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

中国真菌学杂志　 2015, Vol. 10 　Issue (6): 321-327.

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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

张梦翔^1,2, 陆克乔^1,2, 夏丹^1,2, 夏雪³, 施高翔^1,2, 邵菁^1,2, 吴大强^1,2, 汪天明^1,2, 汪长中^1,2

1. 安徽中医药大学中西医结合临床学院, 合肥 230038;
2. 安徽省中医药科学院中西医结合研究所, 合肥 230038;
3. 安徽医科大学口腔医学院, 合肥 230032

收稿日期:2015-07-01 出版日期:2015-12-28 发布日期:2015-12-28
通讯作者: 汪长中,E-mail:ahwcz63@sina.com E-mail:ahwcz63@sina.com
作者简介:张梦翔,男(汉族),硕士研究生在读.E-mail:tojosaki@126.com
基金资助:
国家自然科学基金(81573725),安徽省高校自然科研重点项目(KJ2015A030),安徽中医药大学校级科研基金(2014zr026)

The inhibitory effects of Butyl alcohol extract of BaiTouWeng decoction on biofilm formation of Candida albicans

ZHANG Meng-xiang^1,2, LU Ke-qiao^1,2, XIA Dan^1,2, XIA Xue³, SHI Gao-xiang^1,2, SHAO Jing^1,2, WU Da-qiang^1,2, WANG Tian-ming^1,2, WANG Chang-zhong^1,2

1. School of Integrated Traditional and Western Medicine, Anhui University of Chinese Medicine, Hefei 230038, China;
2. Institute of Integrated Traditional and Western Medicine, Anhui Academy of Chinese Medicine, Hefei 230038, China;
3. School of Stomatology, Anhui Medical University, Hefei 230032, China

Received:2015-07-01 Online:2015-12-28 Published:2015-12-28

摘要/Abstract

摘要：

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。

关键词: 白头翁汤正丁醇提取物, 白念珠菌, 外阴阴道念珠菌病, 生物膜

Abstract:

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 methods 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 biofilm.Confocal laser scanning microscopy (CLSM) was applied to determine the thickness of the biofilm.The quantification real-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 SMIC₈₀s of BAEB against the biofilm of C.albicans strains are determined as ≥ 1024 μg/mL.Time-kill curve results indicate that BAEB has a promise antifungal effect at concentrations of 512 and 1 024 μg/mL.Crystal violet staining results show that the biomass of C.albicans is reduced by BAEB at 512 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 morphology of biofilm is also affected by BAEB.The thickness of C.albicans biofilm is reduced by BAEB according to CLSM results.Furthermore,qRT-PCR results indicate that expression of UME6 is significantly down-regulated by BAEB at 256,512,1 024 μg/mL,and HSP90 is up-regulated at 512 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.

Key words: Butyl alcohol extract of BaiTouWeng decoction (BAEB), Candida albicans, vulvovaginal candidiasis (VVC), biofilm

中图分类号:

R379.4

张梦翔, 陆克乔, 夏丹, 夏雪, 施高翔, 邵菁, 吴大强, 汪天明, 汪长中. 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[J]. 中国真菌学杂志, 2015, 10(6): 321-327.

ZHANG Meng-xiang, LU Ke-qiao, XIA Dan, XIA Xue, SHI Gao-xiang, SHAO Jing, WU Da-qiang, WANG Tian-ming, WANG Chang-zhong. The inhibitory effects of Butyl alcohol extract of BaiTouWeng decoction on biofilm formation of Candida albicans[J]. Chinese Journal of Mycology, 2015, 10(6): 321-327.

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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

The inhibitory effects of Butyl alcohol extract of BaiTouWeng decoction on biofilm formation of Candida albicans

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