和厚朴酚通过ROS的积累和破坏细胞膜杀死白色念珠菌

Honokiol kills Candida albicans through ROS accumulation and cell membrane destruction

摘要:

[目的]研究在体外情况下和厚朴酚对白色念珠菌的抑制作用及其可能机制。[方法]采用微量稀释法测定和厚朴酚对白色念珠菌的最低抑菌浓度（MIC₈₀）和最低杀菌浓度（MFC）；用透射电镜观察不同浓度和厚朴酚对白色念珠菌超微结构的影响；采用Annexin V-FITC/PI染色法分析不同浓度和厚朴酚对白色念珠菌细胞凋亡的影响；用DCFH-DA染色法测定不同浓度和厚朴酚对白色念珠菌细胞内活性氧积累的影响；用JC-1染色法分析不同浓度和厚朴酚对白色念珠菌线粒体膜电位的影响；用碘化丙啶染色、考马斯亮蓝G-250染色检测和厚朴酚对白色念珠菌细胞膜通透性的影响；通过测定加入麦角甾醇后，和厚朴酚对白色念珠菌的抑制作用的变化，检测和厚朴酚对白色念珠菌细胞膜的影响。[结果]和厚朴酚对白色念珠菌具有很强的抑制作用，MIC和MFC分别为16 μg/mL和32 μg/mL。对白色念珠菌细胞壁、细胞膜和胞浆均有明显的影响。和厚朴酚是通过增加活性氧的产生和破坏线粒体功能来诱导白念珠菌的细胞凋亡和坏死。它也影响细胞膜的通透性，这可能和细胞壁的破坏和与麦角固醇的结合有关。[结论]和厚朴酚通过产生活性氧并伴随着一系列的细胞损伤这种复杂的机制从而对白色念珠菌产生抑制作用，使和厚朴酚成为一种潜在的抗真菌药物。

Abstract:

[Objective] To investigate the inhibitory effect of honokiol on Candida albicans in vitro. [Methods] The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of magnolol for C. albicans were determined by microdilution method. Transmission electron microscopy was used to examine the effect of different concentrations of honokiol on the ultrastructure of C. albicans. Apoptosis of C. albicans caused by honokiol was analyzed by Annexin V-FITC/propidium iodide (PI) double staining and the accumulation of reactive oxygen species (ROS) in C. albicans cells was determined by DCFH-DA staining. The effects of honokiol on mitochondrial membrane potential and cell membrane permeability of C. albicans were analyzed by JC-1 staining and PI staining respectively. [Results] Honokiol had strong inhibitory effect on C. albicans, with MIC₉₀ of 16 µg/mL and MFC of 32 µg/mL. Honokiol affected cell wall, cell membrane, and cytoplasm of C. albicans, induced both apoptosis and necrosis in C. albicans, probably through ROS production and disruption of mitochondrial function. It also changed the permeability of cell membrane, leaded to the cell wall damage and the binding of ergosterol. [Conclusion] Inhibition of C. albicans by honikiol involves multiple mechanisms including ROS production accompanied by a series of cellular damages. Honikiol appears to be a potential antifungal drug candidate.