穿心莲内酯联合氟康唑抗耐药白假丝酵母菌作用机制研究

目的探讨穿心莲内酯联合氟康唑抗耐药白假丝酵母菌作用及其机制。方法采用微量稀释法检测穿心莲内酯（AG）及联合氟康唑（FLC）对耐药白假丝酵母菌的MIC；采用罗丹明6G（Rh6G）检测AG对耐药白假丝酵母菌CDR外排功能的影响；利用罗丹明123评估AG对耐药白假丝酵母菌MDR外排功能的影响：采用二氢罗丹明检测AG单用及联合FLC对耐药白假丝酵母菌活性氧（ROS）的影响；采用实时荧光定量PCR（qRT—PCR）检测AG联合FLC对耐药白假丝酵母菌外排泵相关基因CDR1、CDR2和MDR1表达的影响。结果AG联合FLC抗耐药白假丝酵母菌呈相加作用；AG对CDR外排功能无影响；AG可抑制MDR外排功能；AG联合FLC能显著提高耐药白假丝酵母菌细胞ROS水平；AG与FLC联合作用于耐药白假丝酵母菌可下调CDR1和MDR1的表达量，上调CDR2的表达量。结论AG联合FLC抗耐药白假丝酵母菌具有相加作用，其机制可能与抑制外排泵及相关基因表达，提高胞内ROS水平有关。

Mechanisms of the combination effects of Andrographolide and Fluconazole

Objective To investigate effects of Andrographolide combination with fluconazole on fluconazole-resist- ance in Candida albicans and its mechanism. Methods The antifungal activities of AG and FLC and determine minimum inhibitory concentration （MIC） were evaluated by mierodilution method on a fluconazole-resistant Candi- da albicans isolate 215 ; The efflux of Rhodamine 6G and Rhodamine 123 were observed in Candida albicans mem- brane; The levels of reactive oxygen species （ROS） with DHR staining was measured by flow eytometry; Efflux-re- ated genes expression of CDR1 ,CDR2 and MDR1 were inspected by the real-time fluorescence quantification PCR. Results The antifungal effects showed significantly enhanced by AG combination with FLC, and the combination showed clear collaboration; AG could not affect the efflux of rhodamine 6G, and it dramatically reduce efflux of rhodamine 123 and promote the production of intraeelluar ROS with dose-dependent mode; And expressions of CDR1 and MDR1 were significantly downregulated, while the expression of CDR2 was upregulated by qRT-PCR.Conclusion The study indicates that AG can enhance the antifungal activity of FLC by inhibiting the ef？ ux of FLC from C. albicans and raising the levels of ROS.