水飞蓟宾对表皮葡萄球菌的抑菌机制研究
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摘要
目的:探讨水飞蓟宾对表皮葡萄球菌的抑菌机制。方法:测定表皮葡萄球菌的培养上清液蛋白浓度、菌体可溶性蛋白、DNA和RNA量及拓扑异构酶Ⅰ和Ⅱ活性,观察水飞蓟宾对表皮葡萄球菌细胞膜完整性、菌体蛋白合成、核酸合成和拓扑异构酶活性的影响;结晶紫染色法半定量测定表皮葡萄球菌生物膜量,SYTO9/PI荧光核酸染色剂结合激光共聚焦显微镜检测生物膜内死、活细菌及生物膜结构,观察水飞蓟宾对表皮葡萄球菌生物膜的影响。结果:浓度为1MIC(50μg/ml)和2MIC(100μg/ml)的水飞蓟宾作用0～9h,表皮葡萄球菌培养上清液蛋白浓度与对照组蛋白浓度无差异。受50、100μg/ml水飞蓟宾作用12h的表皮葡萄球菌菌体可溶性蛋白电泳图谱,较对照组蛋白条带更细、更浅,甚至缺失。受1MIC和2MIC水飞蓟宾作用6～36h的表皮葡萄球菌核酸量显著低于对照组,细菌DNA荧光强度分别降低16%及21%以上,RNA荧光强度分别降低15%及21%以上。在50、100μg/ml水飞蓟宾环境中,表皮葡萄球菌的拓扑异构酶Ⅰ和Ⅱ对p BR322 DNA的解旋作用减弱,与对照组比较,缺口或线性DNA(FormⅡ)显著减少,超螺旋DNA (FormⅠ)增多。不同阶段(6h、24h和48h)表皮葡萄球菌生物膜受1MBIC(200μg/ml)和2MBIC(400μg/ml)水飞蓟宾作用24h,生物膜进一步形成量较对照组显著减少,生物膜内死菌增多,生物膜结构变简单、厚度变薄,且随着药物浓度增加,作用越显著。结论:水飞蓟宾可抑制表皮葡萄球菌的菌体蛋白合成、核酸合成、拓扑异构酶活性和生物膜生长,可抑制(或杀灭)生物膜内细菌,破坏细菌生物膜结构。对细菌细胞膜无影响。
Objective: To investigate the antibacterial mechanism of Silibinin on Staphylococcus epidermidis. Methods: Culture supernatant protein concentration,bacterial soluble protein,amount of DNA and RNA,topoisomerase I and II activity of Staphylococcus epidermidis were determined. Effect on biofilm of Staphylococcus epidermidis was determined by crystal violet staining semi-quantitatively test,dead and living bacteria and biofilm structure were detected by SYTO9/PI fluorescent nucleic acid stain combined with Confocal Laser Scanning Microscope. Results: After treated with Silibinin at concentrations of 1MIC( 50μg/ml) and 2MIC for 0-9 h,the protein concentrations of culture supernatant of Staphylococcus epidermidis were not significantly different from those in the control group. The soluble protein electrophoresis patterns of Staphylococcus epidermidis,which were treated with 1 MIC and 2MIC Silibinin for 12 h,were thinner,shallower,and even absent than the control protein strip. After 6 ～ 36 h treatment with 1MIC and 2MIC Silibinin,the nucleic acid contents of Staphylococcus epidermidis were significantly lower than those in the control group. The fluorescence intensities of bacterial DNA were decreased more than 16% and 21%,and those of RNA were decreased more than 15% and 21%,respectively. Topoisomerase I and II of S. epidermidis attenuated the unwinding effect on p BR322 DNA. Compared with the control group,the gap or linear DNA( Form II) was significantly reduced,and the super-helical DNA( Form I) was increased. Staphylococcus epidermidis biofilms at different stages( 6h,24 h and 48h) were treated with 1MBIC( 200μg/ml) and 2MBIC Silibinin for 24 h. Compared with the control group,The formations of biofilms were significantly reduced,and the number of dead bacteria in the biofilm was increased. The biofilm structure became simpler and the thickness became thinner,in concentration-effect relationship. Conclusion: Silibinin can inhibit the bacterial protein synthesis,nucleic acid synthesis,topoisomerase activity and biofilm growth of Staphylococcus epidermidis,which can inhibit( or kill) bacteria in biofilm and destroy bacterial biofilm structure,and shows no effect on bacterial cell membranes.
Objective: To investigate the antibacterial mechanism of Silibinin on Staphylococcus epidermidis. Methods: Culture supernatant protein concentration,bacterial soluble protein,amount of DNA and RNA,topoisomerase I and II activity of Staphylococcus epidermidis were determined. Effect on biofilm of Staphylococcus epidermidis was determined by crystal violet staining semi-quantitatively test,dead and living bacteria and biofilm structure were detected by SYTO9/PI fluorescent nucleic acid stain combined with Confocal Laser Scanning Microscope. Results: After treated with Silibinin at concentrations of 1MIC( 50μg/ml) and 2MIC for 0-9 h,the protein concentrations of culture supernatant of Staphylococcus epidermidis were not significantly different from those in the control group. The soluble protein electrophoresis patterns of Staphylococcus epidermidis,which were treated with 1 MIC and 2MIC Silibinin for 12 h,were thinner,shallower,and even absent than the control protein strip. After 6 ～ 36 h treatment with 1MIC and 2MIC Silibinin,the nucleic acid contents of Staphylococcus epidermidis were significantly lower than those in the control group. The fluorescence intensities of bacterial DNA were decreased more than 16% and 21%,and those of RNA were decreased more than 15% and 21%,respectively. Topoisomerase I and II of S. epidermidis attenuated the unwinding effect on p BR322 DNA. Compared with the control group,the gap or linear DNA( Form II) was significantly reduced,and the super-helical DNA( Form I) was increased. Staphylococcus epidermidis biofilms at different stages( 6h,24 h and 48h) were treated with 1MBIC( 200μg/ml) and 2MBIC Silibinin for 24 h. Compared with the control group,The formations of biofilms were significantly reduced,and the number of dead bacteria in the biofilm was increased. The biofilm structure became simpler and the thickness became thinner,in concentration-effect relationship. Conclusion: Silibinin can inhibit the bacterial protein synthesis,nucleic acid synthesis,topoisomerase activity and biofilm growth of Staphylococcus epidermidis,which can inhibit( or kill) bacteria in biofilm and destroy bacterial biofilm structure,and shows no effect on bacterial cell membranes.
引文
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[2]Evren E,Yurtcu E. In vitro effects on biofilm viability and antibacterial and antiadherent activities of silymarin. Folia Microbiol(Praha),2015,60(4)∶351~356.
[3]Lee D G,Kim H K,Park Y,et al. Gram-positive bacteria specific properties of silybin derived from Silybum marianum. Arch Pharm Res,2003,26(8)∶597~600.
[4]袁琳慧,王迪,谢鲲鹏,等.穿心莲和水飞蓟抑制MRSA41577外排系统的作用机制.中国生化药物杂志,2016,36(11)∶27~29.
[5]游庭活,刘凡,温露,等.黄酮类化合物抑菌作用研究进展.中国中药杂志,2013,38(21)∶3645~3649.
[6]师伟.中药有效成分抑菌机制研究.科技信息,2011,(5)∶37.
[7]靳蕊,徐敏纹,刘莹,等.黄酮类化合物的抑菌作用及其机制的研究.继续医学教育,2016,30(8)∶152~154.
[8]杜文静,谢亚斌,葛雅琨,等.水飞蓟宾对金黄色葡萄球菌和大肠杆菌的体外抑菌作用的研究.吉林化工学院学报,2016,33(7)∶66~69.
[9]周本宏,松长青,姜姗,等.地榆鞣质提取物的抗菌活性及对金黄色葡萄球菌的抑菌机制研究.中国药师,2016,2(19)∶464~469.
[10]Booyens J,Labuschagne M C,Thantsha M S. In Vitro Antibacterial Mechanism of Action of Crude Garlic(Allium sativum)Clove Extract on Selected Probiotic Bifidobacterium Species as Revealed by SEM,TEM,and SDS-PAGE Analysis. Probiotics Antimicrob Proteins,2014,6(2)∶82~87.
[11]李昌勤,赵琳,薛志平,等.隐丹参酮抑菌作用机制研究.中国药学杂志,2012,47(21)∶1706~1710.
[12]Wang Q,Wang H,Xie M. Antibacterial mechanism of soybean isoflavone on Staphylococcus aureus. Arch Microbiol,2010,192(11)∶893~898.
[13]Sullivan D M,Glisson B S,Hodges P K,et al. Proliferation dependence of topoisomerase II mediated drug action. Biochemistry,1986,25(8)∶2248~2256.
[14]王倩,谢明杰.木犀草素对金黄色葡萄球菌的抑菌活性及其机制.微生物学报,2010,50(9)∶1180~1184.
[15]李燕,李冬冬,陶传敏,等.表皮葡萄球菌生物膜形成及相关基因的检测及评价.中华医院感染学杂志,2010,20(4)∶473~476.
[16]Li G,Qiao M,Guo Y,et al. Effect of subinhibitory concentrations of chlorogenic acid on reducing the virulence factor production by Staphylococcus aureus. Foodborne Pathog Dis,2014,11(9)∶677~683.
[17]王鑫,张瑞,李雅静,等.白藜芦醇对金黄色葡萄球菌标准株抑菌作用及超微结构的影响.现代检验医学杂志,2016,(3)∶55~58.
[18]王海涛,王倩,谢明杰.大豆异黄酮对金黄色葡萄球菌的抑菌机制研究.中国农学,2009,42(7)∶2586~2591.