温肾养血方调控PI3K/Akt/FoxO3A信号通路改善高龄雌鼠子宫内膜容受性的作用机制

Mechanism of Wenshen Yangxue Prescription Improving Endometrial Receptivity of Aged Female Mice by Regulating PI3K/Akt/FoxO3A Signaling Pathway

目的:基于网络药理学和实验验证,探讨温肾养血方提高高龄雌鼠子宫内膜容受性的有效成分、作用靶点及可能的作用机制。方法:基于BATMAN-TCM和整合药理学平台对温肾养血方中的中药成分及药物靶标进行检索;以"anovulatory sterility"和"anovulatory infertility"为关键词,通过人类孟德尔遗传数据库(OMIM)和GeneCards数据库,对排卵障碍性不孕症靶标进行搜集;基于STRING数据库中的蛋白质-蛋白质相互作用(PPI)数据库,并通过Cytoscape构建温肾养血方成分靶标及排卵障碍性不孕症靶标核心网络;基于DAVID数据库中基因本体(GO)和京都基因与基因组百科全书(KEGG)对共有靶标进行功能和通路分析,并建立"中药-成分-靶标-通路"分子网络,对核心靶点进行动物实验验证。结果:共获得化学成分253个,药物靶标326个,疾病靶标819个,共有靶标74个。GO和KEGG对共有靶标进行功能和通路分析结果显示,共有靶标主要参与了一氧化氮生物合成过程的正调控、细胞增殖的正调控、对雌二醇的反应、老化、氧化应激反应、血管生成等生物学过程。对GO过程中氧化应激反应以及KEGG前20中的5个重要通路进行分析,通过构建"中药-成分-靶标-生物学过程/通路"多维网络发现,共20种中药41种化学成分,这些成分通过影响35个靶标参与了低氧诱导因子-1(HIF-1)信号通路,肿瘤坏死因子(TNF)信号通路,叉头框蛋白O(FOXO)信号通路,Toll样受体(TLR)信号通路,磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路。动物实验结果显示,温肾养血方可提高高龄雌性ICR小鼠子宫磷脂酰肌醇3-激酶(PI3K),磷酸化(p)-PI3K,Akt,p-Akt,叉头框蛋白O3A(FoxO3A)和p-FoxO3A的表达量。结论:温肾养血方通过影响Akt1,双氧化酶-2(DUOX2),表皮生长因子受体(EGFR),血红素加氧酶-1(HMOX1),髓过氧化物酶(MPO)等靶标干预氧化应激及PI3K/Akt/FoxO3A信号通路提高高龄雌鼠子宫内膜容受性。

Objective:To explore the effective components,targets,and possible mechanisms of Wenshen Yangxue prescription in improving endometrial receptivity of aged female mice based on network pharmacology and experimental verification.Method:Based on Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine(BATMAN-TCM)and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine,the components and targets of Wenshen Yangxue prescription were retrieved,and the targets of ovulatory dysfunctional infertility were collected from the Online Mendelian Inheritance in Man(OMIM) and Gene Cards with "anovulatory sterility" and "anovulatory infertility" as keywords.The protein-protein interaction(PPI)network was constructed based on STRING and the core targets of Wenshen Yangxue prescription against ovulatory dysfunctional infertility were screened by Cytoscape,followed by Gene Ontology(GO)term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the core targets in DAVID database.Then,the "medicinal-component-target-pathway" network was established and the core targets were verified by animal experiment.Result:A total of 253 components and 326 targets of Wenshen Yangxue prescription,819 disease targets,and 74 common targets were screened out.The common targets were mainly involved in the biological processes such as positive regulation of nitric oxide biosynthetic process,positive regulation of cell proliferation,response to estradiol,aging,response to oxidative stress,and angiogenesis.The GO term of response to oxidative stress and five of the top 20 KEGG pathways were analyzed.According to the "medicinal-component-target-biological process/pathway" network,41 chemical components in 20 medicinals participated in hypoxia inducible factor-1(HIF-1)signaling pathway,tumor necrosis factor(TNF)signaling pathway,forkhead box O(FOXO)signaling pathway,Toll-like receptor(TLR)signal pathway,and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway by affecting 35 targets.The results of animal experiment showed that the prescription could increase the expression of PI3K,phosphorylated PI3K(p-PI3K),Akt,phosphorylated Akt(p-Akt),forkhead box O3A(FoxO3A),and phosphorylated FoxO3A(p-FoxO3A)in uterus of aged female ICR mice.Conclusion:Wenshen Yangxue prescription interferes with oxidative stress and PI3K/Akt/FoxO3A signaling pathway by influencing Akt1,dual oxidase 2(DUOX2),epidermal growth factor receptor(EGFR),heme oxygenase-1(HMOX1),myeloperoxidase(MPO),and other targets,thereby improving endometrial receptivity of aged female mice.