致病性大肠杆菌的定植对肉牛肠黏膜屏障及短链脂肪酸代谢通路的影响机制

Since food poisoning caused by E.coli O157: H7was found so far, food safety and infection issues happen much more frequently, making human realized that they had never seen such severe pathogenic microorganisms before, the source of the pathogen is difficult to find, people were unable to take effective preventions. To this end, the project team hypothesized that the pathogenic E.coli escapes from the antagonism of antimicrobial peptidesreleased from the animal intestinal tract which destroys the bacterial cell membrane，leading to intestinal infection ,destruction of the intestinal microflora stability and damage of intestinal mucosal barrier ,causes calf disease eventually .Moreover, the mechanism of pathogenicity E.coli colonization on intestinal SCFAs metabolism pathway and metabolic absorption mechanism of N nutrients has not been elucidated.This article is to reveal the effect of pathogenic E.coli colonizes on intestinal microbial community function and metabolic network, TLR4/NF-κB signaling pathway in intestinal mucosal barrier damage, SCFAs metabolic pathway and the biological activity of N metabolites on intestinal epithelial absorption and intestinal health. Explore the interaction mechanism and causal relationship of E.coli effective colonization, intestinal microecologysystem, SCFAs and N nutrient metabolic products. Provide theoretical basis for restraining intestinal pathogenic bacteria and reducing C/N.

自E. coli O157:H7引起的食物中毒,食品安全和感染问题频发，使人类意识到从未见过的致病微生物如此厉害，更让人头疼的是病原菌的源头难以查出，并且无法采取有效的预防措施。为此，项目组假设：致病性E. coli从肠道释放的破坏细菌细胞膜抗菌肽的对抗中逃逸，破坏肠道微生态系统的稳定和损伤肠黏膜屏障使犊牛致病。然而，致病性E. coli的定植对肠道SCFAs代谢通路、N营养素代谢吸收机理尚未阐明。拟揭示致病性E. coli的定植对肠道微生物群落功能以及群落中各成员之间的代谢网络、TLR4/NF-κB信号通路在肠黏膜屏障损伤、SCFAs代谢通路的影响机理及N代谢产物的生物活性对肠道上皮的吸收及肠道健康的作用机理，并探索致病性E. coli的有效定植、肠道微生态系统、SCFAs和N营养素代谢产物的生物活性形成互动作用机制及因果关系，为寻找抑制肠道致病菌和减排C/N提供理论依据。

致病性大肠杆菌是引起犊牛腹泻的主要细菌。它侵入犊牛身体，并释放肠道毒素，导致犊牛腹泻。我们通过人工干预肠道微生物菌群的定植，建立了犊牛腹泻模型，使用ELISA、qPCR和16SrDNA 高通量测序技术来研究致病性E.coli的定植对肠道微生物、TLR4/NF-κB信号通路在肠黏膜屏障损伤、SCFAs代谢通路的影响机理及N代谢产物的生物活性对肠道上皮的吸收及肠道健康的作用机理。我们的研究表明:（1）致病性E.coli O1定植后破坏犊牛肠道微生物动态平衡，降低肠道微生物多样性，抑制抗菌物质的产生和破坏免疫系统。通过增加回肠黏膜不动杆菌属、假单胞菌属和Lelliottia和结肠黏膜肠杆菌科、伯克氏菌科等有害菌的丰度，降低结肠黏膜瘤胃菌科和毛螺菌科等有益菌的丰度刺激肠道黏膜免疫，促发炎症反应，破坏肠黏膜生物内环境稳态;（2）致病性E.coli O1诱发腹泻导致犊牛肠道黏膜通透性增加，损伤肠黏膜屏障，破坏THI细胞和TH2细胞间的平衡，降低犊牛血清IgA、IgG和IgM的生物合成，降低血清中SOD、GSH-Px及 CAT的含量，破坏免疫与抗氧化功能;（3）致病性E.coli O1通过TLR4/NF-κB信号通路调节紧密连接蛋白的表达，通过上调TLR4表达和激活NF-κB信号通路来降低肠道屏障蛋白（claudin-1、occluding和ZO-1）的含量，促炎因子（TNF-α、IL-2和IL-6）mRNA表达显著升高，从而导致肠道炎症，引起肠黏膜屏障损伤；（4）致病性E.coli O1使犊牛直肠粪便中SCFAs含量显著降低（P＜0.05），SCFAs代谢通路中脂肪受体（GPR41、GPR43）mRNA表达显著降低（P＜0.05），脑肠肽激素（GLP-1和PYY）分泌减少，动物能量代谢和肠道完整性受到影响；（5）致病性E.coli O1对犊牛血糖和血脂代谢造成消极影响，显著升高血清谷丙转氨酶(ALT)与谷草转氨酶(AST)的水平（P＜0.05），对犊牛的肾功能及肝脏造成损伤；（6）致病性E.coli O1显著增加血清中5-羟色胺（5-HT）的含量，5-HT信号的改变间接引起犊牛肠道功能改变以及肠道炎症易感性增加。我们的研究结果为进一步研究致病性E.coli诱导的犊牛腹泻相关微生物学的分子机制奠定了理论基础，也为控制致病性E.coli引起的腹泻病和培育健康犊牛具有深远意义。