异亮氨酸经Beta-防御素2介导调控断奶仔猪小肠黏膜屏障功能的作用机制研究

Diarrhea and mortality of weanling piglets are a major cause of economic loss in swine production. The immature intestinal barrier is the leading agent associated with diarrhea of piglet. Our previous study in piglets demonstrated that isoleucine, as a functional amino acid, could induce intestinal β-defensin 2 expression and increase gut mucosal barrier function against the infection of Escherich coli. However, the mechanism of isoleucine on mucosal barrier is unknown. In this study, the weanling piglet and the intestinal epithelial cell and tissue are utilized as the in vivo and in vitro models, respectively. By using the techniques of lactulose/mannitol, quantitative real-time PCR, immunohistochemistry, immunocytochemistry, co-immunoprecipitation and Western Blot, we will study the effect of isoleucine on the synthesis of mucin and the function of tough-junctions in intestine regulated by β-defensin 2, and the protective effect of isoleucine on intestinal barrier in weanling piglets attacked by Escherich coli. Moreover, we will initially determine the mechanism of isoleucine regulating intestinal mucosal barrier through β-defensin 2 by the research of inhibitor and protein interaction under Escherich coli infection. The research will systemically demonstrate the effects of isoleucine on intestinal mucosal barrier in weanling piglets and the corresponding mechanisms. This study will provide rationale and guideline for enhancing the capability of disease resistant in piglets by nutritional regulation therapy. This is also great value for the application of isoleucine in intestinal diseases of infant.

断奶仔猪肠道内外环境复杂多变增加肠道屏障压力，自身免疫功能不完善加剧了仔猪腹泻和死亡，为养猪生产造成巨大损失。申请者前期研究发现，异亮氨酸作为重要的功能性氨基酸可刺激肠道中Beta-防御素2的表达，并提高肠道对大肠杆菌的屏障功能，但异亮氨酸调节肠道黏膜屏障的调控机制仍不清楚。本项目拟以断奶仔猪和肠上皮细胞/组织为体内和体外模型，结合乳果糖/甘露醇、RT-PCR、免疫组织化学、免疫细胞化学、免疫共沉淀和Western blot等技术，研究异亮氨酸经β-防御素2介导对肠道黏液蛋白和紧密连接蛋白的影响，并在大肠杆菌感染的情况下，检测异亮氨酸对细菌增殖、黏附和炎症反应的作用，同时结合β-防御素与免疫蛋白的互作和抑制剂研究，初步探究异亮氨酸保护肠道黏膜屏障的作用机制，为改善仔猪抗病能力的营养调控技术提供理论基础和指导，并对异亮氨酸在人类肠道营养中的功能研究提供借鉴。

本项目通过体内和体外试验研究了异亮氨酸对仔猪生长性能和肠道免疫屏障功能的影响，并通过体内外模型研究异亮氨酸调节肠道上皮细胞表达防御素的分子机制。体内试验轮状病毒攻毒试验结果表明，异亮氨酸可有效提高仔猪的生长性能，抵御轮状病毒的感染，异亮氨酸促进了肠道中防御素、免疫球蛋白的表达，促进了紧密连接蛋白的合成，改善了肠道菌群数量和种类结构，提高肠道屏障功能。以猪肠上皮细胞系IPEC-J2为体外模型，研究了BCAA对猪β防御素表达的时间效应、剂量效应和组合效应。结果显示，在处理0~12 h之内，异亮氨酸对IPEC-J2细胞中防御素表达没有显著影响，处理24 h时，异亮氨酸对防御素表达的刺激作用显著呈现。剂量试验中，异亮氨酸促进IPEC-J2表达防御素的作用最强，浓度为0.8 mM时促进作用最大（P < 0.05）。时间效应表明，异亮氨酸、亮氨酸和缬氨酸处理IPEC-J2细胞0到6 h之内，ERK1/2的磷酸化水平增高（P < 0.05）。抑制剂试验中，抑制剂PD98059和Splitomicin都显著降低了异亮氨酸对β防御素的促进作用。PD98059阻断了异亮氨酸诱发的ERK1/2和90RSK的磷酸化，Sirt1抑制剂也可阻断异亮氨酸对ERK1/2和90RSK的促磷酸化作用（P < 0.05）。本试验发现脂多糖（lipopolysaccharide，LPS）可促进IPEC-J2中pBD-2和pBD-3的表达（P < 0.05），但ERK1/2磷酸化水平降低（P < 0.05）。综上，本研究内容说明异亮氨酸能够通过Sirt1/ERK/90RSK通路促进猪肠上皮细胞表达防御素，同时异亮氨酸可提高肠道免疫防御功能，抵御微生物的入侵，进而保证断奶仔猪的健康和生长。