苦瓜皂苷L调控PI3K/AKT通路促进内皮祖细胞增殖能力和内皮功能的实验研究

Experimental Study on the Regulation of PI3K/AKT Pathway by Momordicoside L Promoting Endothelial Progenitor Cell Proliferation and Endothelial Function

目的:探讨苦瓜皂苷L对内皮祖细胞(EPC)增殖能力和内皮功能的作用及机制。方法:体外分离、培养、鉴定外周血EPC。使用不同浓度苦瓜皂苷L处理EPC 24 h后,应用细胞计数试剂盒(CCK-8)法检测各组细胞活力。使用AutoDock Tools 5.6软件,分别以磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(AKT)为受体,以药物分子苦瓜皂苷L为配体采用盲对接法进行分子对接。使用PI3K/AKT抑制剂LY294002和最佳使用浓度的苦瓜皂苷L单独或同时处理EPC 24 h后检测各组细胞活力和磷酸化PI3K(p-PI3K)、磷酸化AKT(p-AKT)及磷酸化内皮型一氧化氮合酶(p-eNOS)表达水平。结果:与对照组比较,苦瓜皂苷L呈剂量依赖性地促进EPC增殖,且40μmol/L的苦瓜皂苷L是最佳药物浓度。与对照组比较,LY294002可下调EPC细胞活力,苦瓜皂苷L可上调细胞活力。与苦瓜皂苷L组比较,共同使用苦瓜皂苷L和LY294002可降低细胞活力。分子对接结果显示,苦瓜皂苷L与PI3K和AKT均存在直接作用,形成稳定的锁钥结构。与对照组比较,LY294002可显著下调p-PI3K、p-AKT和p-eNOS蛋白水平;苦瓜皂苷L可上调p-PI3K、p-AKT和p-eNOS蛋白水平。与苦瓜皂苷L组比较,使用苦瓜皂苷L和LY294002可降低p-PI3K、p-AKT和p-eNOS蛋白水平。结论:苦瓜皂苷L通过PI3K/AKT信号通路靶向调节EPC的增殖能力,进而上调内皮型一氧化氮合酶(eNOS)活化水平以促进EPC内皮功能。

Objective:To explore the effect of Momordicoside L on endothelial progenitor cells(EPC)proliferation and endothelial function and its mechanism.Methods:Peripheral blood EPCs were isolated,cultured,and identified in vitro.After EPC was treated with different concentrations of Momordicoside L for 24 h,cell viability was detected by cell count(CCK-8).By AutoDock Tools 5.6 software,the molecular docking was performed by blind docking method with phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT)as receptors,and the drug molecule Momordicoside L as ligand.The cell viability and the expressions of phosphorylated PI3K(p-PI3K),phosphorylated AKT(p-AKT)and phosphorylated endothelial nitric oxide synthase(p-eNOS)in each group were detected,when EPC was treated with PI3K/AKT inhibitor LY294002 and the optimal concentration of Momordicoside L alone or simultaneously for 24 h.Results:Compared with control group,Momordicoside L could promote EPC proliferation in a dose-dependent manner,and 40μmol/L was the optimal concentration.Compared with the control group,LY294002 could down-regulate EPC cell viability,and Momordicoside L could up-regulate EPC cell viability.Compared with Momordicoside L group,the co-administration of Momordicoside L and LY294002 decreased cell viability.Molecular docking results showed that Momordicoside L had direct interaction with PI3K and AKT,and could form a stable lock key structure.Compared with the control group,LY294002 significantly down-regulated the protein levels of p-PI3K,p-AKT and p-eNOS.Momordicoside L up-regulated the protein levels of p-PI3K,p-AKT and p-eNOS.Compared with the Momordicoside L group,the administration of Momordicoside L and LY294002 decreased the protein levels of p-PI3K,p-AKT and p-eNOS.Conclusion:Momordicoside L could regulate the proliferation ability of EPC through the PI3K/AKT signaling pathway,and then up-regulate the activation level of endothelial nitric oxide synthase(eNOS)to promote endothelial function of EPC.