盐酸小檗碱对白色念珠菌菌态转换的影响研究
|
摘要
为探讨盐酸小檗碱对白色念珠菌酵母相向菌丝相转换的抑制作用和分子机制,采用微量稀释法测定盐酸小檗碱抑制白色念珠菌生长的MIC值,倒置荧光显微镜观察菌丝动态形成过程;hochest染色法观察不同浓度盐酸小檗碱对菌丝形成的影响;RT-PCR检测在不同浓度盐酸小檗碱作用下白色念珠菌菌丝形成关键基因EFG1、HWP1、ECE1、ALS1表达水平的变化。结果显示,盐酸小檗碱对白色念珠菌的MIC值为32μg/mL,同时白色念珠菌在4 h芽管形成明显,8 h转化为菌丝;hochest染色结果显示:128和32μg/mL的盐酸小檗碱可以通过抑制菌丝的形成以及降低细胞密度,从而达到抑制白色念珠菌发生菌态转换的目的,并且128μg/mL盐酸小檗碱的抑制作用明显优于32μg/mL盐酸小檗碱和4μg/mL氟康唑,而8μg/mL盐酸小檗碱抑制作用不明显;RT-PCR结果表明,128μg/mL盐酸小檗碱可以抑制白色念珠菌4 h干预组菌丝形成关键基因HWP1和ECE1的表达、6 h干预组HWP1、ECE1和ALS1的表达,差异有统计学意义(P<0.05);32μg/mL盐酸小檗碱可以抑制白色念珠菌4 h干预组菌丝形成关键基因EFG1和ALS1的表达,6 h干预组EFG1、HWP1和ALS1的表达,差异具有统计学意义(P<0.05),但8μg/mL盐酸小檗碱无明显抑制作用。结果表明,盐酸小檗碱可以明显抑制白色念珠菌的菌态转换,其作用机制可能与菌丝形成关键基因EFG1、HWP1、ECE1、ALS1的表达下调相关。
The inhibition effect of berberine hydrochloride(BERHY) on the yeast-to-hyphae phase conversion and molecular mechanism against Candida albicans was investigated. The MIC(minimum inhibitory concentration) value of BERHY that inhibited the growth of C.albicans was tested adopting microdilution, and the process of dynamic formation of C.albicans hyphae was observed under inverted fluorescence microscope; Then hyphae of C.albicans in different concentration of BERHY of were observed by Hoechst staining, and the key genes EEG1, HWP1, ECE1, and ALS1 in hyphal formation under the effect of BERHY in different concentration were detected by RT=PCR. The results showed that the MIC of BERHY was at 32 μg/mL, and meantime the tube formation of C.albicans was obvious at 4 h, and converted into hyphae at 8 h. The results of Hoechst staining showed that BRTHY at 128 μg/mL and 32 μg/mL can inhibit the hyphal formation and lead to decrease the cell density, thus accomplish the target of suppression of the yeast-to-hyphae conversion of C. albicans, in addition, the effect of BERHY at 128 μg/mL was obviously better than 32 μg/mL and fluconazole at 4 μg/mL, and the inhibition effect of BERHY at 8 μg/mL was not obvious. The RT-PCR results showed that the BERHY at 128 μg/mL can significantly inhibit the expression of HWP1 and ECE1 at 4 h and the expression of HWP1,ECE1 and ALS1 at 6 h, with statistically significant differences(P<0.05). The BERHY at 32 μg/mL can significantly inhibit the expression of EFG1 and ALS1 at 4 h and the expression of EFG1,HWP1 and ALS1 at 6 h, with statistically significant differences(P<0.05), however, the BERHY at 8 μg/mL has no obvious inhibition effect. To conclude, BERHY can significantly inhibit the yeast-to-hyphal conversion of C.albicans, and the mechanism may correlate to the down-regulation expression of the key genes(EFG1, HWP1, ECE1 and ALS1) of hyphal formation.
The inhibition effect of berberine hydrochloride(BERHY) on the yeast-to-hyphae phase conversion and molecular mechanism against Candida albicans was investigated. The MIC(minimum inhibitory concentration) value of BERHY that inhibited the growth of C.albicans was tested adopting microdilution, and the process of dynamic formation of C.albicans hyphae was observed under inverted fluorescence microscope; Then hyphae of C.albicans in different concentration of BERHY of were observed by Hoechst staining, and the key genes EEG1, HWP1, ECE1, and ALS1 in hyphal formation under the effect of BERHY in different concentration were detected by RT=PCR. The results showed that the MIC of BERHY was at 32 μg/mL, and meantime the tube formation of C.albicans was obvious at 4 h, and converted into hyphae at 8 h. The results of Hoechst staining showed that BRTHY at 128 μg/mL and 32 μg/mL can inhibit the hyphal formation and lead to decrease the cell density, thus accomplish the target of suppression of the yeast-to-hyphae conversion of C. albicans, in addition, the effect of BERHY at 128 μg/mL was obviously better than 32 μg/mL and fluconazole at 4 μg/mL, and the inhibition effect of BERHY at 8 μg/mL was not obvious. The RT-PCR results showed that the BERHY at 128 μg/mL can significantly inhibit the expression of HWP1 and ECE1 at 4 h and the expression of HWP1,ECE1 and ALS1 at 6 h, with statistically significant differences(P<0.05). The BERHY at 32 μg/mL can significantly inhibit the expression of EFG1 and ALS1 at 4 h and the expression of EFG1,HWP1 and ALS1 at 6 h, with statistically significant differences(P<0.05), however, the BERHY at 8 μg/mL has no obvious inhibition effect. To conclude, BERHY can significantly inhibit the yeast-to-hyphal conversion of C.albicans, and the mechanism may correlate to the down-regulation expression of the key genes(EFG1, HWP1, ECE1 and ALS1) of hyphal formation.
引文
[1] Nobile CJ,Johnson AD.Candida albicans Biofilms and Human Disease[J].Annu Rev Microbiol,2015,69:71-92.
[2] Zeng B,Li J,Wang Y,et al.In vitro and in vivo effects of suloctidil on growth and biofilm formation of the opportunistic fungus Candida albicans[J].Oncotarget,2017,8(41):69972-69982.
[3] Li D,Bernhardt J,Calderone R.Temporal expression of the Candida albicans genes CHK1 and CSSK1,adherence,and morphogenesis in a model of reconstituted human esophageal epithelial candidiasis[J].Infect Immun,2002,70:1558-1565.
[4] Desai JV,Mitchell AP,Andes DR.Fungal biofilms,drug resistance,and recurrent infection [J].Cold Spring Harb Perspect Med,2014,4(10):a019729.
[5] 张文静.丹参酮ⅡA磺酸钠对放射性心肌细胞损伤的保护作用及机制研究[D].兰州:甘肃中医药大学,2016.
[6] CLSI.Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts[M].4th Ed.CLSI Standard M27.Wanyne,PA:Clinical Laboratory Standards Institute 2017.
[7] 段万石.脂肪乳对中心静脉导管表面混合生物膜形成的影响[D].昆明:昆明医科大学,2016.
[8] Li Y,Ma Y,Zhang L,et al.In vivo inhibitory effect on the biofilm formation of Candida albicans by liverwort derived riccardin D[J].PLoS One ,2012,7(4):e35543.
[9] Chang W,Li Y,Zhang L,et al.Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production[J].Plos one,2012,7(7):e41624.
[10] Kong E,Jabra-Rizk MA.The great escape:pathogen versus host [J].PLoS Pathog,2015,11(3):e1004661.
[11] Ku M,Baek YU,Kwak MK,et al.Candida albicans glutathione reductase downregulates Efg1-mediated cyclic AMP/protein kinase A pathway and leads to defective hyphal growth and virulence upon decreased cellular methylglyoxal content accompanied by activating alcohol dehydrogenase and glycolytic enzymes[J].Biochim Biophys Acta,2017,1861(4):772-788.
[12] Sun L,Liao K,Wang D.Effects of Magnolol and Honokiol on Adhesion,Yeast-Hyphal Transition,and Formation of Biofilm by Candida albicans[J].PLoS One,2015,10(2):e0117695.
[13] 王彩瑞.4种中药提取物体外抗白色念珠菌的敏感性研究[D].大连:大连医科大学,2010.
[14] 张世川,李亚楠,魏雪仙,等.京万红组方成分体外抗白色念珠菌抑菌效应探讨[J].中国实验方剂学杂志,2015,21(19):111-115.
[15] 敖薪,王娟,刘莲,等.治疗中心静脉导管感染抗生素锁定技术指标的量化研究[J].中华医院感染学杂志,2017,27(20):4626-4629.
[2] Zeng B,Li J,Wang Y,et al.In vitro and in vivo effects of suloctidil on growth and biofilm formation of the opportunistic fungus Candida albicans[J].Oncotarget,2017,8(41):69972-69982.
[3] Li D,Bernhardt J,Calderone R.Temporal expression of the Candida albicans genes CHK1 and CSSK1,adherence,and morphogenesis in a model of reconstituted human esophageal epithelial candidiasis[J].Infect Immun,2002,70:1558-1565.
[4] Desai JV,Mitchell AP,Andes DR.Fungal biofilms,drug resistance,and recurrent infection [J].Cold Spring Harb Perspect Med,2014,4(10):a019729.
[5] 张文静.丹参酮ⅡA磺酸钠对放射性心肌细胞损伤的保护作用及机制研究[D].兰州:甘肃中医药大学,2016.
[6] CLSI.Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts[M].4th Ed.CLSI Standard M27.Wanyne,PA:Clinical Laboratory Standards Institute 2017.
[7] 段万石.脂肪乳对中心静脉导管表面混合生物膜形成的影响[D].昆明:昆明医科大学,2016.
[8] Li Y,Ma Y,Zhang L,et al.In vivo inhibitory effect on the biofilm formation of Candida albicans by liverwort derived riccardin D[J].PLoS One ,2012,7(4):e35543.
[9] Chang W,Li Y,Zhang L,et al.Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production[J].Plos one,2012,7(7):e41624.
[10] Kong E,Jabra-Rizk MA.The great escape:pathogen versus host [J].PLoS Pathog,2015,11(3):e1004661.
[11] Ku M,Baek YU,Kwak MK,et al.Candida albicans glutathione reductase downregulates Efg1-mediated cyclic AMP/protein kinase A pathway and leads to defective hyphal growth and virulence upon decreased cellular methylglyoxal content accompanied by activating alcohol dehydrogenase and glycolytic enzymes[J].Biochim Biophys Acta,2017,1861(4):772-788.
[12] Sun L,Liao K,Wang D.Effects of Magnolol and Honokiol on Adhesion,Yeast-Hyphal Transition,and Formation of Biofilm by Candida albicans[J].PLoS One,2015,10(2):e0117695.
[13] 王彩瑞.4种中药提取物体外抗白色念珠菌的敏感性研究[D].大连:大连医科大学,2010.
[14] 张世川,李亚楠,魏雪仙,等.京万红组方成分体外抗白色念珠菌抑菌效应探讨[J].中国实验方剂学杂志,2015,21(19):111-115.
[15] 敖薪,王娟,刘莲,等.治疗中心静脉导管感染抗生素锁定技术指标的量化研究[J].中华医院感染学杂志,2017,27(20):4626-4629.