DRA相关肠道菌群失调在调控肠道炎症中的作用及机制探讨

Recent data suggest that ulcerative colitis (UC) susceptibility genes play an important role in dysbiotic gut microbiota associated pathogenesis of UC. Ion transporter down regulated in adenoma (DRA) is one of the UC susceptibility genes. Our previous data suggested that DRA acted reciprocally with intestinal inflammation. Moreover, down regulation of DRA resulted in gut microbiota dysbiosis characterized by disequilibrium of Firmicutes/Bacteroidetes ratio (F/B ratio). However, whether DRA-related F/B ratio disequilibrium plays role in regulating intestinal inflammation and the underlying mechanism is yet to be clear. Studies have shown that the concentration of bacterial signal molecules autoinducer 2 (AI-2) alters while the number and density of bacteria in different species changed. AI-2 subsequently acts on the host gut immune system and participate in gut inflammatory reaction. Therefore, this project aims to investigate the regulatory effect of DRA-associated gut microbiota dysbiosis on intestinal inflammation and the underlying mechanism as follow: Firstly, the relation among DRA expression, intestinal F/B ratio and AI-2 concentration is evaluated in colonic biopsies and stool specimens of UC patients and healthy control. Secondly, the regulatory mechanism of DRA deficiency on gut flora F/B ratio disequilibrium is studied in transgenic mice. The effect of imbalanced F/B ratio on the concentration of AI-2 and the immunological pathway of AI-2 in regulating intestinal inflammation is investigated at the cellular level. Finally, the effect of DRA upregulation during intestinal inflammation on intestinal flora and inflammation is studied, so as to provide theoretical and experimental evidence for seeking new potential therapeutic target of UC.

新近研究指出溃疡性结肠炎（UC）易感基因在肠道菌群影响UC的发病模式中意义重大。申请者前期研究发现，作为UC易感基因，腺癌中低表达离子转运体（DRA）与肠道炎症存在交互作用，且其下调可导致肠道厚壁菌/拟杆菌（F/B）比例失调。同时研究显示，不同种属间细菌数量及密度改变可使细菌间信号分子AI-2浓度变化，作用于宿主肠道免疫系统，参与炎症反应。然而DRA相关肠道F/B比例失调是否通过改变AI-2浓度在DRA调控肠道炎症中发挥重要作用及其调控途径未明。故本项目拟首先在UC患者标本中观察DRA表达与肠道F/B比例变化、AI-2浓度相关性；其次应用转基因小鼠研究DRA缺失影响F/B比例的机制，并研究F/B比例失调对AI-2浓度的调节及AI-2调控肠道炎症的免疫学途径；最后在肠道炎症中上调DRA表达，评估其对肠道菌群的调控作用及对肠道炎症的影响及机制，为寻找UC新的潜在干预靶点提供理论及实验依据。

肠道菌群在肠道炎症的发生、发展中具有重要作用。本项目从三部分探讨肠道菌群失调在调控肠道炎症中的作用及机制：第一部分、DRA相关肠道菌群失调在调控肠道炎症中的作用及机制探讨。结果显示，DRA基因缺陷小鼠结肠上皮细胞及肠腔内pH发生显著变化，其幼鼠黏蛋白构成明显改变，结肠杯状细胞数量明显下降、黏蛋白基因表达明显改变，且DRA基因敲除小鼠远段结肠肠道菌群构成明显改变，变形菌门及柔壁菌门比例明显增加。主成分分析结果显示，DRA基因敲除小鼠盲肠、近端结肠及远段结肠肠道菌群构成明显变化。同时，柱前衍生化高效液相色谱分析显示，IBD患者（含UC及CD）粪便AI-2水平较健康对照无明显差异。综合结果显示，DRA缺陷可导致肠道黏液屏障功能障碍，肠道菌群失衡，可能在肠道炎症发生中具有重要调控作用。第二部分、肠道细菌及其代谢产物可能是IBD发病的重要因素之一，既往研究发现粪便胆汁酸浓度在IBD患者中明显增加，抗生素可以通过调节肠道微生物群影响次级胆汁酸的代谢，但尚不清楚这些改变对结肠炎发展的影响。本研究结果表明，抗生素处理可以减轻DSS诱导的小鼠实验性结肠炎的严重程度，降低小鼠的疾病活动程度，降低炎症的组织学评分和炎症因子mRNA表达水平，减少紧密连接的损伤。抗生素使用可以导致肠道细菌和胆汁酸代谢的改变，两者的变化具有一定相关性。本研究为抗生素对DSS诱导结肠炎的保护作用机制提供了新的见解。第三部分、抑制MyD88扰乱肠道微生物群并激活NLRs途径而无法改善DSS诱导的结肠炎。在既往的研究中，髓样分化因子88（MyD88）蛋白参与众多免疫途径，但在IBD的发病机制中的作用尚不完全明确。本研究从肠道微生物群和先天免疫系统的串扰方面出发，旨在探讨MyD88对肠道炎症的影响及其内在机制。本项研究表明，抑制MyD88蛋白表达可降低急性DSS诱导结肠炎时NF-κB信号的活化水平，损害机体抗感染免疫防御，进而引起肠道菌群失调，激活NOD-样受体（NLR）信号通路，诱发肠道炎症，本项研究为MyD88在肠道炎症中的作用及调控机制提供了新的见解。