中药蛴螬多肽Probrelin抗白色念珠菌活性研究

Activity of the antimicrobial peptide probrelin from traditional Chinese medicine grub against Candida albicans

摘要:

[目的] 研究蛴螬多肽Probrelin对白色念珠菌的抗菌活性。[方法] 采用肉汤稀释法测定蛴螬多肽Probrelin对正常菌株及临床耐药菌株的最小抑菌浓度，同时结合平板计数法测定最小杀真菌浓度；通过不同浓度多肽处理后经平板计数绘制时间-杀菌动力学曲线；通过PI吸收实验检测多肽对白色念珠菌细胞膜完整性的影响；通过核酸阻滞实验检测多肽与核酸间是否具有结合作用；通过扫描电子显微镜检测多肽对白色念珠菌形态的影响；通过结晶紫染色法检测多肽对生物膜生成及成熟生物膜的影响；通过显微镜观察多肽对白色念珠菌菌丝形成的影响；通过棋盘法检测多肽与抗真菌药物间的相互效应；通过小鼠皮下感染模型检测多肽在生理条件下的抗白色念珠菌活性。[结果] 蛴螬多肽Probrelin对正常菌株及临床耐药菌株的最小抑菌浓度均为100 μg/mL，最小杀真菌浓度为100-200 μg/mL，且对白色念珠菌的杀菌动力学具有时间和浓度依赖性；该多肽以浓度依赖性的方式影响白色念珠菌细胞膜的完整性，并通过破坏白色念珠菌细胞壁的结构影响其形态，但与核酸间不具有结合作用；该多肽既可抑制白色念珠菌生物膜的形成，又可清除成熟生物膜，同时还可抑制白色念珠菌菌丝的形成；该多肽与抗真菌药物Clotrimazole间具有协同效应；在小鼠皮下感染模型中，该多肽可以有效杀灭白色念珠菌，进而抑制感染。[结论] 蛴螬多肽Probrelin对白色念珠菌具有良好的抑制杀灭活性，可以作为新的药物分子或模板分子用于抗白色念珠菌药物的研发。

关键词:中药;抗微生物多肽;白色念珠菌;生物膜;协同效应

Abstract:

[Objective] To investigate the activity of the peptide probrelin from traditional Chinese medicine grub against Candida albicans. [Methods] The broth microdilution method was used to determine the MIC of probrelin against normal and clinical resistant strains of C. albicans, and the MFC were determined by plate count method. Time-kill curves were drawn by plate counting after treatment with different concentrations of probrelin. The influences of probrelin on the integrity of the cell membrane and morphology of C. albicans were evaluated by PI absorption experiment and scanning electron microscope, respectively. The binding effect between probrelin and nucleic acid was evaluated by DNA gel retardation assay. Crystal violet staining method was used to evaluate the influences of probrelin on the formation of biofilm and mature biofilm of C. albicans. And the influences of probrelin on the hyphae formation of C. albicans were also tested. Checkerboard method was used to determine the interaction between probrelin and antifungal drugs. And a mouse subcutaneous infection model was used to tested the activity of probrelin against C. albicans under physiological conditions. [Results] The MICs of probrelin against normal and clinical resistant strains of C. albicans were 100 μg/mL, and the MFCs were 100-200 μg/mL. Probrelin showed time-and concentration-dependent antifungal activity against C. albicans, as well as on the integrity of the cell membrane, and could destroy the cell wall, but did not bind with nucleic acids. Probrelin not only inhibited the formation of the biofilm of C. albicans, but also disrupted the mature biofilm, and could also inhibit the hyphae formation. Furthermore, probrelin showed synergistic effect with the antifungal drug Clotrimazole, and could effectively clear C. albicans cells in the mouse subcutaneous infection model. [Conclusion] Probrelin showed good activity against C. albicans, and had the potential for the development of anti-C. albicans drugs.

Key words:traditional Chinese medicine;antimicrobial peptides;Candida albicans;biofilm;synergistic effect