盐酸小檗碱对菌丝相白念珠菌细胞壁完整性的影响

Effect of berberine hydrochloride on cell wall integrity of Candida albicans hypha

探讨盐酸小檗碱对菌丝相白念珠菌细胞壁完整性的影响。以微量液基稀释法检测盐酸小檗碱对白念珠菌标准株与临床株的最低抑菌浓度(MIC);spot assay检测白念珠菌落形成;XTT法检测白念珠菌丝代谢;荧光显微镜观察白念珠菌丝活力;扫描电镜观察白念珠菌丝形态;透射电镜观察白念珠菌丝细胞壁结构;流式细胞术检测白念珠菌细胞壁β-葡聚糖暴露程度;qRT-PCR法检测白念珠菌丝特异性基因ECE1及β-葡聚糖合酶调控基因FKS1和FKS2的表达。结果显示,盐酸小檗碱对白念珠菌临床株及标准株的MIC为64~128μg·mL^(-1);512μg·mL^(-1)盐酸小檗碱干预下能抑制白念珠菌落生长,64~512μg·mL^(-1)盐酸小檗碱能抑制菌丝代谢,512μg·mL^(-1)盐酸小檗碱能抑制白念珠菌丝活力,256~512μg·mL^(-1)盐酸小檗碱能抑制形态转化,512μg·mL^(-1)盐酸小檗碱能明显损伤细胞壁结构,128~512μg·mL^(-1)盐酸小檗能促进细胞壁β-葡聚糖暴露,512μg·mL^(-1)盐酸小檗碱能下调白念珠菌丝特异性基因ECE12.72倍及β-葡聚糖合成酶调控基因FKS1与FKS2分别3.49,3.26倍。研究表明,一定浓度的盐酸小檗碱可能通过下调白念珠菌丝特异性基因及β-葡聚糖合成酶相关基因的表达,从而破坏白念珠菌细胞壁的结构并促使β-葡聚糖暴露,进而影响整个细胞壁的完整性。

The aim of this paper was to investigate the effect of berberine hydrochloride on the cell wall integrity of Candida albicans hypha.The minimal inhibitory concentration(MIC)of berberine hydrochloride against clinical and standard C.albicans strains was detected by micro liquid-based dilution method;the effect of berberine hydrochloride on the colony formation of C.albicans SC5314 was investigated by spot assay;the effect of berberine hydrochloride on the metabolism of C.albicans SC5314 hypha was checked by XTT reduction assay,and the viability of C.albicans SC5314 hypha was tested by fluorescent staining assay.The effect of berberine hydrochloride on the morphology of C.albicans SC5314 hypha was examined by scanning electron microscope.The changes in the cell wall of C.albicans SC5314 hypha after berberine hydrochloride treatment were detected by transmission electron microscopy.The effect of berberine hydrochloride onβ-glucan from C.albicans SC5314 was detected by flow cytometry.The effect of berberine hydrochloride on hy-pha-specific gene ECE1 andβ-glucan synthase genes FKS1 and FKS2 in C.albicans was examined by qRT-PCR.The results showed that berberine hydrochloride showed a strong inhibitory effect on both clinical and standard strains of C.albicans,and the MIC was 64-128μg·mL^(-1).Spot assay,XTT redunction assay and fluorescent staining assay showed that with the increase of berberine hydrochloride concentration,the viability of C.albicans SC5314 gradually decreased.The transmission electron microscopy scanning assay showed that this compound could cause cell wall damage of C.albicans.The flow cytometry analysis showed the exposure degree of C.albicansβ-glucan.The qRT-PCR further showed that berberine hydrochloride could significantly down-regulate hypha-specific gene ECE1 andβ-glucan synthase-related gene FKS1 and FKS2.In conclusion,this compound can down-regulate C.albicans andβ-glucan synthase-related gene expressions,so as to destroy the cell wall structure of C.albicans,exposeβ-glucan and damage the integrity of the wall.