从外排泵基因调控作用研究半夏泻心汤提取液逆转幽门螺杆菌多重耐药的机制

Helicobacter pylori (Hp) is a major pathogenic factor of gastrointestinal diseases, in which failing to effect a radical cure of Hp is mainly due to Hp resistance, especially multi-drug resistance. Our previous studies found that Banxiaxiexin decoction extract act on reversing multi-drug resistance of Helicobacter pylori (Hp MDR), and the effect is better than berberine whose chemical composition. However, the mechanism remains unclear. This study, we intent to investigate the issue of the molecular mechanism of Banxiaxiexin decoction extract against multi-drug resistance Hp base on previous achievements. We will first induce Hp MDR by Banxiaxiexin decoction extract in vivo and in vitro, then harvesting MIC-lowering strain against antibiotics. After pre-treatment and post-treatment of the extract, some efflux pump target genes in Hp MDR such as hefA are determined the protein levels and expressions at mRNA as well as gene mutations using the Western Blot, RT-PCR and DNA sequencing technologies , annotate new efflux pump genes by Genomics methods , identify the regulatory association between efflux pump genes and related genes, while drug accumulation content in bacteria is tested by fluorescence spectrophotometer method, cell structure is examined through transmission electron microscope, and cell component is examined by Raman spectroscopy, respectively, then analyze the relationship betweenthe efflux pump gene and cell structure and component, identify the targets about reversing multi-drug resistance. We expect to clarify the molecular mechanism of Banxiaxiexin decoction extract reversing the Hp MDR from the perspective of efflux pump gene regulation, thereby offering the valuable experimental evidences for clinical effect a radical cure Hp.

幽门螺杆菌（Hp）是胃肠疾病的主要致病因子，根治Hp失败的主要原因是Hp耐药、尤其多重耐药。课题组前期研究发现半夏泻心汤对多重耐药Hp（Hp MDR）有逆转作用，而且比其成分黄连素等效果更佳，但机制尚未清楚。本项目在前期研究基础上，拟采用半夏泻心汤提取液在体内、外诱导Hp MDR，获得抗生素MIC降低的Hp，用Western Blot、RT-PCR和DNA测序等技术检测提取液诱导前后菌株外排泵hefA等基因的蛋白质水平、mRNA表达量和基因突变位点，用基因组学方法注释新的外排泵基因，确定参与调控的外排泵基因及基因之间的调控关系；用荧光分光光度计、透射电镜和拉曼光谱分别检测提取液诱导前后菌株体内药物蓄积水平、菌体结构和成分的变化，分析外排泵基因与菌体结构、成分的关系，确定逆转耐药靶点，以期从外排泵基因调控角度阐明半夏泻心汤提取液逆转Hp多重耐药的分子机制，为临床根治Hp提供重要的实验依据。

幽门螺杆菌（Hp）是胃肠疾病的主要致病因子，根治Hp失败的主要原因是Hp耐药、尤其多重耐药。本研究采用半夏泻心汤提取液在体内、外诱导HP_BsR，获得抗生素MIC降低的HP_BsS，用Western Blot、RT-PCR等技术检测提取液诱导前后菌株外排泵hefA等基因的蛋白质水平、mRNA表达量和基因突变位点，确定参与调控的外排泵基因及基因之间的调控关系；用多功能酶标仪、透射电镜分别检测提取液诱导前后菌株体内药物蓄积水平、菌体结构变化，用转录组、蛋白组检测确定逆转耐药靶点.本研究初步明确了半夏泻心汤提取液对 Hp MDR 的具有逆转耐药作用的机制是：sRNA基因HP1181、HP1184、gene1315等基因参与外排泵基因hefA、hefC、msbA 和 spab 等基因调控，减少药物外排，增加药物体内蓄积水平，抑制幽门螺杆菌生长，但作用机制可能还有鞭毛基因和毒力岛基因有关。希望通过本研究，能为临床根治Hp提供重要的实验依据。