约氏乳杆菌通过EGFR/Akt通路调节ETEC腹泻仔猪肠上皮细胞内质网应激性凋亡的机制研究

Enterotoxigenic Escherichia coli (ETEC) infection often results in piglet diarrhea, which threatens the healthy development of pig industry as well as food and sanitation safety. Our previous studies show that administration of Lactobacillus ameliorates ETEC-induced diarrhea in piglets, which is related with activating EGFR/Akt pathway and inhibiting the ETEC-induced intestinal epithelial cell (IEC) endoplasmic reticulum stress and apoptosis. However, the molecular mechanism still remains unclear. Thus, in the present study, the in vivo piglet diarrhea and the in vitro cell models with ETEC infection will be established. 1. We will examine the positioning of endoplasmic reticulum stress proteins Bip, CHOP and apoptotic cells in the small intestine and analyze the effects of Lactobacillus johnsonii on endoplasmic reticulum stress (PERK、ATF6、IRE1 pathways) and apoptosis (BCL-2, BIM, caspase-3/9) in IECs of piglets with ETEC-related diarrhea. 2. Next, we will study the potential role of endoplasmic reticulum stress in ETEC-induced IEC apoptosis and how Lactobacillus johnsonii regulates it by using endoplasmic reticulum stress inhibitor. 3. Last, we will explore the regulating mechanism of EGFR/Akt pathway in the probiotic effect of Lactobacillus johnsonii. This study will reveal the mechanism of Lactobacillus johnsonii regulating endoplasmic reticulum stress-induced IEC apoptosis in piglets with ETEC-related diarrhea via EGFR/Akt pathway and provide the theoretical basis for rational use of probiotic in preventing piglet diarrhea.

肠毒性大肠杆菌（ETEC）常导致仔猪腹泻，威胁养猪业健康发展与食品卫生安全。前期研究发现：乳杆菌改善仔猪ETEC腹泻，与其激活EGFR/Akt，抑制ETEC引起的肠上皮细胞（IEC）内质网应激和凋亡有关，但其分子机制还不清楚。在此基础上，本项目拟建立仔猪ETEC腹泻及细胞感染模型，1. 检测内质网应激相关Bip、CHOP蛋白及凋亡细胞在小肠中定位，分析约氏乳杆菌对ETEC腹泻仔猪IEC内质网应激（PERK、ATF6、IRE1通路）和凋亡（BCL-2、BIM、caspase-3/9）的影响；2. 应用内质网应激抑制剂，研究内质网应激在ETEC引起的IEC凋亡中的作用及约氏乳杆菌对其调节作用；3. 应用RNAi技术，探究EGFR/Akt对约氏乳杆菌作用的调控机制。揭示约氏乳杆菌通过EGFR/Akt通路调节ETEC腹泻仔猪IEC内质网应激性凋亡的机制，为合理利用益生菌防治仔猪腹泻提供理论依据。

肠毒性大肠杆菌（ETEC）是导致仔猪腹泻的常见致病菌，严重威胁养猪业健康发展。我们前期研究发现，乳酸杆菌改善仔猪ETEC腹泻可能与其调控肠上皮细胞内质网应激和凋亡有关。本项目首先对约氏乳杆菌ZLJ010进行全基因组分析，并构建了包含8株不同来源约氏乳杆菌的泛基因组数据库进行比较基因组分析，发现ZLJ010特异性携带大量黏附、氨基酸转运与代谢、细菌素合成相关基因，有助于其适应猪复杂的肠道环境，并持久黏附于肠上皮细胞发挥抗菌、免疫调控功能。进一步进行猪群ZLJ010饲喂试验，并构建了小鼠ETEC感染模型，结果表明，ZLJ010可显著降低粪便中致病性大肠杆菌数量，改善肠道菌群紊乱，增加肠道中拟杆菌、双歧杆菌、梭菌、乳酸杆菌属等有益菌丰度，改善猪生长性能，减弱ETEC导致的肠道上皮损伤。最后建立猪肠上皮J2细胞ETEC感染模型，发现活ZLJ010可抑制ETEC在J2细胞上的黏附，而热灭活、紫外灭活或ZLJ010上清均不具备抑制ETEC黏附的能力。ETEC感染过程中，ZLJ010激活EGFR/Akt信号通路，减弱ATF6α、IRE1α-XBP1和PERK-eIF2α介导的内质网应激，抑制细胞凋亡，但加入EGFR/Akt或内质网应激化学抑制剂后，ZLJ010不能抑制ETEC导致的细胞内质网应激及凋亡。我们结果表明，约氏乳杆菌ZLJ010通过占位效应以及调控肠道菌群结构阻止ETEC在肠上皮细胞的黏附，同时通过激活膜表面EGFR/Akt信号通路，抑制内质网应激介导的细胞凋亡，维持肠上皮屏障功能，最终改善ETEC导致的猪肠上皮损伤及腹泻。