硒蛋白S介导内质网应激CHOP信号抑制乳腺金葡菌性炎症损伤调控机制

Staphylococcus aureus (S. aureus) mastitis has seriously restricted the development of dairy farming, causing food safety problems. The occurrence of the disease is closely related to the animal autoimmune status. Trace element selenium and selenoproteins show important regulation effect on cow immune status, which can effectively improve the mammary gland immunocompetence to defend against S. aureus and other pathogen. The selenoprotein S (SelS) was confirmed the regulation of endoplasmic reticulum stress and inflammatory response, but the specific mechanism is still unclear. The project was on the foundation of immunology research basis of the endoplasmic reticulum stress participated in inflammation. The subject is performed on the S. aureus infection model of dairy cow in vivo and vitro. The molecular biology techniques are used in present study, including Gene knockout and overexpression, immunofluorescence labeling, western blot, qRT-PCR, etc. With the above techniques, we study the effect of SelS on IRE1, PERK, ATF6 endoplasmic reticulum stress regulation pathway, CHOP molecular signal, inflammatory signaling pathway and inflammatory factors, etc. The purpose of this research was to analyze the influence of S. aureus-induced endoplasmic reticulum stress on dairy mastitis, confirm the role of SelS in regulating endoplasmic reticulum stress-CHOP signal, elucidate that the mechanism of SelS regulated the mammary gland inflammatory process with endoplasmic reticulum stress-CHOP signal transduction in dairy cow. This project will be further to reveal the pathogenesis, explore a support on the new strategy and evidence for the prevention of S. aureus mastitis in dairy cow.

金黄色葡萄球菌（金葡菌）性乳腺炎严重制约奶牛业发展，同时引发食品安全问题。该病发生与动物免疫状态密切相关。微量元素硒及硒蛋白参与奶牛免疫调控，可提高乳腺组织对病原的防御能力。研究证实，硒蛋白S（SelS）调节内质网应激反应，对炎症应答具有调控作用，但具体机制还不清楚。本项目以内质网应激参与炎症免疫应答为研究基础，通过建立奶牛金葡菌性乳腺炎体内外模型，利用基因沉默与过表达、免疫荧光、蛋白免疫印迹、荧光定量PCR等分子生物学技术，研究SelS对IRE1、PERK、ATF6内质网应激调控通路、CHOP分子信号、炎症信号通路及炎性因子等的调节作用。分析金葡菌感染诱发内质网应激对乳腺炎症损伤的影响，明确SelS调控内质网应激CHOP信号的作用靶点，阐明SelS通过内质网应激CHOP信号对乳腺炎症损伤的调控机制，为金葡菌性乳腺炎找寻新的防治靶点及防控措施提供理论支持及帮助。

金黄色葡萄球菌（金葡菌）性乳腺炎不仅造成巨大经济损失，同时引发食品安全问题，危害消费者健康，严重制约奶牛养殖业发展。微量元素硒及硒蛋白对于奶牛免疫状态具有重要调节作用，其中硒蛋白S（SelS）参与内质网相关蛋白降解，调节内质网应激反应对炎症发挥调控作用。本项目以兽医学及营养免疫学为理论指导，利用RNA干扰、免疫荧光、蛋白免疫印迹等现代分子生物学技术，通过建立体内外金葡菌乳腺炎模型，结合乳腺组织病理学观察、炎性因子分析确定炎症反应情况，同时从基因及蛋白水平分析SelS表达情况，确定SelS与乳腺炎症反应的相关性。分离培养奶牛乳腺上皮细胞，进行SelS过表达及基因沉默，给予金葡菌刺激后，从基因及蛋白水平分析SelS在金葡菌感染乳腺炎症反应过程中的作用。证实硒蛋白S在奶牛金葡菌诱发乳腺炎的发生过程中发挥重要调控作用。该调控作用与日粮摄入硒含量的变化密切相关。通过动物体内实验及体外细胞实验对内质网应激反应关键蛋白葡萄糖调节蛋白及下游CHOP分子及内质网应激反应相关的IRE1、PERK和ATF6三个通路的分析。同时以细胞程序性死亡相关分子BAX、BCL-2、caspase-3、6、7、9等作为CHOP蛋白分子调控的效应检测分子。全面分析SelS对金葡菌诱发乳腺组织细胞内质网应激反应及下游CHOP分子的调控作用。同时对SelS过表达或基因沉默的奶牛乳腺上皮细胞加入CHOP封闭肽，结合金葡菌刺激及内质网应激激动剂。对NF-κB及MAPKs信号通路关键节点分子及炎性因子进行分析；阐明SelS通过内质网应激CHOP信号作用于炎症信号通路激活及凋亡相关分子，进而抑制金葡菌对乳腺细胞的炎性损伤的作用机制，为金葡菌性乳腺炎防治找寻新的靶点提供理论支持及帮助。围绕着奶牛生产过程中硒有效利用，结合临床奶牛乳腺健康及乳汁分泌情况，选取了不同形式的硒进行补充，为有效地硒蛋白制剂研发、建立奶牛乳腺炎发生的早期诊断防控措施提供指导和帮助。