硒调控SOCS/Nrdp1分子介导的抗奶牛乳腺炎症损伤作用机制研究

The researches showed that Staphylococcus aureus（S. aureus）induced mammitis could be restrained and alleviated with selenium (Se). S. aureus induced mammary inflammatory injury via TLR2 signal transduction pathway in dairy cow. SOCS and Nrdp1 were the negative regulation molecules of TLR2 signal transduction. The question that Se resists mammary inflammatory injury and regulats TLR2 signal transduction via SOCS and Nrdp1 is still unknown. This subject is performed on the S. aureus infection model in vivo and in vitro. The dairy cows are fed with Se supplement diet, and the mammary cells are cultured by Se supplement medium. The SOCS and Nrdp1 were performed gene silencing and overexpression. The techniques are used in present study including co-immunoprecipitation, laser scanning confocal microscope, western blot, qRT-PCR, immunofluorescence labeling, et al. With the above techniques, we analyze the condition of TLR2 signal transduction joint molecules, downstream signaling pathways and inflammatory factors. The purpose of this research was to confirm and elucidate that the mechanism of Se resisting mammary inflammatory injury via SOCS and Nrdp1 and TLR2 signal transduction in Dairy Cow. This project will be further reveal pathogenesis of S. aureus bovine mastitis, offering the new strategy and evidence for the prevention and control of this disease.

研究表明硒可有效降低奶牛金葡菌性乳腺炎的发生，缓解临床症状。金葡菌通过激活TLR2信号转导通路引发奶牛乳腺炎症损伤，SOCS、Nrdp1分子对TLR2信号转导发挥负调控作用，可抑制炎症信号的传递。硒是否通过调控SOCS、Nrdp1影响TLR2信号转导从而降低奶牛乳腺炎症损伤尚不清楚。本课题用加硒饲料饲喂奶牛，加硒培养基培养乳腺细胞，然后通过金葡菌感染建立体内外研究模型。对SOCS、Nrdp1分子应用基因沉默和过表达技术进行处理，通过免疫共沉淀、激光共聚焦显微镜、蛋白免疫印迹、荧光定量PCR、免疫荧光标记等技术，对TLR2信号转导关键接头分子、下游信号通路和炎症因子进行检测分析，确证并阐明硒通过SOCS、Nrdp1调控TLR2信号转导抑制金葡菌感染奶牛乳腺炎症损伤的作用机制。本项目的实施将有助于进一步揭示奶牛金葡菌性乳腺炎的发病机制，为该病的防治提供新的思路和依据。

乳腺炎是造成奶牛业巨大损失和危害的重要疾病。金黄色葡萄球菌(以下简称“金葡菌”)是奶牛乳腺炎的主要致病菌之一。金葡菌性乳腺炎不仅导致奶牛泌乳量下降、使用年限缩短，还影响乳品质量，引发食品卫生安全问题，危害消费者健康。奶牛产后泌乳期机体能量代谢增强。若此阶段饲料质量较差或营养不均衡，会造成奶牛免疫力降低，对金葡菌的防御能力降低，易发乳腺炎，炎症损伤严重。在饲粮中适量添加微量元素硒可显著降低乳腺炎的发病率。奶牛金葡菌性乳腺炎患病率与牛乳中硒含量水平呈负相关，饲粮中添加硒可降低奶牛乳腺病原菌感染机率，同时降低泌乳早期牛乳中较高的体细胞数。但硒降低乳腺炎的发生及抑制乳腺炎症损伤的作用机制目前还不清楚。据此本项目主要研究硒调控SOCS/Nrdp1分子影响TLR2信号转导，抑制金葡菌感染造成的奶牛乳腺炎症损伤作用机制。通过向正常及加硒饲料饲喂奶牛的乳导管灌注金葡菌建立动物疾病模型进行体内实验，分离健康奶牛乳腺上皮细胞进行体外感染实验，研究硒通过SOCS/Nrdp1调控TLR2信号转导降低金葡菌感染奶牛乳腺炎性损伤的作用机制。结果表明补充硒可有效控制金葡菌感染所造成的乳腺炎症损伤，缺硒则会促进损伤。硒对于乳腺炎症损伤的调控可通过影响 TLR2信号转导通路中SOCS、Nrdp1负调控分子的表达来完成，进而对下游Mal、MyD88、IRAK4、TRAF6等分子的信号传递，抑制NF-κB和MAPKs信号通路的激活，抑制促炎因子TNF-α、IL-6、IL-1β和IL-18的过度分泌，从而阻止炎症损伤的发生。本项目研究深入阐明硒在金葡菌性奶牛乳腺炎过程中的抗炎作用及机制，为有效防治奶牛金葡菌性乳腺炎这类感染性疾病，提供新的思路和实验依据。同时进一步丰富了硒的生物学作用，有助于营养免疫学在动物疾病研究领域的拓展及应用提供理论指导和帮助。