硒对金黄色葡萄球菌感染乳腺TLR2/NOD2信号转导通路调控机制的研究

Selenium is one of the important microelements in immune regulation, and previous research confirmed that supplemental selenium decreased the incidence of mastitis. Since Staphylococcus aureus is one of the major causal agent for mastitis and TLR2 and NOD2 are PRRS related to gram-positive bacterium, we ask in this study whether the signal transduction pathways (TLR2 and NOD2) take part in the mammary infection induced by S.aureus with or without the supplemental selenium ? Based on this hypothsis, two S. aureus infection models (in vivo rat mammary and in vitro mammary epithelial cells ) were established to investigate the selenium and its inhibitors on signal transductions pathways of TLR2 and NOD2. QT-PCR and Western blot were utilized to detect the key genes in the signal transduction pathways of TLR2 and NOD2, including TLR2,MyD88,NOD2,RIP2,NF-κb,TNF-α,and IL-1β.In addition, cytokine profiles IL-1、IL-6、TNF-α、TGF-?、IL-8 and IL-10 in serum were detected by ELISA and T lymphocytes subtypes were determined by flow cytometry.Research on function of transduction pathways of TLR2 and NOD2 in selenium influencing the immune balance during S.aureus-causing mastitis assists us to understand the mechanisms of immune regulation with selenium supplement.

硒是重要的免疫调节微量元素，补硒后，能明显降低乳房炎的发病率。而金黄色葡萄球菌感染乳腺后常致持续感染。TLR2和NOD2是与革兰氏阳性菌有关的模式识别受体，其信号通路是否在硒对金黄色葡萄球菌感染乳腺的中起到作用？基于此，本项目建立金黄色葡萄球菌感染大鼠乳腺和乳腺上皮细胞的体内和体外模型，通过添加硒和阻断TLR2和NOD2信号通路，采用QT-PCR、Western blot观察乳腺组织和上皮细胞中TLR2/MyD88/NF-κB 信号通路和NOD2/RIP2/NF-κB信号通路关键因子及TNF-α/IL-1β的mRNA和蛋白表达水平的动态变化。采用 ELISA和流式细胞术，分别检测IL-1β、TNF-α、TGF-ß、IL-8、IL-10的蛋白水平和血液中T细胞亚群的动态变化，探讨NOD2/TLR2信号转导通路在硒对金葡菌感染乳腺中的作用机制，为通过补硒调节免疫提供基础研究。

硒是重要的免疫调节微量元素，补硒后，能够明显降低乳腺炎的发病率，而金黄色葡萄球菌是乳腺炎重要的致病菌，本项目通过体内外研究揭示了金黄色葡萄球菌感染乳腺过程中，硒在免疫调节中的作用机制。结果如下：体内实验显示金黄色葡萄球菌感染大鼠乳腺后，TLR2和NOD2 mRNA表达量显著上升，启动先天性免疫，大量炎性细胞在趋化因子（CXCL1等）的作用下向乳腺炎症部位集中，促进更多炎性细胞因子（TNF-α和IL-1β等）释放，免疫细胞和炎性细胞因子相互协同，防御入侵的金黄色葡萄球菌，并造成乳腺局部组织损伤。而金黄色葡萄球菌性乳腺炎大鼠模型饲补适量的硒后，TLR2蛋白表达量和基因表达量下降，且具有浓度依赖性。硒能够抑制多数（TNF-α、IL-1β、IL-6和IL-8等）促炎因子的合成和释放，且有浓度依赖性。同时，亚硒酸钠对乳腺炎大鼠氧化性损伤具有保护作用，这种作用是通过提高全身性（肝脏、血液）抗氧化能力来发挥的。体外实验结果证实硒通过对奶牛乳腺上皮细胞TLR2和Nod2信号通路的调控，减弱了NF-κB和MAPK信号通路的转导，降低了TNF-α、IL-1β和IL-6以及转录激活蛋白AP-1的表达，表现出减轻金黄色葡萄球菌诱导的奶牛乳腺上皮细胞炎性损伤的作用，其中4μM和8 μM的硒作用最强。巨噬细胞预孵育硒12 h，可显著降低金黄色葡萄球菌诱导的巨噬细胞炎症反应，下调炎症细胞因子的基因和蛋白表达。硒的这种作用可能是通过对NF-κB和MAPK信号通路的调控实现的。