›› 2017, Vol. 60 ›› Issue (10): 1105-1113.doi: 10.16380/j.kcxb.2017.10.001

• 研究论文 •    下一篇


任小双, 张兰*, 张燕宁, 毛连纲, 蒋红云*   

  1. (中国农业科学院植物保护研究所, 农业部作物有害生物综合治理综合性重点实验室, 北京 100193)
  • 出版日期:2017-10-20 发布日期:2017-10-20

MPTP dependency in mitochondrion-mediated apoptosis induced by camptothecin in Spodopetera exigua (Lepidoptera: Noctuidae)

REN Xiao-Shuang, ZHANG Lan*, ZHANG Yan-Ning, MAO Lian-Gang, JIANG Hong-Yun*   

  1. (State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China)
  • Online:2017-10-20 Published:2017-10-20

摘要: 【目的】线粒体通透性转换孔(MPTP)的开放可以导致线粒体膜通透性改变,与细胞凋亡关系密切。本研究旨在探索MPTP在喜树碱诱导的昆虫细胞凋亡中的作用,以进一步揭示喜树碱(CPT)诱导昆虫细胞凋亡的机制。【方法】环孢菌素A(CsA)为MPTP开放抑制剂,通过预加入20 μmol/L CsA,应用流式细胞仪测定其对CPT和羟基喜树碱(HCPT)诱导的甜菜夜蛾Spodoptera exigua细胞(IOZCAS-SPEX-Ⅱ)凋亡作用的影响,包括细胞内Ca2+浓度变化,线粒体膜电位变化以及活性氧簇(ROS)变化,从而分析MPTP在CPT和HCPT诱导细胞凋亡的作用。【结果】结果显示,10 μmol/LCPT和HCPT处理IOZCAS-SPEX-Ⅱ细胞6 h和12 h时,与0.1% DMSO对照组相比,甜菜夜蛾细胞发生凋亡,胞质Ca2+浓度增大,线粒体膜电位降低或丧失,ROS增加,即CPT和HCPT诱导甜菜夜蛾细胞发生凋亡,为线粒体内途径。但经过20 μmol/L CsA预处理2 h后再加入CPT和HCPT处理6 h,与0.1% DMSO组相比,细胞凋亡率、胞质Ca2+浓度、线粒体膜电位及ROS产生均无显著差异(P>0.05),即CsA抑制了MPTP的开放,从而抑制了CPT和HCPT诱导的甜菜夜蛾细胞凋亡;而加入CPT和HCPT处理12 h时,CsA对MPTP开放的抑制作用显著降低,与单CPT和HCPT处理组相比,细胞凋亡率、胞质Ca2+浓度、线粒体膜电位及ROS差异不显著,即CPT和HCPT诱导的细胞凋亡如常发生。【结论】本研究证实喜树碱和羟基喜树碱诱导甜菜夜蛾细胞凋亡线粒体途径具有MPTP开放依赖性,且首次明确这种依赖性具有时间性。

关键词: 甜菜夜蛾, 线粒体通透性转换孔, 细胞凋亡, 线粒体膜电位; 环孢菌素A, 喜树碱

Abstract: 【Aim】 The opening of the mitochondrial permeability transition pore (MPTP) leads to the changes of mitochondrial membrane permeability and is closely related to cell apoptosis. This study aims to explore the role of MPTP in the process of apoptosis induced by camptothecin (CPT) in Spodoptera exigua so as to further reveal the mechanisms of CPT-induced apoptosis in insects. 【Methods】 Effects of cyclosporine A (CsA), an inhibitor of MPTP, on the CPT-and 10-hydroxycamptothecin (HCPT)-induced apoptosis in IOZCAS-SPEX-Ⅱ cells of S. exigua were detected by flow cytometry. The detected parameters included the apoptosis ratio, intracellular Ca2+ concentration, mitochondrial membrane potential and reactive oxygen species (ROS), which would show the role of MPTP in the apoptosis induced by CPT and HCPT. 【Results】 After 10 μmol/L CPT and HCPT pre-treatment for 6 and 12 h, IOZCAS-SPEX-Ⅱ cells were apoptosized through a mitochondira-mediated pathway characterized by the increase of cytoplasmic Ca2+ concentration and ROS, and the reduced mitochondrial membrane potential in IOZCAS-SPEX-Ⅱ cells as compared with the control group (0.1% DMSO). However, after the pretreatment of 20 μmol/L CsA for 2 h, there were no significant differences between the control group of 0.1% DMSO and the CPT and HCPT treated groups (CsA+CPT or CsA+HCPT, 6 h) in the apoptosis ratio, cytoplasmic Ca2+ concentration, ROS generation, and the mitochondrial membrane potential, suggesting that CsA inhibited CPT- and HCPT-induced apoptosis by inhibiting MTPT open in IOZCAS-SPEX-Ⅱ cells. Interestingly, the inhibitory effects of CsA on MPTP were not detected after CPT and HCPT treatment for 12 h. 【Conclusion】 This study confirms that CPT- and HCPT-induced apoptosis in IOZCAS-SPEX-Ⅱ cells of S. exigua is through MPTP-dependent mitochondrial pathway during the initial several hours of treatment.

Key words: Spodoptera exigua, mitochondrial permeability transition pore (MPTP), mitochondrial membrane potential, apoptosis, cyclosporine A, camptothecin