粉防己碱抑制耐氟康唑热带念珠菌外排泵机制的研究

Mechanism underlying inhibitory effect of tetrandrine on efflux pump in fluconazole- resistant Candida tropicalis

目的探讨粉防己碱抑制耐氟康唑热带念珠菌外排泵的机制。方法通过MTT法确定24h粉防己碱对耐氟康唑热带念珠菌的无细胞毒性剂量，并以此剂量处理5株耐氟康唑热带念珠菌。粉防己碱组（20mg／L）及对照组（PBS）加入不同浓度的氟康唑（10～50mg/L）处理48h以测定MIC值。半定量PCR分析粉防己碱（20mg/L）作用24h前后，耐氟康唑热带念珠菌外排泵基因CDR1和MDR1转录水平的变化；流式细胞术检测粉防己碱（20mg／L）作用24h后对耐氟康唑热带念珠菌罗丹明123的蓄积能力的影响。结果粉防己碱对耐氟康唑热带念珠菌的最大无细胞毒性剂量为20mg／L。以此浓度处理5株耐氟康唑热带念珠菌，与对照组相比，MIC的范围由64（64～128）mg/L减少到32（24-64）mg/L（P〈0．05）。粉防己碱可以将耐氟康唑热带念珠菌CDR1和MDR1基因mRNA水平的表达由0．905±0．026及b．649±0．028分别下调至0．715±0．131和0．342±0．068（均P〈0．05）。粉防己碱作用后的耐氟康唑热带念珠菌对罗丹明123的蓄积量提升至（37．7±3．5）％，高于对照组的（16．3±2．2）％（P〈0．05）。结论粉防己碱可以减少耐氟康唑热带念珠菌外排蛋白的表达，增加细胞内药物的蓄积，从而抑制氟康唑的耐药作用。

Objective To explore the mechanism underlying inhibitory effect of tetrandrine on the efflux pump in fluconazole-resistant Candida tropicalis. Methods The non-cytotoxic dose of tetrandrine to Cadida tropicalis at 24 h was determined by MTT method. Tetrandrine by the determined dose was applied to five strains of fluconazole-resistant Candida tropicalis. The tetrandrine-treated group （20 mg/L） and the control group （ PBS ） of Candida tropicalis were then added with various concentrations of fluconazole （ 10- 50 mg/L） for 48 h to determine the minimal inhibitory concentration （MIC）. Semi-quantitative reverse transcription-PCR was used to examine the relative transcriptional level shift of CDR1 and MDR1 genes in these fluconazole- resistant strains before and after treatment with 20 mg/L tetrandrine for 24 h. Flow-cytometry was used to determine the effect of tetrandrine on rhodamine 123 accumulation capacity in Candida tropicalis at 24 h. Results The maximum non-cytotoxic dose of tetrandrine to Candida tropicalis was 20 mg/L as determined by MTT assay. Compared with the control group, in the 5 strains of fluconazole- resistant Candida tropicalis treated with maximum non-cytotoxic dose, the MIC of fluconazole decreased from 64 （range 64-128） mg/L to 32 （range 24-64） mg/L （P〈0.05）. Reverse transcription-PCR showed that the mRNA expression levels of CDR1 and MDR1 genes was reduced from 0.905±0.026 and 0.649±0.028 to 0.715 ±0.131 and 0.342±0.068, respectively （all P〈0.05 ）. After treatment with tetrandrine, the rhodamine 123 accumulation of fluconazole-resistant Candida tropicalis was elevated to （37.7±3.5）%, compared with （16.3±2.2）% in the controls （P〈0.05）. Conclusion Tetrandrine may down-regulate the expression of efflux pump genes and increase intracellular drug accumulation in fluconazole-resistant Candida tropicalis, thereby inhibiting the fluconazole resistance in this fungus.