摘要
[目的]探究木香内酯诱导人脑星形胶质母细胞瘤细胞凋亡的作用机制。[方法]通过CCK8法检测木香内酯对U87细胞增殖作用的影响;采用Annexin V-FITC/PI双染法检测木香内酯对U87细胞凋亡率的影响,采用JC-1染色法检测木香内酯对U87细胞线粒体膜电位的影响;采用DCFH-DA探针检测木香内酯对U87细胞中ROS含量的影响;采用生化法检测木香内酯对U87细胞氧化应激指标丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、微量还原型谷胱甘肽(GSH)和一氧化氮(NO)含量的影响;采用实时荧光定量聚合酶链式反应(RT-qPCR)检测木香内酯对U87细胞Caspase-3、Bax、Bcl-2的mRNA表达水平的影响;采用分子对接寻找木香内酯与Caspase-3、Bax、Bcl-2的具体结合位点。[结果]木香内酯呈剂量依赖性抑制U87细胞的生长,细胞半抑制浓度IC_(50)为11.27μmol/L;细胞凋亡水平检测结果显示木香内酯20μmol/L组U87细胞凋亡率增加,差异具有统计学意义(P<0.001);线粒体膜电位水平检测结果显示木香内酯5μmol/L组、10μmol/L组、20μmol/L组U87细胞线粒体膜电位降低,差异具有统计学意义(P<0.001);氧化应激检测水平显示,木香内酯呈剂量依赖性上调U87细胞中ROS、MDA、NO水平;下调SOD、GSH水平,差异具有统计学意义(P<0.05);木香内酯20μmol/L组U87细胞Caspase-3、Bax mRNA表达水平均上调,差异具有统计学意义(P<0.01),而Bcl-2 mRNA表达水平下调,差异具有统计学意义(P<0.05)。分子对接结果显示木香内酯分别与Caspase-3、Bcl-2、Bax氨基酸残基ARG-164,LEU-168,ARG-66形成稳定的结合能。[结论]木香内酯能明显抑制U87细胞生长和介导细胞高氧化应激水平,降低线粒体膜电位并促进细胞凋亡。
[Objective]To explore the effects of micheliolide(MCL)on the apoptosis of human astrocytoma cells(U87)and its mechanism.[Methods]CCK-8 assay was used to detect the inhibitory effect of MCL on the growth of U87 cells;Annexin V-FITC/PI double staining were performed to detect the apoptosis of U87 cells induced by MCL.Mitochondrial membrane potential of U87 cell effected by MCL was measured by mitochondrial membrane potential assay kit with JC-1;2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe was used to detect the level of reactive oxygen species(ROS)in U87 cells effected by MCL;the contents of malondialdehyde(MDA),superoxide dismutase(SOD),trace reduced glutathione(GSH)and nitric oxide(NO)content of MCL on U87 cells were detected by biochemical methods.RT-qPCR was used to detect Caspase-3,Bax,Bcl-2 mRNA expression in in U87 cells effected by MCL.The specific binding sites of MCL with Caspase-3,Bax and Bcl-2 were found by molecular docking.[Results]The growth of U87 cells was inhibited by MCL in a dose-dependent manner,and the IC_(50) values of MCL against U87 cells were around 11.27μmol/L;and the apoptosis level showed that a significant increase in the apoptosis rate of U87 cells when treated with 20μmol/L MCL(P<0.001).The mitochondrial membrane potential of U87 cells was significantly reduced when administered in the 5,10 and 20μmol/L group(P<0.001).The results of oxidative stress index showed that MCL was dose-dependent and upregulated the ROS,MDA,NO content in U87 cells,and down-regulated SOD and GSH(P<0.05).The expression levels of Caspase-3 and Bax mRNA in U87 cells were significantly upregulated(P<0.01)when administered in the 20μmol/L group(P<0.01),while the expression levels of Bcl-2 mRNA were significantly downregulated(P<0.05).The molecular docking results showed that MCL formed stable hydrogen bonding with Caspase-3,Bcl-2 and Bax amino acid residues ARG-164,LEU-168 and ARG-66.[Conclusion]MCL significantly inhibit U87 cell growth,mediate high oxidative stress levels,reduce m
作者
黄文龙
袁泽
彭卫卫
刘国胜
薛英贤
李斐
李宁
刘建生
HUANG Wenlong;YUAN Ze;PENG Weiwei;LIU Guosheng;XUE Yingxian;LI Fei;LI Ning;LIU Jiansheng(The First School of Clinical Medicine,Gannan Medical University,Ganzhou 341000,China;People’s Hospital of Ganzhou City,Ganzhou 341000,China;Clinical Laboratory of PKUCare Luzhong Hospital,Zibo 255499,China;Southwest Medical University,Luzhou 646099,China;Department of Pathology,Zibo Central Hospital,Zibo 255036,China)
出处
《天津中医药》
CAS
2023年第6期782-789,共8页
Tianjin Journal of Traditional Chinese Medicine
基金
江西省中医药管理局科技计划课题(2021A371)。
