中国中药杂志

2021, v.46(24) 6465-6473

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基于网络药理学和体外实验探讨黄芪甲苷通过激活PI3K/AKT信号通路减轻PC12细胞损伤的作用机制
Mechanism of astragaloside Ⅳ alleviating PC12 cell injury by activating PI3K/AKT signaling pathway: based on network pharmacology and in vitro experiments

张天琪;李传成;张铁峰;王明燕;崔赛男;霍青;
ZHANG Tian-qi;LI Chuan-cheng;ZHANG Tie-feng;WANG Ming-yan;CUI Sai-nan;HUO Qing;Shandong University of Traditional Chinese Medicine;Linyi Central Hospital;Guangrao County People′s Hospital;Guangzhou University of Chinese Medicine;Shandong Affiliated Hospital of University of Traditional Chinese Medicine;

摘要(Abstract):

基于网络药理学探讨黄芪甲苷(astragalosideⅣ,AS-Ⅳ)治疗帕金森病(Parkinson′s disease, PD)的分子机制,并通过分子对接和体外实验验证AS-Ⅳ通过激活PI3K/AKT信号通路,减轻PD神经元损伤的潜在价值。借助SwissTargetPrediction数据库、BTMAN-TAM数据库、GeneCards数据库,对AS-Ⅳ作用于PD的靶点进行预测。采用STRING数据库进行蛋白质-蛋白质相互作用(protein-protein interaction, PPI)分析,构建PPI网络。借助DAVID数据库进行基因本体(GO)富集分析与KEGG通路分析。结合GO富集分析和KEGG通路分析结果,该研究选择PI3K/AKT信号通路进行进一步的分子对接和体外实验验证。通过MPP~+构建帕金森病体外细胞模型,采用MTT法测定细胞存活率,实时荧光定量聚合酶链式反应(real-time PCR)和蛋白免疫印迹法(Western blot)检测AS-Ⅳ对PI3K/AKT信号通路相关基因、蛋白表达的影响。网络药理学共得到AS-Ⅳ治疗PD靶点122个,GO富集分析得到GO条目504个,大部分与生物过程和分子功能相关。KEGG通路共筛选出20条相关信号通路,包括神经活性配体-受体相互作用、PI3K/AKT信号通路、GABA能突触、钙信号通路等。分子对接结果表明AS-Ⅳ与PI3K/AKT信号通路上AKT1、AKT2、PIK3CG、PIK3CA有较好的结合力。体外实验表明AS-Ⅳ能有效抑制MPP~+诱导的PC12细胞存活率降低,上调AKT1、PI3K的mRNA表达水平,上调AKT、PI3K磷酸化表达水平。AS-Ⅳ作为中药黄芪的活性成分,可通过多靶点、多通路发挥治疗PD的作用,通过激活PI3K/AKT通路,起到抑制神经元凋亡、保护神经元的作用,为AS-Ⅳ治疗PD提供可靠的理论和实验支持。
In this study, the molecular mechanism of astragaloside Ⅳ(AS-Ⅳ) in the treatment of Parkinson′s disease(PD) was explored based on network pharmacology, and the potential value of AS-Ⅳ in alleviating neuronal injury in PD by activating the PI3 K/AKT signaling pathway was verified through molecular docking and in vitro experiments. Such databases as SwissTargetPrediction, BTMAN-TAM, and GeneCards were used to predict the targets of AS-Ⅳ for the treatment of PD. The Search Tool for the Retrieval of Interacting Genes/Proteins(STRING) was employed to analyze protein-protein interaction(PPI) and construct a PPI network, and the Database for Annotation, Visualization and Integrated Discovery(DAVID) was used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Based on the results of GO enrichment analysis and KEGG pathway analysis, the PI3 K/AKT signaling pathway was selected for further molecular docking and in vitro experiments in this study. The in vitro cell model of PD was established by MPP~+. The cell viability was measured by MTT assay and effect of AS-Ⅳ on the expression of the PI3 K/AKT signaling pathway-related genes and proteins by real-time polymerase chain reaction(RT-PCR) and Western blot. Network pharmacology revealed totally 122 targets of AS-Ⅳ for the treatment of PD, and GO enrichment analysis yielded 504 GO terms, most of which were biological processes and molecular functions. Totally 20 related signaling pathways were screened out by KEGG pathway analysis, including neuroactive ligand-receptor interaction, PI3 K/AKT signaling pathway, GABAergic synapse, and calcium signaling pathway. Molecular docking demonstrated high affinity of AS-Ⅳ to serine/threonine-protein kinases(AKT1, AKT2), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma(PIK3 CG), and phosphoinositide-3-kinase, catalytic, alpha polypeptide(PIK3 CA) on the PI3 K/AKT signaling pathway. In vitro experiments showed that AS-Ⅳ could effectively inhibit the decrease of the viability of PC12 induced by MPP~+ and up-regulate the mRNA expression levels of AKT1 and PI3 K as well as the phosphorylation levels of AKT and PI3 K. As an active component of Astragali Radix, AS-Ⅳ acts on PD through multiple targets and pathways. Furthermore, it inhibits neuronal apoptosis and protects neurons by activating the PI3 K/AKT signaling pathway, thereby providing reliable theoretical and experimental supports for the treatment of PD with AS-Ⅳ.

关键词(KeyWords): 黄芪甲苷;PI3K/AKT信号通路;网络药理学;分子对接;细胞实验
astragaloside Ⅳ;PI3K/AKT signaling pathway;network pharmacology;molecular docking;cell experiment

Abstract:

Keywords:

基金项目(Foundation): 山东省重点研发项目(2018GSF119021)

作者(Author): 张天琪;李传成;张铁峰;王明燕;崔赛男;霍青;
ZHANG Tian-qi;LI Chuan-cheng;ZHANG Tie-feng;WANG Ming-yan;CUI Sai-nan;HUO Qing;Shandong University of Traditional Chinese Medicine;Linyi Central Hospital;Guangrao County People′s Hospital;Guangzhou University of Chinese Medicine;Shandong Affiliated Hospital of University of Traditional Chinese Medicine;

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