白头翁汤正丁醇提取物对白念珠菌 VVC 临床株体外生物膜形成的抑制作用

摘要

Objective To investigate the effects of Butyl alcohol extract of BaiTouWeng decoction (BAEB)on the biofilm formation of Candida albicans clinical strains isolated from vulvovaginal Candidiasis (VVC).Methods Microdilution meth-ods was used to determine the MIC.XTT reduction assay was applied to determine the SMIC80 .Time-kill curve method was applied to detect the effects of BAEB on living cells of Candida albicans .Crystal violet staining method was used to determine the biomass of the biofilm.Scanning electron microscopy (SEM)was applied to observe the morphological changes of the bio-film.Confocal laser scanning microscopy (CLSM)was applied to determine the thickness of the biofilm.The quantification re-al-time PCR (qRT-PCR)was used to detect expression changes of genes (HSP90 ,UME6 and PES1 )of the biofilm treated by BAEB.Results The MICs of BAEB against C .albicans strains are determined as 64~256 μg/mL.The SMIC80 s of BAEB against the biofilm of C .albicans strains are determined as ≥1 024 μg/mL.Time-kill curve results indicate that BAEB has a promise antifungal effect at concentrations of 5 12 and 1 024 μg/mL.Crystal violet staining results show that the biomass of C .albicans is reduced by BAEB at 5 12 and 1 024 μg/mL.SEM results indicate that the formation of C .albicans biofilm carriers is inhibited by BAEB on different adhesion,and the morphol-ogy of biofilm is also affected by BAEB.The thickness of C .albicans biofilm is reduced by BAEB accord-ing to CLSM results.Furthermore,qRT-PCR results indicate that expression of UME6 is significantly down-regulated by BAEB at 256,5 12,1 024 μg/mL,and HSP90 is up-regulated at 5 12 and 1 024 μg/mL of BAEB,and PES1 is not affected by BAEB at any concentration.Conclusion BAEB inhibits effectively the biofilm formation of VVC strains of C . albicans .%目的：探讨白头翁汤正丁醇提取物(Butyl alcohol extract of Bai Tou Weng decoction,BAEB)对分离自外阴阴道念珠菌病(vulvovaginal candidiasis,VVC)的白念珠菌临床分离株(以下简称 VVC 临床株)生物膜形成的影响。方法采用微量稀释法测定 BAEB 对白念珠菌的最低抑菌浓度(Minimal Inhibitory Concentration,MIC)；甲基四氮盐(XTT)还原法测定 BAEB 对白念珠菌生物膜代谢活性的影响,Time-kill 法检测 BAEB 对白念珠菌活菌数的影响；结晶紫染色法测定BAEB 对白念珠菌生物膜生物量(Biomass)的影响；扫描电镜(SEM)观察 BAEB 对白念珠菌生物膜形态结构的影响；激光共聚焦显微镜(CLSM)检测 BAEB 对白念珠菌生物膜荧光信号强度的影响；实时荧光定量 PCR (qRT-PCR)检测生物膜相关基因UME6、PES1和 HSP90的转录水平变化。结果 BAEB 对12株白念珠菌的 MIC 在64~256μg/mL 之间,对白念珠菌生物膜的 SMIC80(抑制80%生物膜形成的最低药物浓度)为1024μg/mL 或以上；Time-Kill 曲线显示在12 h 之后,512、1024μg/mL 浓度的 BAEB 对白念珠菌均具良好的杀伤作用；结晶紫染色法表明512、1024μg/mLBAEB 能够减少其生物膜生物量；SEM 观察到1024μg/mL BAEB 能够有效抑制白念珠菌在不同黏附介质上生物膜的完整度；CLSM 显示512、1024μg/mL 的 BAEB 可以明显降低生物膜荧光信号强度；qRT-PCR 检测显示在256、512、1024μg/mL 的 BAEB 作用下,UME6转录水平分别下调了72%、71%、77%,在512、1024μg/mL 的 BAEB 下 HSP90转录水平上调了2.23和3.31倍,而 PES1未有明显变化。结论 BAEB 可以抑制白念珠菌 VVC 临床株体外生物膜的形成。