溃疡性结肠炎大鼠肠P-糖蛋白变化的肠道菌群介导机制

As a part of the first-line of host defense barrier,intestinal P-glycoprotein plays important roles in diseases pathogenesis as well as drug absorption and exposure. Clinical reports and experimental data revealed diminished P-glycoprotein expressionand function accompanying with gut dysbiosis in inflammatory bowel disease.Yet whether gut dysbiosis is associated with down-regulation of intestinal P-glycoprotein and the underlying mechanisms are unclear. Our previous study observed a down-regulation of expression and activity ofintestinalP-glycoprotein in rats with ulcerative colitis (UC) induced by dextran sulfate sodium and suggested a gut bacteria-involved regulatory mechanism. In this proposal, the regulation of intestinal P-glycoprotein by gut microbiota and the mechanisms will be investigated. First, UC relateddynamic alterations in gut bacteria compositions, bacteria derived proteins, peptides and 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 intestinal P-glycoprotein, local inflammatory cytokines and targeted signaling (PXR and TLR4) pathways measured with western blot and RT-PCR assays, in the dextran sulfate sodium induced colitis rat model. The involvement of gut microbiota in regulating intestinal P-glycoprotein will be evidenced by inoculation of a combination of the most discriminating gut bacteria strains into the germ-free rats.The main regulatory molecules and mechanisms will be further elaborated by culturing the intestinal epithelial Caco-2 cells in presence of different combinations of discriminating bacteria proteins/small molecules and/or blockers or agonists of PXR/TLR4 pathways. The findings obtaining from the proposed study will verify the involvement of gut bacteria in 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 as well as rational clinical decision-making on dose adjustmentof drugswhich is P-glycoprotein substrate (for instance, cyclosporine A)used for UC therapy.This strategy adopted can also be appliedto other diseases, such as diabetes, obesity,of which the pathogenesis is associated with gut dysbiosis.

作为肠屏障的重要组成部分，P-糖蛋白在疾病发生发展及药物吸收中扮演着双重角色。研究发现，炎症性肠病肠P-糖蛋白下调伴随肠道菌失调，但两者是否关联、机制如何尚不清楚。我们前期研究的结果显示，硫酸葡聚糖诱导的大鼠溃疡性结肠炎肠道菌群失调与肠P-糖蛋白下调相关。本研究拟通过基于16S rRNA PCR-DGGE的肠道菌组成表征、基于多种LC-MS技术的肠道菌代谢组学和蛋白质组学研究，明确硫酸葡聚糖诱导溃疡性结肠炎产生的显著差异性菌、蛋白质和小分子代谢物，并与肠局部炎症因子、P-糖蛋白、PXR /TLR4受体信号通路变化进行关联；进一步通过在无菌大鼠肠道定植溃疡性结肠炎差异菌组合以及用差异蛋白质和小分子组合刺激肠上皮细胞株Caco-2引起的P-糖蛋白变化，结合信号通路分析，证实肠道菌对肠P-糖蛋白的调节作用，揭示主要信号调控机制。研究结果对溃疡性结肠炎疾病治疗靶标研究以及合理用药研究提供新思路。

P-糖蛋白是肠屏障的重要组成部分，炎症性肠病中肠P-糖蛋白下调伴随肠道菌失调。本研究综合应用微生物组学、代谢组学、分子生物学、药理学和药代动力学等多学科思路和方法，结合硫酸葡聚糖诱导的大鼠溃疡性结肠炎模型及体外细胞模型，研究肠道菌群失调与肠P-糖蛋白表达和功能变化的关联及肠道菌群介导的调控机制。研究发现：（1）DSS诱导实验大鼠肠道P-糖蛋白表达及功能下调，伴随其上游调控因子核因子PXR和PPAR-γ的下调、菌群组成及功能改变、致炎/抑炎因子升高、以及整体代谢网络迁移；（2）移植结肠炎菌群则能在健康大鼠体内诱发结肠炎症状，引起肠P-糖蛋白的下调以及具有UC倾向的微生物移位和宿主代谢适应，而移植健康大鼠的粪菌能够减轻结肠炎大鼠的临床症状，恢复肠P-糖蛋白的水平，但其肠道菌群及代谢表征并未向供体组明显转移；（3）肠道菌群可溶性因子或外膜囊泡（OMVs）均能下调Caco-2细胞中P-糖蛋白及核因子水平；其机制主要是通过影响TLR4/MAPK/PI3K/NF-κB通路实现的；（4）对粪便菌群16S rRNA基因测序和尿液代谢组学数据的关联分析发现，粪菌移植改变的菌组成和宿主代谢之间具有显著相关性，其中UC的病理效应导致的相关性占8.0-16.2%，而FMT干预导致的相关性占3.9-7%；（5）通过代表性中药干预，可缓解UC临床症状指标，部分恢复或重建菌群平衡，直接和通过菌群介导机制上调肠道P-糖蛋白表达。研究结果对P-糖蛋白介导溃疡性结肠炎的病理机制、治疗靶标以及合理用药研究提供新思路。