇3-激酶调节亚基1(PIK3R1)、细胞核因子κB p65蛋白(RELA)等,富集的关键通路有PI3K/AKT、肿瘤坏死因子、NF-κB等。分子对接发现脉络舒通丸的活性成分与PI3K、AKT1、RELA均有较好的结合能力。体内实验结果表明,脉络舒通丸能降低小鼠脑梗死体积占比,提高脑组织中p-PI3K、p-AKT的蛋白表达水平,降低p-NF-κB蛋白表达。因此,脉络舒通丸中的有效成分可能是通过激活PI3K/AKT信号通路、抑制NF-κB信号通路发挥脑保护作用。

The present study aimed to explore the targets and mechanism of Mailuo Shutong Pills(MSP) in the treatment of ischemic stroke by network pharmacology, and verify the key targets through molecular docking and animal experiment, so as to provide a theoretical basis for the clinical application of MSP. The main chemical ingredients of MSP were obtained by searching against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and relevant literature. The potential targets of the ingredients of MSP in treating ischemic stroke were obtained from SwissTargetPrediction and DisGeNET. Protein-protein interaction(PPI) network was analyzed in STRING and plotted in Cytoscape. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were carried out with DAVID. Molecular docking was simulated to determine the binding activity of active ingredients to key targets in AutoDock Vina. The mouse model of ischemic stroke was established. The mice were classified into a sham group, a model group, and an MSP group. After the administration, cerebral infarction volume was detected by 2,3,5-triphenyltetrazoliumchloride(TTC) staining, and Western blot was performed to determine the levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), nuclear factor-κB(NF-κB) and their phosphorylated proteins. A total of 222 ingredients of MSP were screened out, including beta-sitosterol, quercetin, licochalcone B, and lupiwighteone, which acted on 701 targets. Totally 1 079 targets associated with ischemic stroke were retrieved, among which 192 common targets were shared by MSP and ischemic stroke. The key targets included AKT1, phosphatidylinositol 3-kinase catalytic subunit alpha(PIK3CA), phosphatidylinositol 3-kinase regulatory subunit 1(PIK3R1), and nuclear factor-κB p65 subunit(RELA), which were mainly involved in PI3 K/AKT, tumor necrosis factor(TNF), and NF-κB signaling pathways. The results of molecular docking revealed that PI3 K, AKT1, and RELA had good binding ability to the active ingredients of MSP. The animal experiment results showed that compared with the model group, MSP decreased cerebral infarction volume, down-regulated the expression of p-NF-κB, and up-regulated the expression of p-PI3 K and p-AKT in mouse brain. In summary, the active ingredients in MSP may treat cerebral injury by activating PI3 K/AKT signaling pathway and inhibiting NF-κB signaling pathway.