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| 木香内酯诱导人脑星形胶质母细胞瘤细胞凋亡 |
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黄文龙1,2, 袁泽3,4, 彭卫卫2, 刘国胜2, 薛英贤5, 李斐2, 李宁3, 刘建生2
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1.赣南医学院第一临床医学院, 赣州 341000;2.赣州市人民医院, 赣州 341000;3.北大医疗鲁中医院检验科, 淄博 255499;4.西南医科大学, 泸州 646099;5.淄博市中心医院病理科, 淄博 255036
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| 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 IC50 为 11.27 μmol/L；细胞凋亡水平检测结果显示木香内酯 20 μmol/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 5 μmol/L 组、10 μmol/L 组、20 μmol/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 20 μmol/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。 |
| 关键词: 神经胶质瘤 细胞凋亡 氧化应激 木香内酯 |
| DOI：10.11656/j.issn.1672-1519.2023.06.17 |
| 分类号:R739.41 |
| 基金项目:江西省中医药管理局科技计划课题（2021A371） |
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| Effect of micheliolide on apoptosis of human astrocytoma cells |
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HUANG Wenlong1,2, YUAN Ze3,4, PENG Weiwei2, LIU Guosheng2, XUE Yingxian5, LI Fei2, LI Ning3, LIU Jiansheng2
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1.The First School of Clinical Medicine, Gannan Medical University, Ganzhou 341000, China;2.People's Hospital of Ganzhou City, Ganzhou 341000, China;3.Clinical Laboratory of PKUCare Luzhong Hospital, Zibo 255499, China;4.Southwest Medical University, Luzhou 646099, China;5.Department of Pathology, Zibo Central Hospital, Zibo 255036, China
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| Abstract: |
| [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 IC50 values of MCL against U87 cells were around 11.27 μmol/L；and the apoptosis level showed that a significant increase in the apoptosis rate of U87 cells when treated with 20 μmol/L MCL（P<0.001）. The mitochondrial membrane potential of U87 cells was significantly reduced when administered in the 5，10 and 20 μmol/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 20 μmol/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 mitochondrial membrane potential and promote apoptosis. |
| Key words: glioma apoptosis oxidative stress micheliolide |
