盐酸小檗碱与氟康唑联用对耐药白色念珠菌钙稳态的影响

The effect of berberine hydroch loride combined with fluconazole on the calcium homeostasis of flucon-azole-resistant Candida albicans

目的探讨盐酸小檗碱(berberine hydrochloride,BBH)与氟康唑(fluconazole,FLC)联用对氟康唑耐药白色念珠菌(Candida albicans,CA)的协同抑菌作用及其对细胞内Ca2+稳态的影响。方法棋盘稀释法筛选对白色念珠菌耐药株具有协同抑制作用的最低浓度盐酸小檗碱与氟康唑组合,XTT减低法测定动态抑菌效果。采用倒置荧光显微镜和流式细胞术检测细胞内Ca2+内流情况,RT-qPCR技术检测Ca2+稳态关键基因的表达量,评价两药联用对耐药白色念珠菌Ca2+稳态的影响。试验设两组单用组及不加药(空白)组作对照。结果联合用药的部分抑菌浓度指数最小的药物浓度组合为盐酸小檗碱2μg/ml+氟康唑1μg/ml。两药联用组在0~48h内动态抑菌效果显著优于两药单用组和空白对照组(P<0.05),两药联用具有协同作用(FICI<0.5)。联用组、盐酸小檗碱单用组处理后白色念珠菌细胞内Ca2+内流现象明显,且联用组细胞内Ca2+浓度均比盐酸小檗碱单用组和氟康唑单用组分别高1.07-2、1.17-8.39倍(P<0.05),但氟康唑单用组与空白对照组比较差异无统计学意义(P>0.05)。联用组和盐酸小檗碱单用组液泡钙通道YVC1基因上调,氟康唑单用组YVC1基因下调,联用组YVC1基因表达量比盐酸小檗碱单用组和氟康唑单用组分别高1.62、6.47倍(P<0.05)。联用组和盐酸小檗碱单用组液泡钙泵PMC1基因下调,氟康唑单用组PMC1基因上调,联用组和盐酸小檗碱单用组PMC1基因的表达量比氟康唑单用组分别低80.86%和80.59%(P<0.05)。结论盐酸小檗碱与氟康唑联用对氟康唑耐药白色念珠菌具有协同抑菌作用,盐酸小檗碱可能通过影响白色念珠菌液泡膜上的钙离子调节器Pmc1p和Yvc1p促进质膜内Ca2+内流,破坏钙离子稳态,从而增加耐药株对氟康唑药物的敏感性。

Objective To investigate the synergistic effect of berberine hydrochloride(BBH)combined with fluconazole(FLC)on FLC-resistant Candida albicans and to examine its effect on intracellular Ca2+ homeostasis.Methods The lowest concentration of the drug combination against FLC-resistant C.albicans was screened using the checkerboard dilution method,and dynamic antifungal activity was determined XXT reduction.Intracellular Ca2+ influx was detected using inverted fluorescence microscopy and flow cytometry.RT-qPCR was used to examine the expression of key genes related to Ca2+ homeostasis to investigate the effect of the drug combination on the Ca2+ homeostasis in FLC-resistant C.albicans.The experiment involved groups receiving each drug alone and an untreated control group. Results The drug combination with the lowest FICI value was 2μg/mL of BBH plus 1μg/mL of FLC.Dynamic antifungal activity in the group receiving combined therapy was significantly higher than that in the groups receiving one drug alone and the control group within 0-48 h(P<0.05),indicating that BBH combined with FLC had a strong synergistic effect(FICI<0.5).Intracellular Ca2+ influx was marked in the BBH + FLC group and the BBH group infected with C.albicans.The intracellular Ca2+ concentration in the BBH + FLC group was 1.07-2 times higher than that in the BBH group and 1.17-8.39 times higher than that in the FLC group(P<0.05).The intracellular Ca2+ concentration did not differ significantly between the FLC group and the control group(P>0.05).The vacuolar calcium channel gene YVC1 was up-regulated in the BBH+FLC group and the BBH group,but down-regulated in the FLC group.YVC1 gene expression in the BBH+FLC group was 1.62 higher than that in the BBH group and 6.47 times higher than that in the FLC group.The vacuolar calcium pump gene PMC1 was down-regulated in the BBH+FLC group and the BBH group,but up-regulated in the FLC group.PMC1 gene expression in the BBH+FLC group was 80.86%lower than that in the FLC group,and that gene expression in the BBH group was 80.59%lower than that in the FLC group(P<0.05).Conclusion BBH plus FLC had a synergistic effect on FLC-resistant C.albicans.BBH may increase the intracellular Ca2+ concentration by affecting the calcium regulators Pmc1p and Yvc1p on the vacuole membrane in C.albicans,disrupting calcium homeostasis,and increasing FLC sensitivity in resistant strains.