荆花胃康胶丸调节AcrAB-TolC外排泵以降低幽门螺杆菌耐药性的作用及机制研究

Resistance to antibiotics of H.pylori has been the major reason for treatment failure. Efflux pump system AcrAB-TolC was the main mechanism of the nonspecific and multidrug resistance of H.pylori. Successful inhibition of efflux pump reduced the drug resistance of H.pylori, but no medical preparation was practical clinically. Our preliminary studies found that Jinghua Weikang Capsule (JWC) reduced the minimum inhibitory concentrations of antibiotics against multidrug resistant H.pylori strains in vitro and improved the efficacy in vivo. Clinical trials revealed that JWC could improve the efficacy of triple therapy beyond the background of different antibiotic resistance. Pre-experiments indicated that JWC regulated the expression of hefABC gene of AcrAB-TolC. These results implied that the mechanism of these effects was related to the common pathway of drug resistance, the efflux pump system. Based on the clues from previous researches, this project was designed to clarify the mechanism of JWC reducing antibiotic resistance of H.pylori, by means of testing the changes of minimum inhibitory concentrations of antibiotics before and after JWC application using E-test and dilution methods, exploring the effect of regulating hefABC/DEF/GHI genes of AcrAB-TolC using PCR and NorthernBlot technologies, and excluding the interference from gene mutation of H.pylori strains through gene sequencing. In addition, antibiotic resistant H.pylori strain infected mice model will be established, based on which testing the reducing resistance activity of JWC in vivo. This project will point out the effect and mechanisms of JWC reducing antibiotic resistance of H.pylori, thus provide experimental evidences for clinical application

幽门螺杆菌对抗生素耐药是制约临床疗效的最主要原因，AcrAB-TolC外排泵系统是其产生耐药的主要机制，抑制该系统能够降低耐药性但尚无药物应用于临床。前期基础研究发现荆花胃康胶丸降低抗生素对不同耐药幽门螺杆菌的最低抑菌浓度，临床研究表明其提高耐药幽门螺杆菌感染的根除率，预实验发现该药抑制外排泵编码蛋白hefABC基因表达，提示其降低耐药性效应与调节非特异性耐药途径AcrAB-TolC外排泵有关。本项目基于前期工作基础，在体外通过E-test、琼脂稀释法测定荆花胃康胶丸对菌株耐药性的影响，采用PCR、NorthernBlot手段探讨其对外排泵蛋白编码基因hefABC/DEF/GHI的调节作用，利用基因测序方法排除菌株耐药基因点突对结果判定的干扰；同时建立多药耐药株感染动物模型，验证该药体内外效应的一致性，从而阐述其降低幽门螺杆菌耐药性的作用和机制，为临床中西医结合治疗耐药幽门螺杆菌提供依据。

幽门螺杆菌（HP）是胃癌发生的最主要的、可控的危险因素。HP对抗生素耐药是当前根除治疗失败的最主要原因。主动外排泵基因的过度表达和生物膜的形成是HP产生非特异性耐药的主要机制。本研究以解决HP对抗生素耐药的临床问题为出发点，基于课题组既往临床和基础研究中发现的线索，提出了荆花胃康胶丸通过干预HP主动外排泵系统和生物膜来降低HP耐药性的科学假说。项目通过筛选单药耐药及多药耐药菌株，测定荆花胃康胶丸对各耐药HP菌株的最低抑菌浓度（MIC），并以低于MIC值浓度的荆花胃康胶丸干预多药耐药菌株，测定经药物干预后各抗生素的MIC，qPCR检测荆花胃康胶丸对HP外排泵基因表达的影响，同时进行药物干预前后耐药基因测序分析，结晶紫法、扫描电镜观察荆花胃康胶丸对HP26695-16R生物膜的影响。研究结果共筛选得到7株HP耐药菌株，荆花胃康胶丸对耐药菌株的MIC值为64-1024μg/ml不等。1/2MIC浓度的荆花胃康胶丸可降低甲硝唑对菌株26695-16R的MIC值，并且将甲硝唑MIC值降低到6μg/ml，逆转了HP26695-16R对甲硝唑的耐药性，其效果优于外排泵抑制剂PaβN和CCCP。qPCR结果显示，外排泵抑制剂CCCP和1/2MIC~2MIC浓度的荆花胃康胶丸均可以抑制外排泵基因HP0605和HP0971的表达；CCCP和2MIC浓度的荆花胃康胶丸可以抑制外排泵基因HP1327的表达；动物试验则发现耐药菌株定植数量量少，基于PCR的定量分析为显示出组间差异。结晶紫结果显示CCCP、1/4MIC~2MIC浓度的荆花胃康胶丸及8μg/ml和64μg/ml的甲硝唑均可抑制HP 26695-16R生物膜的形成。扫描电镜结果则显示26695-16R的生物膜结构的破坏程度随荆花胃康胶丸浓度升高而加重，经MIC浓度的荆花胃康胶丸处理后，26695-16R形态变为球状。.本研究结果表明荆花胃康胶丸对甲硝唑不全耐药株具有显著的降低MIC作用，即提高抗生素敏感性甚至逆转耐药，且低剂量下能够降低该菌株主动外排泵系统hefABC基因表达，且该效应未受到持续的甲硝唑耐药位点突变影响。此外，荆花胃康胶丸还能够抑制耐药菌株生物膜的形成，进一步提示还可能通过其他途径降低耐药性。.研究结果为临床中将荆花胃康胶丸应用于含甲硝唑的根除治疗方案，以及提高敏感性和根除率提供了一定的借鉴。