牛蒡叶倍半萜内酯对氧糖剥夺/复氧损伤脑微血管内皮细胞血管新生的作用机制  

Mechanisms of Melitensin on Angiogenesis of Brain Microvascular Endothelial Cells after Oxygen Glucose Deprivation/Reoxygenation Injury

作　　者：黄娜[1,2] 郝单丽 郭辉 刘雅琳[1,2] 王智民[2,3] 代丽萍[1,2] HUANG na;HAO danli;GUO hui;LIU yalin;WANG zhimin;DAI liping(Henan Collaborative Innovation Center for Research and Development on the Whole Industry Chain of Yu-Yao,Henan University of Chinese Medicine,Zhengzhou 450046,China;Henan University of Chinese Medicine,Zhengzhou 450046,China;National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]河南中医药大学豫药全产业链研发河南省协同创新中心,郑州450046 [2]河南中医药大学,郑州450046 [3]中国中医科学院中药研究所中药质量控制技术国家工程实验室,北京100700

出　　处：《世界中医药》2025年第1期25-33,共9页World Chinese Medicine

基　　金：国家自然科学基金面上项目(82374023)——HDAC9靶标驱动的牛蒡叶抗脑缺血再灌注炎症损伤倍半萜的高效发现及机制研究;河南省省级重大科技专项(221100310400)——道地宛药全产业链关键技术研究及应用;2023年度河南省高校科技创新团队(23IRTSTHN028)。

摘　　要：目的:通过网络药理学、分子对接及细胞实验探讨牛蒡叶倍半萜内酯(Melitensin)对脑缺血再灌注损伤后血管内皮细胞血管新生的作用及机制。方法:人脑微血管内皮细胞(HBMECs)进行氧糖剥夺/复氧(OGD/R)处理,细胞随机分为正常组、模型组、丁苯酞组、OGD/R+Melitensin(12.5、25、50μmol/L)组。MTT法检测细胞活力,划痕实验检测细胞迁移率,小管实验检测细胞成管能力,酶联免疫吸附试验检测乳酸脱氢酶(LDH)、肿瘤坏死因子-α(TNF-α)和血管内皮生长因子(VEGF)的水平。借助数据库和在线平台筛选Melitensin防治缺血性脑卒中(IS)的作用靶点,利用String数据库及Cytoscape软件构建Melitensin防治IS核心靶点的蛋白质-蛋白质相互作用(PPI)网络,对靶点基因进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,并对核心通路上的靶点进行分子对接。最后采用荧光定量法(RT-qPCR)、蛋白质印迹法进行体外验证。结果:Melitensin显著提高细胞存活率,促进细胞迁移及管形成,降低LDH、TNF-α水平,增加VEGF水平(P<0.05,P<0.01)。网络药理学分析得到药物-疾病共同靶点62个、核心作用靶点9个,富集的关键通路有VEGF、IL-17、MAPK、TNF等。分子对接显示Melitensin与VEGF、血管内皮细胞生长因子受体2(VEGFR2)有较好的结合能。体外实验验证显示,Melitensin能够上调细胞中血管生成素-1(Ang-1)、VEGF、VEGFR2的mRNA和蛋白表达水平(P<0.05,P<0.01)。结论:Melitensin可能是通过调控VEGF信号通路促进血管生成来发挥治疗IS的作用。Objective:To investigate the effect and mechanisms of melitensin on angiogenesis in endothelial cells after brain ischemia-reperfusion injury through network pharmacology,molecular docking,and cell experiments.Methods:Human brain microvascular endothelial cells(HBMECs)were treated with oxygen-glucose deprivation/reoxygenation(OGD/R).The cells were randomly divided into normal,model,butylphthalide treatment,and OGD/R+melitensin(12.5,25,50μmol/L)groups.Cell viability was measured using the MTT assay,cell migration was evaluated by scratch assay,tube formation ability was assessed using the tube formation assay,and the levels of lactate dehydrogenase(LDH),tumor necrosis factor-α(TNF-α),and vascular endothelial growth factor(VEGF)were detected by enzyme-linked immunosorbent assay(ELISA).Target genes of melitensin for treating ischemic stroke(IS)were identified using databases and online platforms,and the protein-protein interaction(PPI)network was constructed via String and Cytoscape software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were conducted,and molecular docking was performed on key targets in core pathways.Finally,in vitro validation was performed using quantitative real-time PCR(RT-qPCR)and Western blot.Results:Melitensin significantly improved cell viability,promoted cell migration,and enhanced tube formation ability.It also reduced LDH and TNF-αlevels while increasing VEGF levels(P<0.05,P<0.01).Network pharmacology analysis identified 62 common drug-disease targets and 9 core therapeutic targets.Enriched key pathways included VEGF,IL-17,MAPK,and TNF signaling pathways.Molecular docking showed that Melitensin had a good binding affinity with VEGF and VEGF receptor 2(VEGFR2).In vitro experiments confirmed that melitensin upregulated the mRNA and protein expression levels of angiopoietin-1(Ang-1),VEGF,and VEGFR2(P<0.05,P<0.01).Conclusion:Melitensin may exert therapeutic effects on IS by regulating the VEGF signaling pathway and promoting angiogenesis.

关 键 词：Melitensin 氧糖剥夺/复氧 人脑微血管内皮细胞 网络药理学 分子对接 细胞迁移 成管能力 血管新生 

分 类 号：R543.6[医药卫生—心血管疾病]