皮质醇和孕酮对奶牛子宫内膜感染大肠杆菌先天免疫和再生修复的影响机制

In clinical practice, more than 40% of the postpartum dairy cows are susceptible to bacterial infection in uterine, and most are caused by Escherichia coli (E. coli). Evidence from clinical practice indicates that the high levels of cortisol and progesterone contributes to the risk of infection in uterine. However, few studies have reported it in literatures. The study includes experiments in vitro and in vivo. In vitro, endometrial cells including both epithelial cells and stromal cells from cows will be pretreated with different concentrations of hydrocortisone and progesterone, respectively, and then E.coli will be added into the culture medium. In vivo, dairy cows at 7-10 days postpartum will be consecutively injected with hydrocortisone and progesterone, respectively, which will keep relatively high levels of hydrocortisone or progesterone in blood. And then E.coli will be inoculated into the uterine cavities in the dairy cows. With both experiments in vitro and in vivo, the impact of hydrocortisone and progesterone on the innate immune and regeneration in dairy cow endometrium with E.coli infection will be explored by detecting the key factors and cytokines involved in TLR4/MyD88/NF-κB signaling pathways, GILZ protein, key factors of the MAPK-ERK pathway, the cytokines involved in cell proliferation, and the morphological variations of endometrium.

在临床上，40%以上的奶牛在产后易发生子宫腔内细菌感染，且多为大肠杆菌感染。临床资料显示，产后宫腔内感染与产后血液皮质醇和孕酮水平较高有一定的相关性，但至今未见相关的研究报道。本项目研究内容包括体外实验与体内实验。在体外，用不同浓度的氢化可的松和孕酮分别预处理体外培养的奶牛子宫内膜细胞（包括上皮细胞和间质细胞两部分），然后在细胞培养液中加入大肠杆菌。体内实验是选用产后7-10天健康奶牛，分别连续注射氢化可的松和孕酮，使血液皮质醇或孕酮维持在较高的水平，然后在奶牛子宫腔内接种大肠杆菌。利用体外与体内实验，通过检测子宫内膜细胞TLR4/ MyD88/NF-κB 信号通路关键因子及炎性细胞因子、GILZ蛋白、MAPK-ERK信号通路关键因子、细胞增殖相关因子和内膜细胞形态学的变化规律，探究氢化可的松和孕酮对奶牛子宫内膜感染大肠杆菌时先天免疫机制和内膜再生修复机制的影响。

（1）大肠杆菌脂多糖可诱导奶牛子宫内膜细胞NF-κB、MAPK、Wnt/β-catenin和PI3K/AKT通路的活化，促进炎性因子和细胞生长相关因子的表达；皮质醇能够抑制LPS诱导内膜细胞上述通路的活化，减少细胞促炎因子和细胞相关生长因子的表达，缓解炎症反应及其所致的胶原沉积和炎性增生。.（2）孕酮可抑制LPS诱导的奶牛子宫内膜细胞MAPK和NF-κB通路的活化，以及炎性因子的基因表达；中低浓度的孕酮（1、3ng/mL）可促进LPS作用下，细胞向G2期转化，调节细胞相关生长因子的表达以及Wnt/β-catenin和PI3K/Akt通路的活性。孕酮单独作用于奶牛子宫内膜细胞，对细胞MAPK和NF-κB通路和下游炎性因子无影响；低浓度孕酮（1ng/mL）单独作用可促进细胞向G2期转化。提示孕酮可能有助于缓解炎症反应，且低水平孕酮有助于促进子宫内膜炎性增生。.（3）皮质醇能够缓解大肠杆菌诱导的子宫内膜炎山羊体温、心率和呼吸速率的升高，降低外周血白细胞数量以及子宫分泌物中嗜中性粒细胞百分比，抑制子宫内膜的炎症反应，减少子宫内膜上皮细胞的损伤脱落和固有层炎性细胞的浸润。皮质醇抑制子宫内膜组织MyD88蛋白表达以及p65、IκBα、ERK1/2、JNK和p38 MAPK的磷酸化水平，抑制促炎因子的基因表达，提示皮质醇对大肠杆菌性子宫内膜炎的子宫先天免疫反应具有抑制作用，其效应可能是通过抑制MAPK和NF-κB信号通路实现。.（4）孕酮可降低大肠杆菌性子宫内膜炎山羊外周血WBC和子宫分泌物PMN%的水平，抑制生理指标的异常变化，缓解子宫内膜炎症反应，减少上皮细胞脱落和炎性细胞浸润；孕酮可抑制大肠杆菌感染引起的子宫内膜组织促炎因子的基因表达，抑制JNK、ERK、p38、p65蛋白的磷酸化水平，提示孕酮可抑制大肠杆菌所致子宫内膜炎症反应的先天免疫机制，该作用可能是通过抑制MAPK和NF-κB信号通路实现。