氟康唑对热带念珠菌活性氧和线粒体膜电位的影响

Effect of fluconazole on reactive oxygen species and mitochondrial membrane potential of Candida tropicalis

摘要:

为了探讨氟康唑作用机制, 观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygen species, ROS)、线粒体膜电位(Mitochondrial membrane potential, △Ψm)和细胞周期的变化。参照NCCLS M27-A方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC); 热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry, FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm和细胞周期的变化。结果表明, 氟康唑作用后, 热带念珠菌氟康唑耐药株的存活率、ROS、线粒体膜电位△Ψm和细胞周期各期比例均没有明显变化; 而热带念珠菌氟康唑敏感株的存活率和线粒体膜电位△Ψm明显下降, ROS明显升高, 而且大部分热带念珠菌阻滞于G2/M期, 并出现明显凋亡峰, 呈一定的时间剂量依赖关系。自由基清除剂谷胱甘肽抑制热带念珠菌ROS的产生, 阻止细胞周期G2/M期阻滞和降低凋亡。由此可见, 氟康唑可能通过刺激热带念珠菌产生过多ROS, 并使线粒体膜电位△Ψm下降, 从而诱导热带念珠菌凋亡。

Abstract:

To explore fluconazole’s effect mechanism, we investigated the changes of viability rate, reactive oxygen species (ROS), mitochondrial membrane potential (△Ψm) and cell cycle of Candida tropicalis after treatment with fluconazole. The minimum inhibitory concentration (MIC) of the clinical isolates Candida tropicalis to fluconazole were tested by NCCLS M27-A microdilution method. After treatment wth different concentration of fluconazole, viability rate, the intracellular accumulation of ROS, the loss of mitochondrial membrane potential △Ψm and cell cycle of Candida tropicalis were detected with flow cytometry, respectively After treatment with fluconazole, there were no significant variation among viability rate, ROS, mitochondrial membrane potential △Ψm and cell cycle in flu-conazole-resistant strains, but a decrease of mitochondrial membrane potential △Ψm and viability rate, an increase of ROS accumulation were detected in a time-dose-dependent manner in flucona-zole-susceptibile strains. A majority of Candida tropicalis were arrested in G2/M phase and apoptosis peak was seen. Free radicals scavenger glutathione inhibited ROS production, prevented G2/M arrest and decreased apoptosis in fluconazole-susceptibile strains. According to it, fluconazole maybe induce intracellular accumulation of ROS and decrease of mitochondrial membrane potential △Ψm, which could result in apoptosis of Candida tropicalis.