60%以上仔猪断奶腹泻与ETEC引起的肠黏膜损伤有关，“肠道干细胞-杯状细胞-MUC2”轴是肠黏膜抵抗病原菌感染的重要路径。预备试验表明，止泻中药石榴皮中的鞣质可缓解ETEC诱导的肠黏膜损伤及干细胞和杯状细胞损失，尚不清楚ETEC损伤肠黏膜及石榴皮鞣质干预的机制。本项目以断奶仔猪和肠道干细胞为材料，采用流式细胞、蛋白定量、基因编辑等技术，先从形态结构、屏障功能、紧密连接蛋白等多角度，明确石榴皮鞣质对ETEC诱导的肠黏膜损伤的缓解作用。然后测定肠道干细胞和杯状细胞比例及MUC2表达和分泌，揭示ETEC和石榴皮鞣质对“肠道干细胞-杯状细胞-MUC2”轴的影响。最后过表达β-catenin/Klf4，进而探明ETEC和石榴皮鞣质通过β-catenin/Klf4信号发挥作用的分子机制。研究结果将从肠道干细胞角度阐明ETEC损伤仔猪肠黏膜的机制及石榴皮鞣质的干预作用，为研发新型饲料添加剂提供新思路。

It has been reported that more than 60% of post-weaning diarrhea in piglets is related with intestinal mucosal injury caused by Enterotoxigenic Escherichia coli (ETEC) infection. And a significant defensive way of intestinal mucosal against pathogen is via “intestinal stem cells-goblet cells-MUC2 (ISCs-GCs-MUC2)” axis. Besides, pomegranate peel is a kind of antidiarrheal traditional Chinese medicine, whose tannins are vital bioactive substances to protect intestinal mucosa from pathogen infection. Our preliminary studies showed that pomegranate peel tannins (PPT) alleviated the loss of ISCs and GCs and intestinal mucosal damage induced by ETEC. But it is not clear how ETEC causes intestinal mucosal damage and whether PPT could play a role through "ISCs-GCs-MUC2" axis. .The project will be carried out in weaned piglets (in vivo) and ISCs (ex vivo). Flow cytometry, protein quantification, RNA interference and other technical approaches are about to be used. Firstly, the effects of PPT on alleviation of ETEC induced intestinal mucosal injury will be discussed from the aspects of morphological structure, trans-epithelial electrical resistance, permeability of FITC-dextran (4400 Da) and tight junction proteins. Moreover, the proportions of ISCs and GCs and the levels of MUC2 will be measured in vivo and ex vivo to reveal the effects of ETEC on "ISCs-GCs-MUC2" axis and the intervention of PPT. Finally, we’ll use PLKO.1-puro-β-catenin lentivirus and PLKO.1-puro-Klf4 lentivirus to modifyβ-catenin expression and Klf4 expression in ISCs, respectively, and then elucidate the mechanism of ETEC suppressing "ISCs-GCs-MUC2" axis throughβ-catenin/ Klf4 signaling and the alleviative effects of PPT. .In summary, the results will elucidate the mechanism of ETEC damage to intestinal mucosa in piglets and the intervention of PPT from the perspective of ISCs, which will also provide a new idea for the development of new feed additives for the treatment of intestinal mucosal injury.