茶皂素诱导活性氧产生介导白色念珠菌线粒体功能障碍

doi:10.5246/jcps.2021.11.077

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (11): 895-903.DOI: 10.5246/jcps.2021.11.077

茶皂素诱导活性氧产生介导白色念珠菌线粒体功能障碍

李诗慧¹, 郭可蒙¹, 尹妮颖¹, 单明珠¹, 祝垚², 李颖¹^,^*()

1. 徐州医科大学医学技术学院, 徐州市实验室诊断学重点实验室, 江苏徐州 221004
2. 徐州医科大学遗传学系, 江苏徐州 221004

收稿日期:2021-05-18 修回日期:2021-06-21 接受日期:2021-08-12 出版日期:2021-11-28 发布日期:2021-11-28
通讯作者: 李颖
作者简介:
+ Tel.: +86-18605167210, E-mail: sdwhrsly@163.com
基金资助:
Jiangsu Province College Students Innovation and Entrepreneurship training program (Grant No. 201910313016Z).

Teasaponin induces reactive oxygen species-mediated mitochondrial dysfunction in Candida albicans

Shihui Li¹, Kemeng Guo¹, Nining Yin¹, Mingzhu Shan¹, Yao Zhu², Ying Li¹^,^*()

1 Medical Technology School of Xuzhou Medical University, Xuzhou Key Laboratory of Laboratory Diagnostics, Xuzhou 221004, China
2 Department of Genetics, Xuzhou Medical University, Xuzhou 221004, China

Received:2021-05-18 Revised:2021-06-21 Accepted:2021-08-12 Online:2021-11-28 Published:2021-11-28
Contact: Ying Li

摘要/Abstract

摘要：

白色念珠菌是目前最常见的共生真菌, 在免疫功能低下的个体中很容易引起侵袭性真菌感染。茶皂素(TS)是一种安全的天然产物, 已有报道可以抑制白色念珠菌的丝状生长。本研究发现TS对白色念珠菌具有中度的杀菌作用, 并进一步探索其杀菌机制。采用微量肉汤稀释法测定最小杀菌浓度(MFC)。采用菌落计数法, 测定TS对白色念珠菌的杀菌曲线。采用2,7-二氯二氢荧光素双乙酸(DCFH-DA)染色法分析TS对白念珠菌内活性氧(ROS)含量的影响, 使用罗丹明123染色法测定线粒体膜电位。使用ATP检测试剂盒测定TS处理后白念珠菌内ATP水平。结果表明, TS诱导的ROS积累和线粒体功能障碍导致白念珠菌死亡。进一步的研究表明,维生素C可以加强TS的抗菌能力,相反维生素E拮抗TS杀伤白念珠菌的功能, 这可以指导临床TS的应用。研究结果初步阐明了TS杀伤白念珠菌的机制, 并提供了一个治疗临床念珠菌药物选择。

关键词: 白色念珠菌, 茶皂素, 杀菌活性, 活性氧, 线粒体

Abstract:

Candida albicans is the most prevalent commensal fungus and readily causes invasive fungal infection in immunocompromised individuals. Teasaponin (TS), a natural product generally regarded as safe, has been reported to inhibit filamentation of C. albicans. This study found that TS could exert moderate fungicidal activity against C. albicans, and the mode of action was further explored. The minimum fungicidal concentration (MFC) was determined by the broth microdilution method. The colony counting method was used to determine the time-killing curve of TS against C. albicans in every 2 h. The effect of TS on the content of intracellular reactive oxygen species (ROS) in C. albicans was analyzed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) staining, and the mitochondrial membrane potential (mtΔψ) was determined by rhodamine123 (RH123) staining. An ATP assay kit was utilized to determine the intracellular ATP levels after TS treatment. Results showed that TS-induced ROS accumulation and mitochondrial dysfunction contributed to the death of C. albicans cells. Further research demonstrated that VC could reinforce the fungicidal ability of TS. On the contrary, VE could antagonize the function of TS against C. albicans, which might guide the clinical application of TS. The results preliminarily elucidated the potential mechanisms of TS against C. albicans and might provide a potential option for the treatment of clinical Candida.

Key words: Candida albicans, Teasaponin, Fungicidal activity, Reactive oxygen species, Mitochondria

Supporting:

李诗慧, 郭可蒙, 尹妮颖, 单明珠, 祝垚, 李颖. 茶皂素诱导活性氧产生介导白色念珠菌线粒体功能障碍[J]. 中国药学(英文版), 2021, 30(11): 895-903.

Shihui Li, Kemeng Guo, Nining Yin, Mingzhu Shan, Yao Zhu, Ying Li. Teasaponin induces reactive oxygen species-mediated mitochondrial dysfunction in Candida albicans[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(11): 895-903.

图/表 4

Figure 1. The fungicidal activity of TS against C. albicans. (A) MIC and MFC of TS against different species of Candida. (B) The time-killing curves of wild-type C. albicans strain YEM30 under TS (64 μg/mL) and AMB (2 μg/mL) treatment were tested by colony counting method. Bars mean standard deviation.

Figure 2. Effects of TS on intracellular ROS production. (A) The relative fluorescence intensity of cells treated with DCFH-DA staining was measured with a fluorescence photometer. The data represent the mean, and the error bar represents the standard deviation. TS concentrations of 16, 32 and 64 μg/mL were statistically different from the control group. Similarly, there was a statistically significant difference between 64 μg/mL TS with Tu supplementation and 64 μg/mL TS. **Means statistically significant difference (P < 0.01). (B) Fluorescence microscope observation of C. albicans YEM30 after DCFH-DA staining with different treatments. The scale bar represents 20 μm.

Figure 3. Destruction of TS on C. albicans mitochondrial. (A) Fluorescence microscope observation of C. albicans YEM30 after DCFH-DA staining with different treatments. The scale bar represents 20 μm. (B) Relative fluorescence intensity was measured to indicate the mitochondrial membrane potential by fluorescence spectroscopy. The expression of fluorescence intensity in the TS-treated groups was significantly higher compared with the control group (P < 0.01). A significant difference could be observed between 64 μg/mL TS with Tu supplementation and 64 μg/mL TS groups. (C) Intracellular ATP levels of YEM30 in different treatment states were determined using ATP kits. Levels of ATP in C. albicans YEM30 after TS treatment. ATP production was evaluated by fluorometric detection. Each column represents the mean of each group, and the error bar represents the standard deviation. Compared with the control group, the ATP level of the cells exposed to 32, 64 and 128 μg/mL was significantly different. *Indicates that the difference was statistically significant (P < 0.05).

Figure 4. Effect of different antioxidant Tu, VC and VE on the fungicidal activity of TS. YEM30 pretreated with Tu (A), VC (B) or VE (B) was exposed to 64 μg/mL TS at 30 oC for 3 h. The number of viable cells was then determined by the colony counting method. At the concentration of 64 μg/mL, the survival rate of C. albicans with Tu, VC or VE was statistically different from the control group (P < 0.05). The bars indicate standard deviations.

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茶皂素诱导活性氧产生介导白色念珠菌线粒体功能障碍

Teasaponin induces reactive oxygen species-mediated mitochondrial dysfunction in Candida albicans

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