辣椒素经由紧密连接蛋白调控肉牛唾液腺分泌功能的机制研究

亚急性瘤胃酸中毒（SARA）是严重危害我国高产奶牛和育肥肉牛健康的一种营养代谢病。唾液分泌量是缓冲瘤胃pH的重要生理机制，紧密连接（TJ）蛋白介导的旁细胞途径对唾液腺分泌功能具有重要调控作用。申请人前期发现，辣椒素可提升肉牛的瘤胃pH值，降低腮腺组织中TJ蛋白的表达。但辣椒素经TJ蛋白介导的旁细胞途径调控肉牛唾液分泌的机制尚不明确。本项目拟采用体内辣椒素饲喂肉牛试验，明确辣椒素对肉牛唾液分泌量和瘤胃pH的影响；进一步通过体外腮腺上皮细胞试验，应用免疫荧光、RT-PCR、Western-blot和Ussing Chamber等技术，阐明辣椒素对TJ蛋白表达和分布的调控作用；应用RNA干扰和质粒转染技术，选择性敲低或过表达TJ蛋白，明确TJ蛋白在旁细胞途径中的作用；围绕RhoA-ROCK信号通路，揭示辣椒素经由TJ蛋白调控肉牛唾液腺分泌功能的分子机制，为防治SARA提供新思路。

Subacute ruminal acidosis (SARA) is one of the prevalent metabolic disorder of high-producing dairy cows and beef cattle. Saliva is an important physiological mechanism for buffering the ruminal pH. The tight junction (TJ) protein-mediated paracellular route plays an important role in the regulation of salivary gland secretion. In our previous work, we found that capsaicin elevated the ruminal pH and decreased the expression of tight junction (TJ) proteins in parotid gland tissue of beef cattle. However, the mechanism of capsaicin regulating salivary secretion of beef cattle via the TJ protein-mediated paracellular route is still unclear. In the proposed project, beef cattle will be used to investigate the effects of capsaicin addition on ruminal pH and saliva secretion. Furthermore, the parotid epithelial cells will be cultured to evaluate the effects of capsaicin on expression and distribution of TJ proteins using immunofluorescence, RT-PCR, Western-blot and Ussing chamber. The role of TJ proteins on paracellular permeability is clarified by using small molecule RNA interference and plasmid transfection to selectively knock down or overexpress TJ proteins. We will further illustrate the molecular mechanism of capsaicin regulating salivary gland secretion through TJ proteins , especially focusing on RhoA/ROCK pathways, and providing a new strategy for prevention of the SARA.