Sun Lingmei, Liao Kai, Hang Chengcheng, Wang Dayong
Department of Pharmacology, Medical School of Southeast University, Nanjing, China.
Department of Pathology and Pathophysiology, Medical School of Southeast University, Nanjing, China.
PLoS One. 2017 Feb 13;12(2):e0172228. doi: 10.1371/journal.pone.0172228. eCollection 2017.
To investigate the effects of honokiol on induction of reactive oxygen species (ROS), antioxidant defense systems, mitochondrial dysfunction, and apoptosis in Candida albicans.
To measure ROS accumulation, 2',7'-dichlorofluorescein diacetate fluorescence was used. Lipid peroxidation was assessed using both fluorescence staining and a thiobarbituric acid reactive substances (TBARS) assay. Protein oxidation was determined using dinitrophenylhydrazine derivatization. Antioxidant enzymatic activities were measured using commercially available detection kits. Superoxide dismutase (SOD) genes expression was measured using real time RT-PCR. To assess its antifungal abilities and effectiveness on ROS accumulation, honokiol and the SOD inhibitor N,N'-diethyldithiocarbamate (DDC) were used simultaneously. Mitochondrial dysfunction was assessed by measuring the mitochondrial membrane potential (mtΔψ). Honokiol-induced apoptosis was assessed using an Annexin V-FITC apoptosis detection kit.
ROS, lipid peroxidation, and protein oxidation occurred in a dose-dependent manner in C. albicans after honokiol treatment. Honokiol caused an increase in antioxidant enzymatic activity. In addition, honokiol treatment induced SOD genes expression in C. albicans cells. Moreover, addition of DDC resulted in increased endogenous ROS levels and potentiated the antifungal activity of honokiol. Mitochondrial dysfunction was confirmed by measured changes to mtΔψ. The level of apoptosis increased in a dose-dependent manner after honokiol treatment.
Collectively, these results indicate that honokiol acts as a pro-oxidant in C. albicans. Furthermore, the SOD inhibitor DDC can be used to potentiate the activity of honokiol against C. albicans.
研究厚朴酚对白色念珠菌活性氧（ROS）生成、抗氧化防御系统、线粒体功能障碍及细胞凋亡的影响。
采用2',7'-二氯荧光素二乙酸酯荧光法检测ROS积累。通过荧光染色和硫代巴比妥酸反应性物质（TBARS）测定评估脂质过氧化。使用二硝基苯肼衍生化法测定蛋白质氧化。使用市售检测试剂盒测量抗氧化酶活性。采用实时RT-PCR检测超氧化物歧化酶（SOD）基因表达。为评估其抗真菌能力及对ROS积累的作用，同时使用厚朴酚和SOD抑制剂N,N'-二乙基二硫代氨基甲酸盐（DDC）。通过测量线粒体膜电位（mtΔψ）评估线粒体功能障碍。使用Annexin V-FITC细胞凋亡检测试剂盒评估厚朴酚诱导的细胞凋亡。
厚朴酚处理后，白色念珠菌中的ROS、脂质过氧化和蛋白质氧化呈剂量依赖性发生。厚朴酚导致抗氧化酶活性增加。此外，厚朴酚处理诱导白色念珠菌细胞中SOD基因表达。而且，添加DDC导致内源性ROS水平升高，并增强了厚朴酚的抗真菌活性。通过测量mtΔψ的变化证实了线粒体功能障碍。厚朴酚处理后，细胞凋亡水平呈剂量依赖性增加。
总体而言，这些结果表明厚朴酚在白色念珠菌中作为促氧化剂起作用。此外，SOD抑制剂DDC可用于增强厚朴酚对白色念珠菌的活性。
