黄连通过肠道菌调节肠外排蛋白P-gp修复UC肠屏障损伤的作用机制研究

Clinical reports and experimental data revealed gut dysbiosis is associated with down-regulation of intestinal P-glycoprotein (P-gp), resulting in gut barrier dysfunction in UC. As the main active component of Huang Lian (Rhizoma Coptidis), berberine (BBR) is paid more and more attention increasingly for its good therapeutic effect to ulcerative colitis (UC). However, the molecular mechanisms underlying its action remain elucidation. Our previous study showed that BBR markedly alleviated the inflammatory responses, restored the intestinal flora imbalance, and enhanced P-gp expresion in DSS-induced UC rats. In this proposal, we hypothesis that the mechanism underlying its action may involve enhancement of intestinal barrier by up-regulating P-gp via a gut microbiota-mediated pathway. First, intestinal P-gp, local inflammatory cytokines and targeted signaling (NF-κB and PXR) pathways will be measured with western blot and RT-PCR assays, in normal, UC and BBR groups. Then the UC and BBR related dynamic alterations in gut bacteria compositions, bacteria derived small molecules will be characterized using 16S rRNA based PCR-DGGE analysis and LC-MS based proteomics and metabonomics techniques, respectively, and be associated with the changes in P-gp and targeted signaling. The main regulatory molecules and mechanisms underlying protection effect of BBR on DSS induced UC rats, will be further elaborated by culturing the intestinal epithelial Caco-2 cells in presence of different combinations of discriminating bacteria small molecules and/or blockers of PXR/ NF-κB pathways. The findings obtaining from the proposed study will provide research basis for UC therapy by BBR, and verify the involvement of gut bacteria in BBR regulating intestinal P-glycoprotein, identify the main bacteria strains and molecules that elicit the regulation, as well as the main pathways. The knowledge will provide new targets for UC therapy, new ideas for development of innovative drug, and research basis of Chinese medicine treatment for UC in clinic.

肠道菌群紊乱导致肠外排转运体P-gp下调与溃疡性结肠炎（UC）发病机制密切相关，黄连治疗UC效果显著，有调节肠道菌作用。本项目结合前期研究提出：其活性成分小檗碱（BBR）通过肠道菌调节肠P-gp，修复肠黏膜屏障的新设想。通过葡聚糖硫酸钠诱导UC大鼠模型，采用RT-PCR、蛋白免疫印迹等技术明确BBR对肠P-gp表达及相关信号通路（PXR、NF-κB）蛋白和基因的调节；通过基于16S rRNA PCR-DGGE肠道菌组成表征技术、基于多种LC-MS技术的菌代谢组学，明确BBR对肠道菌调节作用，筛选菌差异小分子，并与P-gp及相关信号通路蛋白变化关联；采用Caco-2细胞与差异小分子孵育，结合信号通路阻断分析证实BBR通过菌代谢物对P-gp的调节作用，并揭示主要信号调控机制。本研究将阐明黄连治疗UC新机制，为黄连临床治疗UC提供依据，为UC治疗靶点的探索提供新方法，为开发UC创新药提供新思路。

肠道菌群紊乱导致肠外排转运体P-gp下调与溃疡性结肠炎（UC）发病机制密切相关，黄连治疗UC效果显著，有调节肠道菌作用。本项目的假说：黄连活性成分小檗碱（BBR）通过肠道菌调节肠P-gp，修复肠黏膜屏障的新设想。研究通过葡聚糖硫酸钠成功诱导UC大鼠模型，采用RT-PCR、蛋白免疫印迹等技术明确了BBR对肠P-gp表达及相关信号通路（PXR、NF-κB）蛋白和基因的调节。BBR的治疗改善了DSS诱导结肠炎的症状，减弱炎症标志物（骨髓过氧化物酶、肿瘤坏死因子-A、白细胞介素-1B和-6），并且以剂量依赖的行为上调了P-gp的表达。尽管结肠炎模型中P-gp相关核受体孕烷X受体和转录因子核因子E2相关因子2（Nrf2）的结肠表达下调，但基因和蛋白质表达的分析揭示了BBR治疗只逆转了Nrf2的下调。利用Caco-2细胞进行的体外研究表明，BBR能以剂量和时间依赖的方式上调多药耐药1（MDR1）基因和P-gp蛋白的表达。BBR对MDR1基因的显著上调被Nrf2沉默所消除，这表明Nrf2介导的途径是导致这种激活的原因。荧光分析表明在Caco-2细胞模型中BBR治疗后Nrf2受体基因的活化是剂量依赖性增长，在BBR剂量为2.5微摩尔有显著两倍升高，表明BBR是一个强效Nrf2激动剂。通过基于16S  rRNA PCR-DGGE肠道菌组成表征技术、基于多种LC-MS技术的菌代谢组学，明确了BBR对肠道菌调节作用，筛选菌差异胆汁酸小分子，并与P-gp及相关信号通路蛋白变化进行了关联；采用Caco-2细胞与差异小分子孵育，结合信号通路阻断分析证实BBR通过菌代谢物对P-gp的调节作用，并揭示主要信号调控机制。本研究将阐明黄连治疗UC新机制，为黄连临床治疗UC提供依据，为UC治疗靶点的探索提供新方法，为开发UC创新药提供新思路。