TLR4/NLRP3信号通路在雌激素与孕酮调控湖羊子宫内膜炎中的作用

The incidence of endometritis in milking animals is closely related to the levels of estrogen and progesterone, which is produced by the growing follicle and punctuates with menstrual cycles. However, it is unclear how estrogen and progesterone mediate the occurrence and remediation of endometritis. To reveal the biological mechanisms underlying estrogen and progesterone immune regulation activity, the roles of estrogen and progesterone to prevent uterus from endometritis will be characterized in vivo and in vitro in this study. Firstly, the Hu sheep will be ovariectomized (OVX), then injected with exogenous estradiol and progesterone at varying concentrations. Uterus will be injected with LPS to establish the infection model. The manifestations of disease of uterus and the immune response mediated by TLR4/NLRP3 signaling pathway will be analyzed by histopathology, real-time quantitative PCR, Western blot and ELISA accordingly. The primary endometrial epithelial cell will be used as an in vitro model to explore the roles of estrogen and progesterone in reduction of inflammation. Meanwhile, the receptor antagonists of estrogen and progesterone will be added to the cultures of epithelial cells with or without RNA silenced p38 MAPK network to reveal the signaling of TLR4/NLRP3 on inflammatory factors release as analyzed by flow cytometry. This will provide insight for alternative roles of sex hormones underlying aspects of estrogen and progesterone mediated endometritis remediation in milking animals.

产奶家畜子宫内膜炎的发生与雌激素和孕酮的生理周期性变化密切相关，但雌激素和孕酮是如何介导子宫内膜炎的发生与修复呢？本项目以湖羊为模型，从体内和体外两个方面展开研究。体内实验是先对湖羊实施卵巢摘除术，通过外源注射的方法提高湖羊雌激素和孕酮水平，再进行子宫内注射脂多糖建立感染模型，通过病理切片、实时定量PCR、免疫印迹和ELISA等技术，观察不同激素条件下子宫组织的病理变化及TLR4/NLRP3信号通路介导的子宫先天免疫应答情况。体外实验以湖羊原代子宫内膜上皮细胞为模型，通过添加雌激素和孕酮及ER/PR拮抗剂和用siRNA沉默上皮细胞p38 MAPK等手段，利用流式细胞仪和ELISA等分析方法，探索ER/PR和p38 MAPK在雌激素与孕酮调控TLR4/NLRP3信号通路中的作用。该项目将为预防和控制泌乳动物性激素有关的子宫内膜炎的发生提供理论依据。

产奶家畜子宫内膜炎的发生与卵巢的生理周期性变化密切相关，雌激素是其中一种非常重要的生殖激素，但雌激素是如何介导子宫内膜炎的发生与修复呢？本项目从体内和体外两个方面展开研究。体内实验首先对湖羊实施了卵巢摘除术，通过皮下放置缓释片或肌肉注射17β-雌二醇的方式补充外源雌激素，观察不同激素条件下子宫组织的结构变化及雌激素受体ERα和ERβ的表达情况。结果显示雌激素的缺失在一个发情周期内对肝肾等生化指标没有影响，但会导致子宫内膜上皮的萎缩，下调ERα的表达，而上调ERβ的表达。体外实验以Raw264.7为细胞模型，分别通过添加雌激素、雌激素受体拮抗剂，利用ELISA、荧光定量和细胞免疫荧光等检测的方法，探索了雌激素调剂机体免疫反应的分子机制。结果显示向Raw264.7细胞中加入100nM的雌激素后，可以显著降低LPS引起的IL-1β的蛋白表达，而这种抑制作用主要被ICI182 780和PHTPP所拮抗。研究显示雌激素主要与ERβ结合后，主要通过抑制NF-κB p65进入细胞核和IκBα磷酸化的方式来抑制LPS引起的细胞炎症反应。该项目揭示了雌激素抑制炎症反应的部分分子机制，为预防和控制泌乳动物雌激素相关的子宫内膜炎的发生提供了理论依据。