基于网络药理学和动物实验探讨补肾启智方对血管性痴呆铁死亡的作用机制

Mechanism of Bushen Qizhi Formula on Ferroptosis in Vascular Dementia Based on Network Pharmacology and Animal Experiments

目的:采用网络药理学方法和分子对接技术并结合体内实验,分析并验证补肾启智方调控海马神经元铁死亡改善血管性痴呆(vascular dementia,VD)认知功能的作用机制。方法:在TCMSP数据库、BATMAN-TCM数据库、ETCM数据库筛选补肾启智方的活性成分及其作用靶点;在GeneCards数据库、OMIM数据库、DisGeNET数据库筛选血管性痴呆的潜在靶点;在FerrDB数据库筛选铁死亡的相关靶点,以上所有靶点均经Uniprot数据库规范,并在微生信-免费在线绘制韦恩图——Venn Diagrams网站获得三者的交集靶点。采用Cytoscape 3.9.1软件绘制“补肾启智方药物-活性成分-潜在靶点”网络、“补肾启智方-血管性痴呆-铁死亡靶点”的蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,并进行拓扑学分析以获得核心成分与核心靶点;在DAVID数据库进行交集靶点的基因本体(Gene Ontology,GO)功能富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析。采用AutoDock 4软件进行分子对接,并利用PyMol 2.5软件对分子对接结果进行可视化分析。通过动物实验验证关键靶点及信号通路。将32只SD雄性大鼠随机分为假手术组、模型组、补肾启智方高剂量组(14.4 g·kg^(-1))、补肾启智方低剂量组(3.6 g·kg^(-1)),每组8只。除假手术组外,其余大鼠采用双侧颈总动脉永久结扎(2-vessel occlusion,2-VO)法建立血管性痴呆模型。采用Morris水迷宫实验评估大鼠的空间学习与记忆能力;Western blot法检测大鼠海马组织中缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)、细胞溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)的蛋白表达;生化法检测大鼠海马组织中丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)水平。结果:筛选得到补肾启智方调控海马神经元铁死亡治疗VD的活性成分40种,有效靶点30个,核心成分是槲皮素、茉莉酮、马兜铃酮,核心靶点是肿瘤蛋白P53(tumor protein P53,TP53)、核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,MTOR)、HIF-1α;GO功能富集分析结果显示,补肾启智方可能通过改善细胞对过氧化氢、缺氧、氧化应激的反应及促进血管生成等方面治疗VD,KEGG通路富集分析表明,其可能通过调控缺氧诱导因子1(hypoxia inducible factor-1,HIF-1)信号通路、细胞衰老、活性氧、叉头盒蛋白O(forkhead box protein O,FoxO)信号通路、表皮生长因子受体(epidermal growth factor receptor,EGFR)信号通路等治疗VD。经补肾启智方干预后,VD大鼠的空间学习与记忆能力得到改善,海马组织MDA表达降低(P<0.01)、GSH水平升高(P<0.01),HIF-1α与铁死亡核心调控蛋白SLC7A11和GPX4蛋白表达水平显著升高(P<0.01,P<0.05,P<0.01)。结论:补肾启智方可通过多成分、多靶点改善VD认知功能,其关键机制可能与激活HIF-1α/SLC7A11/GPX4信号通路、抑制海马神经元铁死亡相关。

Objective:To analyze and verify the mechanism of Bushen Qizhi Formula(BSQZF)in improving cognitive function of vascular dementia(VD)by regulating Ferroptosis in hippocampal neurons using network pharmacology and molecular docking technology combined with in vivo experiments.Methods:The active ingredients and their action targets of BSQZF were screened in TCMSP database,BATMAN-TCM database and ETCM database.Genecards database,OMIM database and DisGeNET database were used to screen potential targets of vascular dementia.FerrDB database was used to screen the Ferroptosis related targets,and all the above targets were standardized by Uniprot database.The intersection targets of the three were obtained from the website of Venn Diagrams.Cytoscape 3.9.1 software was used to draw the protein-protein interaction(PPI)network of"BSQZF drugs-active ingredients-potential targets"and"BSQZF-vascular dementia-Ferroptosis targets".Then the topological analysis was performed to obtain the core components and core targets.The DAVID database was used for Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.AutoDock4 software was used for molecular docking,and PyMol2.5 software was used to visualize the molecular docking results.The key targets and signaling pathways were verified by animal experiments.32 SD male rats were randomly divided into sham operation group,model group,BSQZF high-dose group(14.4 g·kg^(-1))and BSQZF low-dose group(3.6 g·kg^(-1)),with 8 rats in each group.Except the sham operation group,the vascular dementia model was established by permanent 2-vessel occlusion(2-VO).Morris water maze test was used to evaluate the spatial learning and memory ability of rats.Western blot was used to detect hypoxia inducible factor-1α(HIF-1α)and solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)in rat hippocampus.The levels of malondialdehyde(MDA)and glutathione(GSH)in hippocampus were detected by biochemical methods.Results:40 active ingredients and 30 effective targets of BSQZF for regulating Ferroptosis of hippocampal neurons in the treatment of VD were screened out.The core ingredients were quercetin,jasmonone and aristolochia,and the core targets were tumor protein P53(TP53),nuclear factor erythroid 2-related factor 2(Nrf2),mammalian target of rapamycin(MTOR),HIF-1α.According to the results of GO enrichment of function analysis,BSQZF could treat VD by improving the response of cells to hydrogen peroxide,hypoxia,oxidative stress and promoting angiogenesis.KEGG pathway enrichment analysis suggests that BSQZF may regulate hypoxia inducing factor 1(HIF-1)signaling pathway,cell senescence,reactive oxygen species,forkhead box protein O(FoxO)signaling pathway,epidermal growth factor receptor(EGFR)signaling pathway,etc.to treat VD.After treatment with Bushen Qizhi Decoction,the spatial learning and memory ability of VD rats was improved.The expression of MDA in hippocampus was decreased significantly(P<0.01),while GSH level was increased significantly(P<0.01).Besides,the expression levels of HIF-1αand ferroptotic core regulatory proteins SLC7A11 as well as GPX4 were significantly increased(P<0.01,P<0.05,P<0.05)Conclusion:Bushen Qizhi Formula can improve the cognitive function of VD through multi-components and multi-targets,and its key mechanism may be related to activation of HIF-1α/SLC7A11/GPX4 signaling pathway and inhibition of Ferroptosis in hippocampal neurons.