黄芩苷对大肠杆菌AcrB外排泵的抑制作用及其机制  

Antimicrobial sensitization of baicalin against multidrug-resistant Escherichia coli

作　　者：赵子玉 张鹏[1] 王春光[1] 张铁[1] ZHAO Ziyu;ZHANG Peng;WANG Chunguang;ZHANG Tie(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 07100l,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001

出　　处：《中国兽医学报》2022年第9期1861-1868,共8页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(21326617D);河北农业大学科学发展基金资助项目(JY2020008)。

摘　　要：AcrAB-TolC外排泵系统是大肠杆菌产生多重耐药性(MDR)的重要生物学基础,内膜转运蛋白(AcrB)在该系统中起着决定性作用。为解决大肠杆菌的多重耐药问题,本试验以大肠杆菌AcrB为靶点通过虚拟筛选工具Autodock Vina进行筛选,得到与AcrB结合较好的中药单体抑制剂-黄芩苷,通过DS Visualizer分析黄芩苷与AcrB蛋白的相关作用力,应用联合抑菌试验验证黄芩苷与抗生素的联合抑菌作用,采用尼罗红外排试验验证黄芩苷对外排泵转运子AcrB的外排抑制作用,以实时荧光定量PCR方法检测黄芩苷对AcrB表达相关基因的影响,最后通过内膜质子梯度影响试验与外膜渗透稳定性试验验证黄芩苷抑制外排泵转运子AcrB的作用机制。结果显示,中药单体黄芩苷与AcrB蛋白结合位点形成氢键,并与对接位点多个氨基酸残基形成了疏水作用力,两者的结合作用力为44.7 kJ/mol;黄芩苷与磷霉素钠、头孢噻肟钠、氯霉素对大肠杆菌E320具有协同作用(FICI≤0.5);黄芩苷具有明显阻滞尼罗红外排作用的趋势且能够通过显著提高AcrB负调控蛋白AcrR、MarR、MppA、RpoS的mRNA表达从而降低AcrB蛋白的表达;0.625 g/L的黄芩苷对内膜质子梯度浓度与外膜渗透性均无明显影响。结果表明,黄芩苷具有成为多重耐药大肠杆菌外排泵抑制剂的潜质。AcrAB-TolC discharge pump system is an important biological basis for multiple drug resistance(MDR) in E.coli,and AcrB plays a decisive role in this system.In order to solve the problem of multiple drug resistance of Escherichia coli,this experiment took Escherichia coli AcrB as the target, by screening with the virtual screening tool Autodock Vina, we found that baicalin, a traditional Chinese medicine monomeric inhibitor could combine with AcrB.The interaction between baicalin and AcrB protein was analyzed by DS Visualizer.Bacteriostatic test was used to verify the combined antibacterial effect of baicalin with antibiotics.Nile infrared exhaust test, impact test and outer membrane lining proton gradient method of seepage stability test were used to detect the inhibition effect of baicalin on efflux pump AcrB.The real-time fluorescent quantitative PCR method was used to detect the effect of baicalin on the expression of AcrB related genes.The results showed that baicalin formed hydrogen bond with AcrB protein binding site, and formed hydrophobic interaction with amino acid residues at the anti-junction site.The binding force was 44.7 kJ/mol.Baicalin, fosfomycin sodium, cefotaxime sodium and chloramphenicol had synergistic effects on E320(FICI≤0.5).Baicalin had obvious tendency to block the infrared emission of Nile, and decreased the expression of AcrB protein by significantly increasing the mRNA expressions of AcrB negative regulatory proteins AcrR,MarR,MppA and RpoS.Baicalin at 0.625 g/L had no significant effect on the proton gradient concentration and the permeability of the outer membrane.The experiment proved that baicalin has the potential to be an efflux pump inhibitor of multidrugresistant Escherichia coli.

关 键 词：黄芩苷 多重耐药 虚拟筛选 ACRB 分级抑菌浓度指数 

分 类 号：S859.3[农业科学—临床兽医学]