Mechanism of action of dietary fibre in the regulation of intestinal epithelial barrier function

膳食纤维调节肠上皮屏障功能的作用机制

• 批准号: RGPIN-2018-04321
• 负责人: MacNaughton, Wallace
• 金额: $ 5.83万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=763348
• 关键词: Mechanism; action; dietary; fibre; regulation

The intestinal epithelium provides an essential, multi-functional barrier that: (1) establishes the osmotic, chemical and electrical potential gradients that facilitate absorption of nutrients, water and electrolytes; (2) secretes mucus, antimicrobial peptides and immunoglobulins that are important for host defence; and (3) prevents excessive mucosal penetration of bacterial and luminal antigens and, consequently, prevents over-activation of the mucosal immune system. This Discovery Grant proposal describes a new research program that will determine the mechanisms underlying how plant- and fungus-based polysaccharides (PS) act as important physiological regulators of the intestinal epithelial barrier. Our preliminary data show that the plant-derived PS, rhamnogalacturonan (RGal), directly alters epithelial behaviour (cell migration, barrier function) in the Caco-2 epithelial cell line, although the exact mechanism of action has not been elucidated. Our hypothesis is that PS enhance intestinal epithelial barrier function either directly or through the gut microbiota, to elicit specific signaling responses that regulate tight junction structure and function to maintain epithelial homeostasis. Three aims will test this hypothesis.Aim 1. Determine the structural and functional changes in intestinal epithelium promoted by PS. Effects of PS on epithelial function (migration, proliferation, death) will be determined with a high-throughput screening system we developed on the IncuCyte platform. Dynamic effects on tight junction structure will be assessed using fluorescence recovery after photobleaching (FRAP) to track PS-induced changes in tight junction protein mobility. Aim 2. Analyze the genes and signalling pathways activated by PS. Using RNAseq, we will identify PS-induced changes in the transcription of genes with known roles in regulating epithelial barrier function. We will then target identified pathways with siRNA, CRISPR-Cas9 or pharmacological inhibitors to determine their functional role in epithelial barrier function. Aim 3. Determine the role of the gut microbiota in PS-induced effects on the epithelial barrier. Through the new Western Canadian Microbiome Centre at University of Calgary, we will have access to germ-free mice that will help us to understand the epithelial effects of PS in the absence of gut microbiota. If a dependence on bacterial metabolism is observed in the barrier-specific effects of PS, we will subsequently investigate the PS metabolites that have relevant biological activity and determine their cellular mechanism of action. Our program will characterize the largely unexplored mechanisms of interactions among PS, the gut microbiome and the epithelium. This information will contribute to food science by mechanistically highlighting the benefits of dietary fibre on epithelial function and overall intestinal homeostasis.

肠上皮细胞提供了一个基本的多功能屏障，其：（1）建立渗透、化学和电势梯度，促进营养素、水和电解质的吸收;（2）分泌粘液、抗微生物肽和免疫球蛋白，这些对于宿主防御是重要的;和（3）防止细菌和管腔抗原的过度粘膜渗透，并因此防止粘膜免疫系统的过度激活。 这项发现补助金提案描述了一项新的研究计划，该计划将确定植物和真菌多糖（PS）作为肠上皮屏障重要生理调节剂的机制。 我们的初步数据表明，植物来源的PS，鼠李糖半乳糖醛酸聚糖（RGal），直接改变上皮细胞的行为（细胞迁移，屏障功能）在Caco-2上皮细胞系，虽然确切的作用机制尚未阐明。我们的假设是，PS直接或通过肠道微生物群增强肠上皮屏障功能，以引起调节紧密连接结构和功能以维持上皮稳态的特异性信号传导反应。三个目标将检验这一假设。测定PS促进的肠上皮结构和功能变化。将使用我们在IncuCyte平台上开发的高通量筛选系统确定PS对上皮功能（迁移、增殖、死亡）的影响。将使用光漂白后荧光恢复（FRAP）跟踪PS诱导的紧密连接蛋白迁移率变化，评估对紧密连接结构的动态影响。目标2.分析PS激活的基因和信号通路。使用RNAseq，我们将确定PS诱导的基因转录的变化，这些基因在调节上皮屏障功能中具有已知的作用。然后，我们将用siRNA、CRISPR-Cas9或药理学抑制剂靶向已鉴定的通路，以确定它们在上皮屏障功能中的功能作用。目标3。确定肠道微生物群在PS诱导的上皮屏障效应中的作用。通过卡尔加里大学新的加拿大西部微生物组中心，我们将获得无菌小鼠，这将有助于我们了解PS在没有肠道微生物群的情况下对上皮细胞的影响。如果在PS的屏障特异性效应中观察到对细菌代谢的依赖性，我们将随后研究具有相关生物活性的PS代谢物，并确定其细胞作用机制。我们的计划将描述PS，肠道微生物组和上皮之间相互作用的基本未探索的机制。这些信息将有助于食品科学的机械突出的好处，膳食纤维上皮功能和整体肠道内稳态。