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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1 024μg/mL或以上;Time-Kill曲线显示在12h之后,512、1 024μg/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、1 024μg/mLBAEB能够减少其生物膜生物量;SEM观察到1 024μg/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、1 024μg/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、1 024μg/mL的BAEB作用下,UME6转录水平分别下调了72%、71%、77%,在512、1 024μg/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。
Objective To investigate the biofilm formation of Candida albicans clinical isolates isolated from vulvovaginal candidiasis (VVC) with Butyl alcohol extract of Bai Tou Weng decoction (BAEB) Impact. Methods Minimal inhibitory concentration (MIC) of BAEB against Candida albicans was determined by microdilution method. The effect of BAEB on metabolic activity of Candida albicans biofilm was determined by methyltetrazolium (XTT) reduction method. Time-kill assay BAEB on the viable count of Candida albicans; the effect of BAEB on the biomass of Candida albicans by crystal violet staining; the effect of BAEB on the morphology of Candida albicans biofilm by scanning electron microscopy (SEM); and the confocal laser scanning microscopy (CLSM) was used to detect the fluorescence intensity of BAEB on Candida albicans biofilm. The transcription level of biomembrane related genes UME6, PES1 and HSP90 was detected by real-time quantitative PCR (qRT-PCR). Results The MICs of BAEB against 12 C. albicans strains ranged from 64 μg / mL to 256 μg / mL. The MICIC values of SMIC80 against C. albicans biofilm were 1024 μg / mL or more for the 12 strains of Candida albicans. The Time-Kill curves It was shown that after 12h, BAEB at concentrations of 512,1 024μg / mL had a good killing effect on Candida albicans; crystal violet staining showed that 512,1 024μg / mL BAEB could reduce its biofilm biomass; the SEM showed that 1024μg / mL BAEB can effectively inhibit the biofilm integrity of Candida albicans on different adhesion media; CLSM showed that BAEB at 512,1 024μg / mL could significantly reduce the fluorescence signal intensity of biofilm; qRT-PCR detection showed that at the range of 256, 512 and 1024μg / mL BAEB, the transcript levels of UME6 were down-regulated by 72%, 71% and 77%, respectively. The transcriptional level of HSP90 was up-regulated by 2.23 and 3.31 times at BAEB of 512 and 1024 μg / mL, respectively, while PES1 had no significant change. Conclusion BAEB can inhibit the formation of biofilm in vitro of Candida albicans VVC clinical strains.