复方黄柏液涂剂对耐甲氧西林金黄色葡萄球菌毒力和生物膜的影响  

Effect of Fufang Huangbai Fluid Paint on Virulence and Biofilm of Methicillin-resistant Staphylococcus aureus

作　　者：李金泽 崔开宇 李东影[2] 马淑骅[2] 贺改英 孙娅楠[2] 王毅[2] 何忠梅[1] 杨伟峰[2] LI Jin-ze;CUI Kai-yu;LI Dong-ying;MA Shu-hua;HE Gai-ying;SUN Ya-nan;WANG Yi;HE Zhong-mei;YANG Wei-feng(Jilin Agricultural University,Changchun 130118,China;Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]吉林农业大学,长春130018 [2]中国中医科学院医学实验中心,北京100700

出　　处：《中国实验方剂学杂志》2022年第8期54-62,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：科技部“中医药现代化研究”重点专项(2019YFC1709305);中国中医科学院科技创新工程中西医结合基础重大攻关项目(CI2021A00601);中国中医科学院医学实验中心基本科研业务项目(zz2018018);李连达院士工作站专项经费项目(HX2017001);北京自然科学基金面上项目(7192141)。

摘　　要：目的:研究复方黄柏液涂剂(FFHBFP)对耐甲氧西林金黄色葡萄球菌(MRSA)毒力和生物膜的抑制作用,发现FFHBFP对MRSA的抗菌作用,为临床指导用药提供理论依据与参考。方法:首先采用微量稀释法和时间-生长曲线测定FFHBFP和万古霉素(VAN)对MRSA的最低抑菌浓度(MIC)和对细菌生长的影响;而后选择亚抑菌浓度(sub-MIC)分别观察FFHBFP和VAN对MRSA毒力因子脂肪酶的抑制能力和恢复过氧化氢(H_(2)O_(2))敏感性能力;采用微孔板法检测FFHBFP和VAN对MRSA生物膜形成期和成熟期的抑制能力;应用扫描电子显微镜(SEM)观察给药前后成熟生物膜在细菌形态方面的变化;结合实时荧光定量聚合酶链式反应(Real-time PCR)检测生药质量浓度50.600 g·L^(-1)FFHBFP对MRSA毒力基因crt M和生物膜形成基因fnb A、ica A表达的影响,最后运用分子对接技术预测FFHBFP中潜在抗菌有效成分与MRSA毒力和生物膜的作用靶点机制。结果:VAN的MIC为2 mg·L^(-1),在1 mg·L^(-1)以下时不影响MRSA生长,而FFHBFP无法测定出MIC,在生药质量浓度101.200~202.400 g·L^(-1)时对生长有抑制作用;相比于空白组和VAN组,sub-MIC(生药质量浓度25.300~50.600 g·L^(-1))具有显著抑制脂肪酶和恢复MRSA对H_(2)O_(2)敏感(P<0.01);微孔板法结果显示FFHBFP(生药质量浓度25.300~202.400 g·L^(-1))对生物膜形成期和成熟期均有明显抑制作用(P<0.05,P<0.01);SEM显示FFHBFP使生物膜结构疏松,菌体大小不一;生药质量浓度50.600 g·L^(-1)质量浓度FFHBFP能显著抑制相关的毒力基因和生物膜形成基因的表达(P<0.05,P<0.01),同时分子对接结果也显示FFHBFP主要抗菌活性成分对作用靶点有较好的结合能力。结论:FFHBFP对MRSA没有直接杀灭的能力,而是通过削弱毒力表达和生物膜形成等致病能力来发挥临床疗效。Objective:To study the virulence and biofilm inhibition effect of Fufang Huangbai Fluid Paint(FFHBFP)on methicillin-resistant Staphylococcus aureus(MRSA),and to explore the antibacterial effect of FFHBFP on MRSA,which provides a theoretical basis and reference for clinical medication.Method:Firstly,the microdilution method and time–growth curve were used to determine the minimum inhibitory concentration(MIC)of FFHBFP and vancomycin(VAN)against MRSA and the effect on bacterial growth.The effects of FFHBFP and VAN on the inhibition of MRSA virulence factor lipase and restoration of hydrogen peroxide(H_(2)O_(2))sensitivity were detected under sub-minimum inhibitory concentration(sub-MIC).The inhibitory effect of FFHBFP and VAN on MRSA biofilm formation and maturation was detected by the microplate method.The morphological changes of mature biofilms before and after administration were observed under a scanning electron microscope(SEM).Real-time polymerase chain reaction(Real-time PCR)was utilized to detect the effect of 50.600 g·L^(-1)concentration of FFHBFP on the expression of MRSA virulence gene crt M and biofilm-forming genes fnb A and ica A.Finally,molecular docking technology was used to predict the mechanism of potential antibacterial active ingredients of FFHBFP in inhibiting the virulence and biofilm of MRSA.Result:The MIC of VAN was 2 mg·L^(-1),and VAN below 1 mg·L^(-1)exerted no effect on MRSA growth.The MIC of FFHBFP was not determined,while the 101.200-202.400 g·L^(-1)original solution inhibited MRSAgrowth.Compared with the blank group and the VAN group,sub-MIC(25.300-50.600 g·L^(-1)original solution)inhibited lipase and recovered MRSA sensitivity to H_(2)O_(2)(P<0.01).The results of the microplate method showed that FFHBFP(25.300-202.400 g·L^(-1)original solution)inhibited biofilm formation and maturation(P<0.05,P<0.01).The SEM exhibited that FFHBFP made the structure of biofilm loose and the size of the bacteria varied.FFHBFP at 50.600 g·L^(-1)concentration can inhibit the expression of relat

关 键 词：复方黄柏液涂剂 耐甲氧西林金黄色葡萄球菌 毒力因子 生物膜 

分 类 号：R2-0[医药卫生—中医学] R22