GPR109A与变形菌互作在断奶仔猪腹泻中的作用研究

Improvement of intestinal barrier is regarded useful to solve post weanling diarrhea in piglets. Proteobacteria is one of the largest bacteria community, including many pathogenic bacteria, such as Escherichia coli, Salmonella, which affect local intestinal inflammation and lead to diarrhea in animal by lipopolysaccharide (LPS) and their metabolites. GPR109A is involved in many physiological processes including regulation of cell proliferation, inflammatory reactions and metabolism. GPR109A plays an important role in regulating intestinal barrier. Our previous studies confirmed that GPR109A affects the community of intestinal bacteria especially lower the abundance of Proteobacteria, thereby regulating the inflammatory response and intestinal barrier function in CLP induced sepsis. Thus, we hypothesized that the bacteria and its metabolites, regulates the expression and activation of GPR109A which contribute to colon health. Activation of GPR109A affect the epithelium metabolism rate (especially fatty acid levels) and immune status in colon, which play a selective role on the intestinal flora. This study intends to explore the mechanisms of interaction between GPR109A and intestinal Proteobacteria. Metabonomics, bacterial genomics, transcriptomics, cell biology, molecular biology, bioinformatics methods, will be used to investigate the effect of Salmonella and Escherichia coli on expression and activation of porcine intestinal GPR109A, to explore the effects of GPR109A activation on intestinal metabolism, immune function and colonization of Salmonella or Escherichia coli. Reveal the role of the interaction in maintaining intestinal health is supposed to provide an effective approach and therapeutic targets to solve post weaning diarrhea in piglets.

沙门氏菌和致病性大肠杆菌等变形菌（Proteobacteria），是断奶仔猪腹泻的重要诱因之一。我们前期研究结果证实，GPR109A在肠道屏障和局部炎症调控中发挥着重要的作用。GPR109A还直接（或间接）地影响着变形菌的丰度，但机制不明。因此，本研究拟从GPR109A与变形菌互作的角度，采用代谢组学、细菌基因组学、转录组学、细胞、分子生物学等研究方法，结合生物信息学手段，探讨沙门氏菌和大肠杆菌对猪肠道GPR109A表达和活化的影响，研究GPR109A的活化对变形菌诱导的炎症信号通路的影响，观察GPR109A对肠道局部代谢水平、免疫功能、屏障功能的调控作用，分析上述改变对沙门氏菌和大肠杆菌的定植、增殖能力的影响。预期结果不仅有利于阐明GPR109A与沙门氏菌和大肠杆菌的互作机制，而且为揭示GPR109A在猪肠道屏障调控中的作用提供实验依据，并且有望为解决断奶仔猪腹泻提供新的思路和治疗靶点。

本研究按照实验计划，重点开展了变形菌与GPR109A互作的关系研究。首次揭示了GPR109A的激活对致病性大肠杆菌为代表的变形菌肠道黏膜下定植的影响及机制。实验结果表明GPR109A-/-鼠粪便中致病菌的丰度显著升高，如变形菌门的沙门氏菌、大肠杆菌等。GPR109A-/-鼠表现出对ETEC的极易感性。与之相反，GPR109A的激活可有效抑制ETEC在肠道的定植和易位、维持肠屏障的完整性。进一步的实验结果表明，GPR109A可能是通过促进肠道IgA的分泌发挥作用。体外实验表明丁酸钠可通过激活IPI-2I细胞内GPR109A和ERK1/2信号通路进而增加pIgR的表达。另一方面，我们探讨了变形菌对宿主GPR109A表达的影响。证实了ETEC通过ERK1/2途径促进巨噬细胞产生IL-1β和TNF-α，间接促进肠上皮细胞中GPR109A的表达，解释了机体在ETEC感染期间调节宿主肠道基因表达稳态的分子机制。此外，利用本课题研究建立的肠道模型研究方法和条件，开展了昆虫饲料添加对ETEC K88引起的断奶仔猪腹泻及肠道屏障功能影响的研究。研究表明饲料中添加黑水虻幼虫粉可以改变仔猪肠道微生物群分布，增加相对益生菌的丰度，改善肠道健康，这种效应是由柠檬酸所介导的。