大蒜素体外抗白念珠菌生物膜作用的实验研究
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摘要
目的探讨大蒜素在体外对于白色念珠菌生物膜作用的抗击作用。方法通过用4-2 048μg/ml二倍倍比稀释的大蒜素或0. 125-1 024μg/ml二倍倍比稀释的氟康唑处理白色念珠菌48 h,24 h或8 h后,采用XTT法检测大蒜素对白色念珠菌的杀伤作用,以及大蒜素造成生物膜80%抑制浓度(SMIC_(80))的测定,采用FUNI免疫荧光染色的方式测定大蒜素对白色念珠菌形成的生物膜厚度的影响,采用q PCR检测大蒜素对白色念珠菌中与生物膜形成相关的基因表达水平。结果大蒜素对于所有白色念珠菌的MIC在32-128μg/ml,SMIC_(80)为64-256μg/ml,均小于对应到氟康唑的作用值。大蒜素对于白色念珠菌标准株SC5314的生物膜形成呈现剂量依赖的抑制效果,当大蒜素浓度为4μg/ml时对生物膜形成的抑制作用不明显,随着剂量的升高开始表现出抑制作用,而当大蒜素浓度升为128μg/ml时,对生物膜形成的抑制作用达到80%,显著高于对照组(大蒜素浓度为0)(P <0. 001);当大蒜素的浓度为16μg/ml及以下时,对于菌株的抑制作用不明显(P> 0. 05),而当大蒜素的浓度升高到128μg/ml时,对菌株的抑制作用表现明显(P <0. 01),并且在第4小时时lg CFU降为4。通过对染色后的荧光强度进行量化来显示生物膜的厚度,可以发现,荧光强度随着大蒜素的浓度升高而不断降低,在大蒜素浓度为128μg/ml时,荧光强度值接近于0。对生物膜形成相关基因表达情况进行q PCR测定,发现经过大蒜素128μg/ml处理8 h后,相比不处理,生物膜相关基因ALS1表达下调了4. 7%,SUN41下调了18. 7%,MP65下调了68. 3%,HWP1下调了77. 4%,GSH下调了38. 4%。结论大蒜素对于白色念珠菌具有抑制作用,并对其表面生物膜的形成具有良好的抵抗作用,效果显著,值得临床参考。
Objective To investigate the effect of allicin against the biofilm of Candida albicans in vitro. Methods Candida albicans was treated with 4-2 048 μg/ml double diluted allicin or 0. 125-1 024 μg/ml double diluted fluconazole for 48 h,24 h or 8 h,respectively,and then the killing effect of allicin and 80% inhibitory concentration on biofilm( SMIC_(80)) were detected by XTT assay,the effect of allicin on biofilm thickness formed by Candida albican was detected using FUNI immunofluorescent staining,and the expression levels of genes related with biofilm formation in Candida albican were detected by q PCR. Results Except the standard strain SC5314 and the strains other than the clinical strains No. 4 and No. 6,the MIC of fluconazole was above 128 μg/ml,which was defined as a drug-resistant strain,and the SMIC_(80) of all strains for fluconazole was 1 024 μg/ml or more. The allicin had an MIC of 32-128 μg/ml for all Candida albicans and 64-256 μg/ml for SMIC_(80),which were smaller than the corresponding values for fluconazole.Allicin had a dose-dependent inhibitory effect on the biofilm formation of Candida albicans SC5314. While the allicin concentration was4 μg/ml,the inhibitory effect on biofilm formation was not obvious,and the inhibition was gradually increased with the increasing dose. When the concentration of allicin increased to 128 μg/ml,the inhibitation on biofilm formation reached 80%,which was significantly higher than that of controls( 0 μg/ml,P < 0. 001). When the concentration of allicin was 16 μg/ml and below,the inhibitory effect on the strain was not significant( P > 0. 05). When the concentration of allicin was increased to 128 μg/ml,the inhibitory effect on the strain was significant( P < 0. 01),and the lg CFU decreased to 4 at 4 h. The thickness of the biofilm was quantified by the fluorescence intensity after staining and the fluorescence intensity decreased with the increasing concentration of allicin. When the allicin concentration was 128 μg/ml,the fluorescence intensity value was close to 0. The q PCR results showed that compared with no treatment,ALS1 expression was reduced by 4. 7% after 128 μg/ml allicin treatment for 8 h,SUN41 was reduced by 18. 7%,MP65 was reduced by 68. 3%,HWP1 was reduced by 77. 4%,and GSH was reduced by 38. 4%. Conclusion Allicin has an inhibitory effect on Candida albicans and has a good resistance to the formation of biofilm on the surface. The effect is significant and worthy of clinical reference.
Objective To investigate the effect of allicin against the biofilm of Candida albicans in vitro. Methods Candida albicans was treated with 4-2 048 μg/ml double diluted allicin or 0. 125-1 024 μg/ml double diluted fluconazole for 48 h,24 h or 8 h,respectively,and then the killing effect of allicin and 80% inhibitory concentration on biofilm( SMIC_(80)) were detected by XTT assay,the effect of allicin on biofilm thickness formed by Candida albican was detected using FUNI immunofluorescent staining,and the expression levels of genes related with biofilm formation in Candida albican were detected by q PCR. Results Except the standard strain SC5314 and the strains other than the clinical strains No. 4 and No. 6,the MIC of fluconazole was above 128 μg/ml,which was defined as a drug-resistant strain,and the SMIC_(80) of all strains for fluconazole was 1 024 μg/ml or more. The allicin had an MIC of 32-128 μg/ml for all Candida albicans and 64-256 μg/ml for SMIC_(80),which were smaller than the corresponding values for fluconazole.Allicin had a dose-dependent inhibitory effect on the biofilm formation of Candida albicans SC5314. While the allicin concentration was4 μg/ml,the inhibitory effect on biofilm formation was not obvious,and the inhibition was gradually increased with the increasing dose. When the concentration of allicin increased to 128 μg/ml,the inhibitation on biofilm formation reached 80%,which was significantly higher than that of controls( 0 μg/ml,P < 0. 001). When the concentration of allicin was 16 μg/ml and below,the inhibitory effect on the strain was not significant( P > 0. 05). When the concentration of allicin was increased to 128 μg/ml,the inhibitory effect on the strain was significant( P < 0. 01),and the lg CFU decreased to 4 at 4 h. The thickness of the biofilm was quantified by the fluorescence intensity after staining and the fluorescence intensity decreased with the increasing concentration of allicin. When the allicin concentration was 128 μg/ml,the fluorescence intensity value was close to 0. The q PCR results showed that compared with no treatment,ALS1 expression was reduced by 4. 7% after 128 μg/ml allicin treatment for 8 h,SUN41 was reduced by 18. 7%,MP65 was reduced by 68. 3%,HWP1 was reduced by 77. 4%,and GSH was reduced by 38. 4%. Conclusion Allicin has an inhibitory effect on Candida albicans and has a good resistance to the formation of biofilm on the surface. The effect is significant and worthy of clinical reference.
引文
[1]陆克乔,张梦翔,施高翔,等.云南重楼正丁醇提取物对白色念珠菌生物膜形成的抑制作用[J].中草药,2016,47(3):440-446.
[2]张梦翔,夏丹,施高翔,等.白头翁汤正丁醇提取物对白念珠菌VVC临床株黏附作用的影响[J].中国真菌学杂志,2015,10(1):25-30.
[3]Lee HS,Kim Y.Paeonia lactiflora inhibits cell wall synthesis and triggers membrane depolarization in Candida albicans[J].World J Microb Biot,27(2):395-404.
[4]卓清,曾丹,周艺,等.大蒜及其提取物在猪生产中的应用[J].饲料工业,2015,36(11):18-21.
[5]孟君,彭秀丽,庄勤等.新、陈大蒜提取物抑菌活性的研究[J].中国调味品,2017,42(11):35-43.
[6]郭永刚,樊克锋.大蒜素对鸡大肠杆菌的体外抑菌试验[J].中兽医学杂志,2015,(4):9-10.
[7]汪天明,严圆圆,施高翔,等.黄连解毒汤乙酸乙酯提取物抑制白念珠菌菌丝的形成[J].中国中药杂志,2014,39(24):4834-4838.
[8]郭兰芳,刘大标,周梦婕,等.粉防己碱对白念珠菌生物膜及芽管形成的影响[J].中国真菌学杂志,2015,10(5):276-278.
[9]王霞,吴大强,施高翔,等.龙胆泻肝汤氯仿提取物对白念珠菌VVC临床株菌丝抑制作用研究[J].中国真菌学杂志,2016,11(6):341-347.
[10]俞丽华,陆克乔,施高翔,等.龙胆泻肝汤不同提取部位对白念珠菌生物膜的抑制作用研究[J].中国中药杂志,2014,39(7):1280-1284.
[11]Ma C,Du F,Yan L,et al.Potent activities of roemerine against Candida albicans and the underlying mechanisms[J].Molecules,2015,20(10):17913-17928.
[12]施高翔,严园园,邵菁,等.穿心莲内酯对白念珠菌生物膜分散的影响[J].中国中药杂志,2014,39(17):3339-3343.
[13]张梦翔,夏丹,施高翔,等.白头翁汤正丁醇提取物对碱性p H条件下白念珠菌VVC临床株形态转化的影响[J].中国中药杂志,2015,40(4):710-715.
[14]张梦翔,陆克乔,夏丹,等.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.
[15]Zhong H,Hu DD,Hu GH,et al.Activity of sanguinarineagainst Candida albicans biofilms[J].Antimicrob Agents Chemother,2017,61(5):e02259-16.
[16]严园园,汪天明,施高翔,等.黄连解毒汤联合氟康唑对耐药白念珠菌麦角甾醇的影响[J].中国中药杂志,2015,40(4):727-732.
[17]施高翔,汪云霞,冯鑫,等.白头翁汤正丁醇提取物诱导白念珠菌生物被膜细胞凋亡[J].中国真菌学杂志,2017,12(1):13-18.
[18]夏丹,张梦翔,施高翔,等.白头翁汤抑制小鼠阴道念珠菌的增殖并降低炎性细胞因子水平[J].细胞与分子免疫学杂志,2016,32(2):153-157.
[19]李亚婷,刘林波,黄燕杰,等.中药有效成分对白色念珠菌生物膜作用的研究现状[J].国际检验医学杂志,2016,37(17):2439-2442.
[20]贺慧娟,谢水祥.中草药抗白色念珠菌的作用机制[J].赣南医学院学报,2017,37(6):979-981.
[21]Hosseini SS,Ghaemi E,Koohsar F.Influence of ZnO nanoparticles on Candida albicans isolates biofilm formed on the urinary catheter[J].Iran J Microbiol,2018,10(6):424-432.
[2]张梦翔,夏丹,施高翔,等.白头翁汤正丁醇提取物对白念珠菌VVC临床株黏附作用的影响[J].中国真菌学杂志,2015,10(1):25-30.
[3]Lee HS,Kim Y.Paeonia lactiflora inhibits cell wall synthesis and triggers membrane depolarization in Candida albicans[J].World J Microb Biot,27(2):395-404.
[4]卓清,曾丹,周艺,等.大蒜及其提取物在猪生产中的应用[J].饲料工业,2015,36(11):18-21.
[5]孟君,彭秀丽,庄勤等.新、陈大蒜提取物抑菌活性的研究[J].中国调味品,2017,42(11):35-43.
[6]郭永刚,樊克锋.大蒜素对鸡大肠杆菌的体外抑菌试验[J].中兽医学杂志,2015,(4):9-10.
[7]汪天明,严圆圆,施高翔,等.黄连解毒汤乙酸乙酯提取物抑制白念珠菌菌丝的形成[J].中国中药杂志,2014,39(24):4834-4838.
[8]郭兰芳,刘大标,周梦婕,等.粉防己碱对白念珠菌生物膜及芽管形成的影响[J].中国真菌学杂志,2015,10(5):276-278.
[9]王霞,吴大强,施高翔,等.龙胆泻肝汤氯仿提取物对白念珠菌VVC临床株菌丝抑制作用研究[J].中国真菌学杂志,2016,11(6):341-347.
[10]俞丽华,陆克乔,施高翔,等.龙胆泻肝汤不同提取部位对白念珠菌生物膜的抑制作用研究[J].中国中药杂志,2014,39(7):1280-1284.
[11]Ma C,Du F,Yan L,et al.Potent activities of roemerine against Candida albicans and the underlying mechanisms[J].Molecules,2015,20(10):17913-17928.
[12]施高翔,严园园,邵菁,等.穿心莲内酯对白念珠菌生物膜分散的影响[J].中国中药杂志,2014,39(17):3339-3343.
[13]张梦翔,夏丹,施高翔,等.白头翁汤正丁醇提取物对碱性p H条件下白念珠菌VVC临床株形态转化的影响[J].中国中药杂志,2015,40(4):710-715.
[14]张梦翔,陆克乔,夏丹,等.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.
[15]Zhong H,Hu DD,Hu GH,et al.Activity of sanguinarineagainst Candida albicans biofilms[J].Antimicrob Agents Chemother,2017,61(5):e02259-16.
[16]严园园,汪天明,施高翔,等.黄连解毒汤联合氟康唑对耐药白念珠菌麦角甾醇的影响[J].中国中药杂志,2015,40(4):727-732.
[17]施高翔,汪云霞,冯鑫,等.白头翁汤正丁醇提取物诱导白念珠菌生物被膜细胞凋亡[J].中国真菌学杂志,2017,12(1):13-18.
[18]夏丹,张梦翔,施高翔,等.白头翁汤抑制小鼠阴道念珠菌的增殖并降低炎性细胞因子水平[J].细胞与分子免疫学杂志,2016,32(2):153-157.
[19]李亚婷,刘林波,黄燕杰,等.中药有效成分对白色念珠菌生物膜作用的研究现状[J].国际检验医学杂志,2016,37(17):2439-2442.
[20]贺慧娟,谢水祥.中草药抗白色念珠菌的作用机制[J].赣南医学院学报,2017,37(6):979-981.
[21]Hosseini SS,Ghaemi E,Koohsar F.Influence of ZnO nanoparticles on Candida albicans isolates biofilm formed on the urinary catheter[J].Iran J Microbiol,2018,10(6):424-432.