赤芍水提物及芍药苷对大肠杆菌抗菌增敏活性的影响  

Antibacterial and sensitizing activity of Paeonia lactiflora aqueous extract and paeoniflorin to Escherichia coli

作　　者：贾泽 王春光 张石磊 张鹏 张铁 JIA Ze;WANG Chun-guang;ZHANG Shi-lei;ZHANG Peng;ZHANG Tie(College of Veterinary Medicine , Hebei Agricultural University ,Baoding , Hebei 071000 ,China)

机构地区：[1]河北农业大学动物医学院

出　　处：《中国兽医学报》2019年第10期2026-2032,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31572560);河北省自然科学基金资助项目(C2018204092)

摘　　要：旨在考察中药赤芍水提物及其主要成分芍药苷对磷霉素钠的抗菌增敏活性,验证芍药苷对外排泵转运子AcrB的抑制作用,推测其抗菌增敏机制。首先,通过微量棋盘稀释法检测赤芍水提物及其主要成分芍药苷与磷霉素钠联合应用对临床分离的禽多重耐药大肠杆菌E320和质控菌株大肠杆菌ATCC35218的抑菌作用;其次,利用YASARA软件,以大肠杆菌多重耐药AcrAB-TolC系统中的外排泵转运子AcrB为靶标,与芍药苷进行分子对接模拟,探讨两者的识别机制;通过尼罗红外排试验验证芍药苷对外排泵的抑制作用;最后,通过实时荧光定量PCR验证芍药苷降低AcrB mRNA的表达量进而发挥抗菌增敏作用。试验结果证实赤芍水提物和芍药苷与磷霉素钠联合用药均具有协同抑制多重耐药大肠杆菌E320的作用(FICI≤0.5);分子对接结果显示芍药苷与AcrB蛋白受体的结合能达到8.333 kJ/mol,作用主要位点是AcrB蛋白受体上Phe628、Phe615、Val612、Ile277、Asn274、Gly179、Phe178等氨基酸残基,分子间主要作用力为疏水作用力;外排抑制试验结果显示芍药苷可以抑制耐药大肠杆菌外排泵活性,明显阻滞尼罗红的外排;实时荧光定量PCR结果显示芍药苷联合磷霉素钠比单独使用磷霉素钠显著降低大肠杆菌AcrB mRNA的表达量。本试验证明芍药苷通过作用外排泵转运子AcrB,使抗生素在菌体内积聚;并能显著降低AcrB mRNA的表达量进而起到抗菌增敏作用,为临床提供一种有效的抗菌增敏剂,对治疗耐药菌引起的细菌感染具有重要意义。To verify the inhibitory effect of paeoniflorin on AcrB of the efflux pumps transporter and presume the mechanism of antimicrobial sensitization.In this study,the antimicrobial sensitivity of Paeonia lactiflora aqueous extract and its main component paeoniflorin with fosfomycin sodium to the Escherichia coli were investigated.Firstly,the bacteriostatic effect of Paeonia lactiflora aqueous extract and paeoniflorin combined with fosfomycin sodium on clinically isolated multiple antibiotic resistant avian Escherichia coli E320 and Escherichia coli ATCC35218 of quality-controlling strain were detected by checkerboard microdilution method;secondly,YASARA software was applied to target the efflux pump transporter AcrB in the multiple antibiotic resistant AcrAB-TolC system of Escherichia coli.Molecular docking simulation of AcrB and paeoniflorin were used to explore the recognition mechanism.Inhibition of paeoniflorin on efflux pump was analyzed by Nile red experiment of discharge.Finally,real-time fluorescent quantitative PCR was used to verify that paeoniflorin reduces the expression of AcrB mRNA and then exerts antibacterial sensitization.The results confirmed that the combination of Paeonia lactiflora aqueous extract and paeoniflorin and fosfomycin has synergistic effect on the inhibition of the multiple antibioticresistant Escherichia coli E320(FICI≤0.5).Molecular docking results showed that the binding energy of paeoniflorin and AcrB protein receptor reached 8.333 kJ/mol.The main sites of action were Phe628,Phe615,Val612,Ile277,Asn274,Gly179,Phe178 and other amino acid residues on the AcrB protein receptor,and the main inner-molecular force is hydrophobic interaction;the results of efflux inhibition test showed that paeoniflorin can inhibit the activity of efflux pump of antibiotic resistant Escherichia coli,significantly block the efflux of Nile red.The results of real-time fluorescence quantitative PCR showed that paeoniflorin combined with fosfomycin sodium significantly decreased the expression of AcrB mRNA

关 键 词：赤芍水提物 芍药苷 抗菌增敏剂 AcrB外排泵 分子对接 

分 类 号：S859.7[农业科学—临床兽医学] S852.61[农业科学—兽医学]