摘要
目的:基于网络药理学和分子对接方法探讨黄芪对早发性卵巢功能不全治疗作用的分子机制。方法:在中药系统药理学数据库TCMSP中筛选黄芪有效成分及药物作用靶点;在Genecards数据库中筛选早发性卵巢功能不全疾病靶点;运用Venny在线工具筛选两者的交互基因;通过Cytoscape3.7.2软件构建“药物-成分-靶点-疾病”网络关系图;采用String数据库获得蛋白互作(PPI)信息,进一步通过CytoNCA插件构建蛋白质相互作用网络,筛核心靶点蛋白;利用DAVID数据库对交互基因进行GO富集和KEGG通路富集;使用分子对接技术进一步明确其发挥治疗作用的分子;最后,采用血清饥饿法建立POI细胞模型,给予黄芪中的有效化合物槲皮素进行干预,通过Tunel染色、蛋白质印迹实验(western blotting,WB)对网络药理学预测结果进行实验验证。结果:通过TCMSP数据库筛选出黄芪的32个有效化合物及552个基因靶点,从疾病数据库中筛选出730个早发性卵巢功能不全的疾病靶点,两者共62个交集基因,其中黄芪中的主要活性成分包括槲皮素(quercetin)、异鼠李素(isorhamnetin)、山奈酚(kaempferol)、丁子香萜(Mairin)、芒柄花黄素(formononetin)和芒柄花黄素(astragaloside IV),10个POI的潜在核心靶基因为ALB、INS、AKT1、ACTB、TP53、TNF、ESR1、CASP3、STAT3和PTEN;潜在靶点基因主要参与调控JAK2/STAT3、PI3K/Akt信号通路;分子对接验证了黄芪主要活性化合物与核心蛋白STAT3和Akt1存在较强的结合作用;细胞实验结果发现,黄芪中的重要活性化合物槲皮素能促进JAK2/STAT3信号通路关键分子JAK2及STAT3的磷酸化水平,改变卵巢颗粒细胞凋亡相关蛋白表达水平。结论:网络药理学和分子对接分析表明中药黄芪能通过调节对雌二醇的反应、抑制凋亡过程以及调控JAK-STAT和PI3K-Akt信号通路治疗早发性卵巢功能不全,其机制可能与激活JAK2/STAT3信号通路,抑制卵巢颗粒细胞凋亡相关。
Objective:This study aims to investigate the molecular mechanisms of Huangqi(Astragalus) in the treatment of premature ovarian insufficiency(POI) using network pharmacology and molecular docking methods.Methods:The active compounds and target proteins of Huangqi were screened from the TCMSP database,while the disease target proteins associated with POI were obtained from the Genecards database.The Venny online tool was used to identify the overlapping genes between the two datasets.Cytoscape software was used to construct a gene regulatory network for the active ingredients of Huangqi.The protein-protein interaction(PPI) network and core genes were selected based on the STRING database and CytoNCA plug-in of Cytoscape.GO(gene ontology) enrichment and KEGG(Kyoto encyclopedia of genes and genomes) pathway enrichment analyses of the overlapping genes were performed using the DAVID database.Molecular docking techniques were used to further elucidate the molecular mechanisms underlying the therapeutic effects.Human ovarian granulosa-like tumor cells(KGN) underwent serum starvation(SS) treatment to induce POI cell models and then received quercetin treatment,and experimentally validated the network pharmacology prediction results by TUNEL staining and Western blotting assay.Results:The TCMSP database provided 32 active compounds and 552 target genes associated with Huangqi,while totally 730 disease target genes associated with POI were screened out from the disease database.Of these,62 genes were found to be shared between the two datasets.The top 6 compounds based on degree values were quercetin,isorhamnetin,kaempferol,mairin,formononetin and astragaloside IV.The 10core genes identified were ALB,INS,AKT1,ACTB,TP53,TNF,ESR1,CASP3,STAT3 and PTEN.These potential targets were mainly involved in regulating the JAK2/STAT3 and PI3K/Akt signaling pathway.Molecular docking results revealed strong binding interactions between the compounds and the key proteins STAT3 and Akt1 in the JAK-STAT and PI3K-Akt signalling pathways.The results of in vitro experiments revealed that quercetin could activate the JAK2/STAT3 pathway and inhibit cell apoptosis.Conclusions:Network pharmacology and molecular docking analysis showed that Astragalus could regulate the response of ovarian cells to estradiol,inhibit apoptosis and regulate the JAK-STAT and PI3K-Akt signalling pathways in the treatment of early onset ovarian dysfunction.
作者
李锦婧
钟婉
黄元华
马燕琳
LI Jinjing;ZHONG Wan;HUANG Yuanhua;MA Yanlin(Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research,Key Laboratory of Reproductive Health Diseases Research and Translation(Hainan Medical University),Department of Reproductive Medicine,Hainan Provincial Clinical Research Center for Thalassemia,National Center for International Research“China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease”by the Ministry of Science and Technology of China,Haikou Key Laboratory for Preservation of Human Genetic Resource,The First Affiliated Hospital of Hainan Medical University,Hainan Medical University,Haikou 571199,China;School of Stomatology,Hainan Medical University,Hainan Medical University,Haikou 571199,China)
出处
《海南医学院学报》
CAS
北大核心
2024年第15期1156-1168,共13页
Journal of Hainan Medical University
基金
国家自然科学基金面上项目(82072880)
国家自然科学基金青年科学基金项目(82202044)
海南省自然科学基金项目(823QN349)
海南省院士创新平台科研项目资金资助
海南省省级临床医学中心建设项目资助。
关键词
网络药理学
分子对接
黄芪
早发性卵巢功能不全
信号通路
细胞凋亡
Network Pharmacology
Molecular Docking
Huangqi
Premature ovarian insufficiency
Signalling pathways
Cell apoptosis
