盐酸小檗碱对白假丝酵母生物膜形成抑制作用的研究

Inhibitory effect of berberine hydrochloride on biofilm formation of Candida albicans

目的探讨盐酸小檗碱对白假丝酵母生物膜表型的抑制作用及其机制。方法采用甲基四氮盐(XTT)减低法、激光共聚焦显微镜和实时定量PCR(RT-qPCR)分别检测不同浓度盐酸小檗碱作用下白假丝酵母生物膜膜内细胞代谢活性、微观形态结构、厚度和生物膜形成关键基因efg1、hwp1、ece1、als1表达水平的变化。结果128μg/ml和32μg/ml盐酸小檗碱对白假丝酵母标准菌株ATCC 10231生物膜膜内细胞活性的抑制率分别为:88.4%和34.3%(4 h),96.3%和47.6%(6 h),对白假丝酵母临床菌株CA 2119的抑制率分别为:77.2%和29.8%(4 h),72.8%和43.2%(6 h);对ATCC 10231生物膜厚度的抑制率分别为:61.4%和41.03%(4 h),53.49%和39.53%(6 h),对CA 2119的抑制率分别为:45.45%和27.27%(4 h),45%和25%(6 h);而8μg/ml盐酸小檗碱抑制效果较差。128μg/ml盐酸小檗碱抑制作用优于4μg/ml氟康唑(P<0.05),但对CA 2119生物膜的抑制效果不及ATCC 10231;RT-qPCR结果表明高浓度和中浓度盐酸小檗碱均能明显抑制ATCC 10231生物膜形成关键基因efg1、hwp1、ece1、als1的表达(P<0.05),但对CA 2119生物膜形成关键基因表达的抑制作用较差。结论盐酸小檗碱通过杀死生物膜中的细胞并破坏其结构来抑制白假丝酵母ATCC 10231和CA 2119早期生物膜的形成,其抑制作用与浓度呈正相关;其机制可能是通过下调菌丝形成关键基因efg1、hwp1、ece1、als1的表达,从而延缓白假丝酵母的形态转化。

OBJECTIVE To explore the inhibitory effect of berberine hydrochloride on biofilm phenotypes of Candida albicans and analyze its mechanisms.METHODS The intramembrane cellular metabolic activity,micromorphology and thickness of biofilms of C.albicans as well as the levels of key genes for biofilm formation,including efg1,hwp1,ece1 and als1,were detected under different concentrations of berberine hydrochloride by respectively using methyltetrazide salt(XTT)reduction method,confocal laser scanning microscopy(CLSM)and real-time quantitative PCR(RT-qPCR).RESULTS The inhibition rates of 128μg/ml and 32μg/ml of berberine hydrochloride against intramembrane cellular activity of biofilm K of C.albican standard strain ATCC 10231 were 88.4%and 34.3%(4 h),96.3%and 47.6%(6 h),respectively;the inhibition rates against the clinical strain C.albicans CA 2119 were 77.2%and 29.8%(4 h),72.8%and 43.2%(6 h),respectively;the inhibition rates against the thickness of ATCC 10231 biofilm were 61.4%and 41.03%(4 h),53.49%and 39.53%(6 h),respectively;the inhibition rates against CA 2119 were 45.45%and 27.27%(4 h),45%and 25%(6 h),respectively;while the inhibitory effect of 8μg/ml of berberine hydrochloride was less satisfied.The inhibitory effect of 128μg/ml of berberine hydrochloride was significantly better than that of 4μg/ml of fluconazole(P<0.05),however,the inhibitory effect on biofilm of CA 2119 was not as good as on ATCC 10231.RT-qPCR showed that both high concentration and medium concentration of berberine hydrochloride could significantly inhibit the expression of key genes for biofilm formation of ATCC 10231 such as efg1,hwp1,ece1 and als1(P<0.05)but had less remarkable inhibitory effect on the expression of key genes for biofilm formation of CA2119.CONCLUSION Berberine hydrochloride can inhibit the formation of biofilm of C.albicans ATCC 10231 and CA 2119 in early stage by killing the cells in the biofilm and destroying its structure,and the inhibitory effect is positively correlated with the concentration.Its possible mechanism is to down-regulate the expression of key genes in hyphae formation efg1,hwp1,ece1 and als1,thereby,retarding the morphological transformation of C.albicans.