黄连解毒汤联合氟康唑对耐药白念珠菌麦角甾醇的影响(1)
[摘要]目的：探讨黄连解毒汤乙酸乙酯提取物(ethyl acetate extract of Huanglian Jiedu decoction，EAHD)联合氟康唑(fluconazole，FLZ)对耐药白念珠菌生物膜麦角甾醇的影响。方法：采用CLSI M27-A3的微量稀释法测定EAHD与FLZ对白念珠菌FLZ耐药株的MIC与SMIC80;棋盘稀释法测定EAHD与FLZ联用对白念珠菌的协同效应;稀释涂布平板法考察EAHD与FLZ联用不同时间对菌体的杀伤作用;分别用HPLC和qRT-PCR法检测耐药白念珠菌生物膜麦角甾醇含量及相关基因的变化。结果：对于9株白念珠菌FLZ耐药株，EAHD对其MIC介于156～1 250 mg·L-1，FLZ对其MIC介于256～2 048 mg·L-1以上，二者联用后MIC显著降低，FICI(部分抑菌浓度指数)最低达0.066;EAHD单用对其SMIC80均高于1 250 mg·L-1，FLZ单用对其SMIC80均高于512 mg·L-1，最高可达2 048 mg·L-1以上，联用组除个别组具有相加作用外也均有协同效应。时间杀菌(time kill，TK)实验显示当作用12 h，二者联用杀菌效应最强。HPLC结果显示联用组、EAHD组与空白组麦角甾醇量相比，分别降低了近85%，50%。qRT-PCR检测FLZ组ERG1，ERG2，ERG6，ERG7，ERG11均上调，ACS1，ACS2，MET6均下调;EAHD组所有基因均下调;联用组ERG2，ERG6，ERG11均显著上调，ERG1，ERG7，ACS1，ACS2，MET6均显著下调。结论：EAHD协同FLZ抗白念珠菌耐药株，可能与抑制麦角甾醇合成相关基因的表达来降低麦角甾醇的合成有关，造成细胞膜损伤，抑制菌体生长。
, http://www.100md.com
[关键词]黄连解毒汤乙酸乙酯提取物(EAHD);耐药白念珠菌;生物膜;协同作用;麦角甾醇
Effect of Huanglian Jiedu decoction in combination with fluconazole
on ergosterol of fluconazole-resistant Candida albicans
YAN Yuan-yuan1，2， WANG Tian-ming1，2， SHI Gao-xiang1，2， ZHANG Meng-xiang1，2，LU Ke-qiao1，2， SHAO Jing1，2， WANG Chang-zhong1，2*
(1.School of Integrated Traditional and Western Medicine， Anhui University of Chinese Medicine， Hefei 230038， China;
, 百拇医药
2. Institute of Integrated Traditional and Western Medicine， Anhui Academy of Chinese Medicine， Hefei 230038， China)
[Abstract]Objective： To investigate the effects of ethyl acetate extract of Huanglian Jiedu decoction (EAHD)， alone and in combination with fluconazole(FLZ) on FLZ-resistant Candida albicans. Method： The minimum inhibitory concentrations (MIC) and sessile MIC80 (SMIC80) of EAHD and FLZ to FLZ-resistant C. albicans were determined by CLSI M27-A3 microdilution method， and the synergy of EAHD combined with FLZ were examined by the checkerboard microdilution assay. Agar plate-method was adopted to observe the rate of antifungal activity according to time-kill curve. HPLC and qRT-PCR were utilized to evaluate the changes of ergosterol content and expressions of related genes， respectively. Result： MICs of EAHD ranged from 156 to 1 250 mg·L-1， those of FLZ from 256 to above 2 048 mg·L-1with FICI approximate 0.066 in combination; SMIC80 of EAHD were higher than 1 250 mg·L-1， SMIC80 of FLZ were higher than 512 mg·L-1 and up to above 2 048 mg·L-1. Combination group also showed synergy effect except one group showing addition effect. The results of T-K experiment also confirmed obviously fungicidal effect when treated for 12 h. When compared with control groups， the ergosterol was reduced 85% and 50% in the treatments of combination and EAHD alone by HPLC， respectively. The expressions of ERG1， ERG2， ERG6， ERG7 and ERG11 were upregulated， and ACS1， ACS2， MET6 were downregulated when exposed to FLZ. The expressions of the above genes were downregulated by treatment of EAHD. The expressions of ERG2， ERG6， ERG11 were upregulated， while ERG1， ERG7， ACS1， ACS2， MET6 were downregulated in combination group. Conclusion： The combination of EAHD and FLZ exhibited synergy against FLZ-resistant C. albicans through decreasing the synthesis of ergosterol， and resulting in the breakage of cell membrane., 百拇医药(严园园 汪天明 施高翔 张梦翔 陆克乔 邵菁 汪长中)
, http://www.100md.com
[关键词]黄连解毒汤乙酸乙酯提取物(EAHD);耐药白念珠菌;生物膜;协同作用;麦角甾醇
Effect of Huanglian Jiedu decoction in combination with fluconazole
on ergosterol of fluconazole-resistant Candida albicans
YAN Yuan-yuan1，2， WANG Tian-ming1，2， SHI Gao-xiang1，2， ZHANG Meng-xiang1，2，LU Ke-qiao1，2， SHAO Jing1，2， WANG Chang-zhong1，2*
(1.School of Integrated Traditional and Western Medicine， Anhui University of Chinese Medicine， Hefei 230038， China;
, 百拇医药
2. Institute of Integrated Traditional and Western Medicine， Anhui Academy of Chinese Medicine， Hefei 230038， China)
[Abstract]Objective： To investigate the effects of ethyl acetate extract of Huanglian Jiedu decoction (EAHD)， alone and in combination with fluconazole(FLZ) on FLZ-resistant Candida albicans. Method： The minimum inhibitory concentrations (MIC) and sessile MIC80 (SMIC80) of EAHD and FLZ to FLZ-resistant C. albicans were determined by CLSI M27-A3 microdilution method， and the synergy of EAHD combined with FLZ were examined by the checkerboard microdilution assay. Agar plate-method was adopted to observe the rate of antifungal activity according to time-kill curve. HPLC and qRT-PCR were utilized to evaluate the changes of ergosterol content and expressions of related genes， respectively. Result： MICs of EAHD ranged from 156 to 1 250 mg·L-1， those of FLZ from 256 to above 2 048 mg·L-1with FICI approximate 0.066 in combination; SMIC80 of EAHD were higher than 1 250 mg·L-1， SMIC80 of FLZ were higher than 512 mg·L-1 and up to above 2 048 mg·L-1. Combination group also showed synergy effect except one group showing addition effect. The results of T-K experiment also confirmed obviously fungicidal effect when treated for 12 h. When compared with control groups， the ergosterol was reduced 85% and 50% in the treatments of combination and EAHD alone by HPLC， respectively. The expressions of ERG1， ERG2， ERG6， ERG7 and ERG11 were upregulated， and ACS1， ACS2， MET6 were downregulated when exposed to FLZ. The expressions of the above genes were downregulated by treatment of EAHD. The expressions of ERG2， ERG6， ERG11 were upregulated， while ERG1， ERG7， ACS1， ACS2， MET6 were downregulated in combination group. Conclusion： The combination of EAHD and FLZ exhibited synergy against FLZ-resistant C. albicans through decreasing the synthesis of ergosterol， and resulting in the breakage of cell membrane., 百拇医药(严园园 汪天明 施高翔 张梦翔 陆克乔 邵菁 汪长中)