基于网络药理学和实验验证分析中风康复胶囊治疗脑梗死作用机制
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R285

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河南省中医药科学研究专项课题(2023ZY2190);河南省高等学校重点科研项目(24B360003)


Analysis of Mechanism of Zhongfeng Kangfu Capsules in the Treatment of Cerebral Infarction Based on Network Pharmacology and Experimental Validation
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    摘要:

    目的:基于网络药理学和实验验证分析中风康复胶囊治疗脑梗死的作用机制。方法:通过TCMSP、 HERB数据库检索中风康复胶囊的中药组分并确定其有效成分,获取有效成分相对应的靶蛋白;运用人类基因数 据库(GeneCards) 获取脑梗死相关疾病靶点;利用STRING数据库分析中风康复胶囊与脑梗死交集靶点的互作 关系,并通过Cytoscape3.9.1软件进行成分-靶点拓扑分析筛选核心靶点;并对核心靶点进行基因本体(GO) 功 能和京都基因与基因组百科全书(KEGG) 通路富集分析,采用Cytoscape3.9.1软件构建“中药材-成分-核心靶 标”网络,筛选中风康复胶囊治疗脑梗死的关键活性成分;利用AutoDock Vina软件对蛋白和成分进行分子对接 验证。采用MCAO法建立大鼠大脑中动脉栓塞的脑梗死模型,采用中风康复胶囊配方干预,以核心靶点信号通路 抑制剂作干预为对照,干预14 d后对各组大鼠进行神经功能缺损评分,酶联免疫吸附法(ELISA) 检测脑梗死相 关炎性因子的水平,HE染色观察脑组织形态学结构,尼氏染色观察各组神经元损伤情况,Real-time PCR法、 Western-blot法检测主动脉的主要核心靶标蛋白表达水平。结果:通过筛选得到中风康复胶囊174种活性成分、 350种中药潜在作用靶点,其中槲皮素、山奈酚、豆甾醇、7-甲氧基-2-甲基异黄酮和β-谷甾醇为排名前5关键活 性成分,药物与疾病交集蛋白中IL-6、TNF、AKT1、IL-1β、TP53、MMP9、PTGS2、CASP3、EGFR、SRC等 51 个为核心靶点。基因富集分析得到1 107条GO 功能条目、197条KEGG 通路,结果表明,磷脂酰肌醇3激 酶(PI3K)-蛋白激酶B(AKT)、IL-17、HIF-1和丝裂原活化蛋白激酶1(MAPK1) 等通路与脑梗死关系密切。分子对 接显示,核心蛋白与相应成分之间均具有良好的结合能力。通过动物实验对重要核心靶点信号通路PI3K-AKT进 行验证,结果表明,与模型组比较,中风康复胶囊高、低剂量组Zealonga评分均降低(P<0.05)。HE染色发现中风 康复胶囊高、低剂量组镜下病理变化明显比模型组减轻,镜下可见细胞大小均匀,结构完整,间质水肿相对较轻。 尼氏染色显示中风康复胶囊高、低剂量组脑组织神经细胞形态有所恢复,尼氏体数量比模型组明显较多。RT-PCR 和Western-blot检测结果发现,与模型组比较,中风康复胶囊高、低剂量组的PI3K、AKT1、TP53表达水平均明显 较低(P<0.05)。ELISA法检测结果显示,与模型组比较,中风康复胶囊高、低剂量组的IL-6、TNF、IL-1β水平 均较低(P<0.05)。结论:从网络药理学角度预测中风康复胶囊治疗脑梗死的分子机制,初步证实其可下调核心靶 点信号通路PI3K-AKT及其下游信号表达治疗脑梗死,为中风康复胶囊的基础及临床研究提供理论和实验依据。

    Abstract:

    Abstract: Objective: To analyze the mechanism of Zhongfeng Kangfu Capsules in the treatment of cerebral infarction based on network pharmacology and experimental validation. Methods:The herbal components of Zhongfeng Kangfu Capsules were retrieved and their active ingredients were identified through TCMSP and HERB databases,and the corresponding target proteins of the active ingredients were obtained. Disease targets related to cerebral infarction were retrieved using the GeneCards database. The interaction relationships between the intersection targets of Zhongfeng Kangfu Capsules and cerebral infarction were analyzed using the STRING database,and component-target topological analysis was performed using Cytoscape3.9.1 software to screen core targets. Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted on the core targets,and a "herbal medicine - component - core target" network was constructed using Cytoscape3.9.1 software to identify key active ingredients of Zhongfeng Kangfu Capsules in the treatment of cerebral infarction. Molecular docking validation was performed using AutoDock Vina software. A rat model of cerebral infarction induced by middle cerebral artery occlusion (MCAO)was established,and the model was intervened with Zhongfeng Kangfu Capsules formula. The intervention was compared with a core target signaling pathway inhibitor as a control, and after 14 days of intervention, neurological deficit scores were assessed in each group of rats. Levels of cerebral infarction-related inflammatory factors were detected by enzyme-linked immunosorbent assay(ELISA),brain tissue morphology was observed by hematoxylin and eosin(HE)staining,neuronal damage was observed by Nissl staining,and the expression levels of major core target proteins in the aorta were detected by real-time PCR and Western blot. Results:A total of 174 active ingredients and 350 potential targets of Zhongfeng Kangfu Capsules were identified, with quercetin, kaempferol, stigmasterol, 7- methoxy-2-methyl isoflavone,and β-sitosterol being the top five key active ingredients. Among the 51 core targets in the drug-disease intersection proteins, IL-6, TNF, AKT1, IL-1β, TP53, MMP9, PTGS2, CASP3, EGFR, and SRC were identified. Gene enrichment analysis yielded 1 107 GO functional entries and 197 KEGG pathways, indicating that the phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), IL-17, HIF-1, and mitogenactivated protein kinase 1(MAPK1)pathways are closely related to cerebral infarction. Molecular docking showed that the core proteins have good binding ability with the corresponding components. Animal experiments validated the important core target signaling pathway PI3K-AKT,and the results showed that compared with the model group,the Zealonga scores of the high- and low-dose groups of Zhongfeng Kangfu Capsules were significantly lower(P<0.05). HE staining revealed that the pathological changes in the high- and low-dose groups of Zhongfeng Kangfu Capsules were significantly less severe than those in the model group, with uniform cell size, intact structure, and relatively mild interstitial edema. Nissl staining showed that the morphology of nerve cells in the brain tissue of the high- and low-dose groups of Zhongfeng Kangfu Capsules was restored,with a significantly higher number of Nissl bodies than in the model group. RT-PCR and Western blot detection results showed that compared with the model group,the expression levels of PI3K,AKT1,and TP53 in the high- and low-dose groups of Zhongfeng Kangfu Capsules were significantly lower(P< 0.05). ELISA detection results showed that compared with the model group,the levels of IL-6,TNF,and IL-1β in the high- and low-dose groups of Zhongfeng Kangfu Capsules were significantly lower(P<0.05). Conclusion:The molecular mechanism of Zhongfeng Kangfu Capsules in treating cerebral infarction was predicted from the perspective of network pharmacology, and it was preliminarily confirmed that it can downregulate the expression of the core target signaling pathway PI3K-AKT and its downstream signals,providing theoretical and experimental basis for the basic and clinical research of Zhongfeng Kangfu Capsules.

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王媛,赵平丽,刘丹丹,秦合伟.基于网络药理学和实验验证分析中风康复胶囊治疗脑梗死作用机制[J].新中医,2025,57(17):211-220

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