【摘要】 [目的]通过超高效液相色谱-四极杆-飞行时间质谱联用技术(ultra-performance liquid chromatography-quadrupole time-offlight mass spectrometry,UPLC-Q-TOF/MS)结合网络药理学及体外实验,探讨大黄抗脑缺血的作用机制。[方法]通过UPLC-QTOF/MS鉴定大黄的入血成分,并通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)和Swiss Target Prediction获取大黄入血成分的潜在靶点,从基因组注释数据平台(Genome Annotation Database Platform,GeneCards)、在线人类孟德尔遗传(Online Mendelian Inheritance in Man,OMIM)和药物靶标数据库(Therapeutic Target Database,TTD)获得缺血性脑卒中(ischemic stroke,IS)的相关靶点。采用Venny 2.1.0获得交集靶点,导入STRING数据库进行蛋白互作(protein-protein interaction,PPI)网络分析,利用Cytoscape 3.7.2软件可视化并鉴定核心靶点,通过Metascape数据库进行基因本体(gene oncology,GO)功能富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析以预测作用机制,通过AutoDock Vina进行分子对接,最后通过体外实验对网络药理学预测结果进行验证。[结果]大黄含药血清(serum containing rhubarb,SR)中共鉴定出15种活性成分,大黄与IS共有87个交集靶点。KEGG富集分析显示,大黄可能通过调控磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K/AKT)和核因子-κB(nuclearfactor-κB,NF-κB)等信号通路来减轻IS脑损伤。分子对接结果表明,核心成分与Toll样受体4(Toll-like receptor 4,TLR4)/髓样分化因子(myeloid differentiation primary response gene 88,MyD88)/NF-κB信号通路关键靶点有较强的结合能力。体外实验发现,SR减少氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的BV2细胞中活性氧(reactive oxygen species,ROS)的产生,提高超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活力,并降低丙二醛(malondialdehyde,MDA)和一氧化氮(nitric oxide,NO)的水平。此外,SR降低OGD/R诱导的BV2细胞中的促炎因子水平,同时提高抗炎因子水平,抑制TLR4、MyD88和NF-κB的蛋白表达。[结论]大黄通过抑制氧化应激和炎症反应,保护OGD/R诱导的BV2小胶质细胞,其作用机制可能与调控TLR4/MyD88/NF-κB信号通路相关。更多还原

【Abstract】 [Objective] To explore the anti-cerebral ischemia mechanism of Rhubarb root and rhizome by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS) combined with network pharmacology and in vitro experiment.[Methods] The active components of Rhubarb root and rhizome in rat serum samples were identified by UPLC-Q-TOF/MS.The potential targets of Rhubarb root and rhizome in blood components were obtained by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Swiss Target Prediction.The related targets of ischemic stroke(IS) were obtained from Genome Annotation Database Platform(GeneCards),Online Mendelian Inheritance in Man(OMIM) database and Therapeutic Target Database(TTD).The intersection targets were obtained by using Venny 2.1.0 software and imported into the STRING database for protein-protein interaction(PPI) network analysis.Cytoscape 3.7.2 software was employed to visualize and identify the core targets.Gene ontology(GO) enrichment function analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signal pathway enrichment analysis were conducted to predict the mechanism of action.Molecular docking was performed by AutoDock Vina.Finally,the network pharmacological prediction results were verified by in vitro experiments.[Results] A total of 15 active components were identified in serum containing Rhubarb root and rhizome(SR),and there were 87 intersection targets between Rhubarb root and rhizome and IS.KEGG enrichment analysis showed that Rhubarb root and rhizome may alleviate IS through phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT) and nuclear factor-κB(NF-kB) signaling pathways.Molecular docking results showed that the core components had a strong binding ability to the key targets of Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/NF-κB signaling pathway.In vitro experiment demonstrated that SR reduced the production of reactive oxygen species(ROS) induced by oxygen-glucose deprivation/reoxygenation(OGD/R) in BV2 cells.It also increased the activity of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px),while decreasing the levels of malondialdehyde(MDA) and nitric oxide(NO).Additionally,SR reduced the levels of proinflammatory factors,increased the levels of anti-inflammatory factors in BV2 cells induced by OGD/R,and decreased the protein expression of TLR4,MyD88 and NF-kB in BV2 cells.[Conclusion] Rhubarb root and rhizome protects BV2 microglia from OGD/Rinduced injury by inhibiting oxidative stress and inflammatory response,and its mechanism may be related to regulation of TLR4/MyD88/NF-κB signaling pathway.更多还原