色氨酸促进断奶仔猪肠黏膜β-防御素表达的信号通路及对致病性大肠杆菌的防御机制研究

β-defensin, which is an important component of the chemical barrier of intestinal mucosa, can effectively prevent the invasion of pathogenic microorganisms. Previous studies showed that tryptophan have special role in promoting the expression of β-defensin in intestinal mucosa, however, the specific mechanism is not clear. The preliminary work of this project demonstrated that tryptophan and its metabolite kynurenine take the responsibility for the up-regulated expression of β-defensin in the porcine intestinal mucosa. According to the finding, the applicant of this project presents two hypothesized signaling pathways. However, the mechanisms of Rheb and AHR and the roles of AKT mediating the signaling pathways for the tryptophan-promoted expression of β-defensin in porcine small intestine epithelial cells need to be further studied. Therefore, combining with in vitro and in vivo tests and applying the technologies of RNA interference and gene overexpression, this project will address the following objectives: (i) to determine the mechanisms of Rheb and AHR mediating the signaling pathways for the tryptophan-promoted expression of β-defensin in porcine small intestine epithelial cells; (ii) to identify the roles of AKT regulating the tryptophan-promoted expression of β-defensin in porcine small intestine epithelial cells; (iii) to verify the defense mechanisms of the tryptophan-promoted expression of β-defensin in piglet small intestinal mucosa against pathogenic Escherichia coli. The implementation of the project finally will reveal the key proteins and conduction pathways that mediate the tryptophan-promoted expression of β-defensin in porcine small intestine epithelial cells, elucidate the defense mechanisms of the tryptophan-promoted expression of β-defensin in piglet intestinal mucosa against pathogenic Escherichia coli, and provide the theoretical references for defending against intestinal pathogen infection and keeping intestinal health of weaned piglets.

β-防御素是肠黏膜化学屏障的重要组成部分，能有效抑制病原菌。已有研究表明，色氨酸具有促进肠黏膜β-防御素表达的特殊功能，但机理不详。申请人前期研究表明，色氨酸自身及其代谢产物犬尿氨酸均可促进肠黏膜β-防御素的表达。根据这一发现，项目组提出两条信号通路假设，其中Rheb与AHR的介导机理及AKT的调控作用有待进一步研究。本项目结合体内与体外试验，以RNA干扰和过表达为调控手段，拟开展以下研究：①Rheb和AHR介导色氨酸促进猪小肠上皮细胞β-防御素表达的机理；②AKT在色氨酸促进猪小肠上皮细胞β-防御素表达过程中的调控作用；③色氨酸促进仔猪小肠黏膜β-防御素表达对致病性大肠杆菌的防御机制。本项目最终揭示介导色氨酸促进猪小肠上皮细胞β-防御素表达的关键蛋白及传导路径，并阐明色氨酸促进仔猪肠黏膜β-防御素表达对致病性大肠杆菌的防御机制，为抵御仔猪肠道致病菌感染及维持肠道健康提供理论参考。

肠黏膜β-防御素能有效防御病原性微生物的侵袭。已有研究表明色氨酸促进肠黏膜β-防御素表达与NF-κB和mTOR信号通路相关，申请人前期研究表明色氨酸自身及其代谢产物犬尿氨酸均可促进肠黏膜β-防御素的表达，但在色氨酸促进仔猪肠黏膜β-防御素表达的信号通路中，Rheb与AHR的介导机理和AKT的调控作用机制仍需进一步研究。本项目以细胞与动物为研究模型，以 RNA干扰为调控手段，开展以下研究：①Rheb和AHR介导色氨酸促进猪小肠上皮细胞β-防御表达的机理；② AKT在色氨酸促进猪小肠上皮细胞β-防御表达过程中的调控作用；③色氨酸促进断奶仔猪肠黏膜β-防御素表达对致病性大肠杆菌的防御机制。结果发现：色氨酸促进猪小肠上皮细胞BD表达的信号通路为（1）Trp-Kyn-AHR- IL-17-PI3K/PDK1-AKT1-IKKα-NF-κB-S6K1/4EBP1-BD；（2）Trp-Rheb-mTOR- S6K1/4EBP1-BD。含0.35%色氨酸的玉米和豆粕日粮能改善断奶仔猪生长性能、肠粘膜屏障完整性和肠道微生物生态，防御产毒性大肠杆菌ETEC的致病作用。本项目揭示介导色氨酸促进猪小肠上皮细胞β-防御素表达的关键蛋白及传导通路，并阐明色氨酸促进仔猪肠黏膜β-防御素表达对致病性大肠杆菌的防御机制，为日粮色氨酸防御致病菌与维持仔猪肠道健康提供理论依据与参考。