Chang Wen-Qiang, Wu Xiu-Zhen, Cheng Ai-Xia, Zhang Li, Ji Mei, Lou Hong-Xiang
Department of Natural Product Chemistry, Shandong University, Jinan, People's Republic of China.
Biochim Biophys Acta. 2011 May;1810(5):569-76. doi: 10.1016/j.bbagen.2011.02.001. Epub 2011 Feb 21.
Retigeric acid B (RAB), a triterpene acid isolated from Lobaria kurokawae exerts antifungal effect. The present study was designed to elucidate the underlying mechanisms by which RAB regulates the proliferation and cell death of Candida albicans.
We measured the metabolic activity of C. albicans with WST1 Cell Proliferation and Cytotoxicity Assay Kit, analyzed the cell cycle by flow cytometry, visualized the ultrastructure by transmission electron microscopy (TEM) and investigated the apoptosis and necrosis induced by RAB using confocal microscopy. The reactive oxygen species (ROS) accumulation was determined by spectrophotometry, flow cytometry and fluorescent microscopy. The mtΔψ was detected using flow cytometry. And the levels of intracellular cAMP and ATP were measured with cAMP ELISA and ATP Assay Kits, respectively.
The proliferation of the yeasts was blocked in G(2)/M phase by a low dose of RAB treatment and in G(1) phase at high concentration. When cultured in phosphate buffered saline (PBS) deprived of energy source, yeasts displayed the phenotype of death caused by accumulated ROS, mtΔψ hyperpolarization and dramatic decrease in ATP level in the presence of high dose of RAB.
RAB inhibits the growth of C. albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB. Our findings provide a novel molecular mechanism for exploring possible applications of lichen derived metabolites in fighting fungal infection in humans.
从黑脉梅花衣中分离得到的三萜酸——网肺衣酸B（RAB）具有抗真菌作用。本研究旨在阐明RAB调节白色念珠菌增殖和细胞死亡的潜在机制。
我们使用WST1细胞增殖和细胞毒性检测试剂盒测量白色念珠菌的代谢活性，通过流式细胞术分析细胞周期，用透射电子显微镜（TEM）观察超微结构，并使用共聚焦显微镜研究RAB诱导的凋亡和坏死。通过分光光度法、流式细胞术和荧光显微镜测定活性氧（ROS）的积累。使用流式细胞术检测线粒体膜电位（mtΔψ）。分别用cAMP ELISA试剂盒和ATP检测试剂盒测量细胞内cAMP和ATP的水平。
低剂量RAB处理可使酵母细胞的增殖在G(2)/M期受阻，高浓度时则在G(1)期受阻。当在缺乏能量源的磷酸盐缓冲盐水（PBS）中培养时，在高剂量RAB存在的情况下，酵母细胞表现出因ROS积累、mtΔψ超极化和ATP水平显著降低而导致的死亡表型。
RAB通过刺激ROS产生和降低细胞内cAMP来抑制白色念珠菌的生长。ROS积累、mtΔψ超极化、ATP耗竭和质膜完整性受损共同介导了RAB诱导的白色念珠菌细胞死亡。我们的研究结果为探索地衣衍生代谢物在对抗人类真菌感染中的可能应用提供了一种新的分子机制。
