甲硫脑啡肽在孕酮抑制奶牛子宫内膜细胞增殖中的作用机制研究

High-level progesterone could induce or exacerbate postpartum uterine bacterial infection in cows, which is related to the inhibitory effect of progesterone on the endometrial regeneration. Met-enkephalin (MENK), an endogenous opioid peptide, is induced by progesterone, and can inhibit cell proliferation. We hypothesize that MENK may participate in the progesterone-induced uterine infection by inhibiting endometrial cell proliferation. Most postpartum uterine bacterial infections in cows are caused by Escherichia coli (E.coli). By using the animal model of postpartum uterine infection with E.coli, the effect of progesterone on uterine MENK level will be measured. Through observation of histological changes and detection of cell proliferation factors and pathways, the effect of opioid antagonists on progesterone-induced inhibition of regeneration will be investigated. These in vivo studies will clarify the role of MENK in endometrial regeneration regulated by progesterone. In vitro, the cell proliferation factors and pathways of bovine endometrial cells (including epithelial cells and stromal cells) treated with MENK will be measured. The specific opioid receptor mediating the inhibition of cell proliferation by MENK will be identified by applying opioid antagonists. Finally through silencing or overexpressing the gene of the specific opioid receptor, the potential mechanism of the inhibitory effect of MENK on cell proliferation will be revealed. This study may accelerate our understanding of the mechanism of postpartum uterine bacterial infection in cows under the influence of high-level progesterone, providing new ideas to the prevention and treatment of postpartum uterine bacterial infection.

高水平孕酮可诱发或加重奶牛产后子宫感染，这与孕酮抑制子宫内膜再生修复有关。甲硫脑啡肽（MENK）是一种内源性阿片肽，可抑制细胞增殖，其分泌受孕酮诱导。我们推测，MENK通过抑制子宫内膜细胞增殖参与高水平孕酮加重子宫感染。大肠杆菌是产后子宫感染的常见菌。通过建立产后子宫大肠杆菌感染模型，研究高水平孕酮对子宫MENK表达的影响，以及阿片受体拮抗剂对子宫内膜组织修复和增殖相关因子和通路的影响，明确MENK在孕酮抑制产后子宫内膜修复中的作用；通过体外培养奶牛子宫内膜细胞（上皮细胞和基质细胞），检测MENK对细胞增殖相关因子和通路的影响；利用阿片受体拮抗剂确定介导MENK抑制细胞增殖的阿片受体类型；最后通过沉默或过表达阿片受体基因，明晰MENK抑制细胞增殖的潜在机制。该研究有助于加深对高水平孕酮诱发或加重奶牛产后子宫感染机制的了解，为防治奶牛产后子宫感染提供新思路。

（1）大肠杆菌脂多糖（LPS）将奶牛子宫内膜基质细胞（BESC）细胞周期阻滞在G0/G1期，抑制细胞迁移率和增殖相关基因表达，激活Wnt/β-catenin通路、抑制PI3K/AKT通路。加入甲硫脑啡肽（MENK）后，细胞周期阻滞缓解，增殖相关基因上调，Wnt/β-catenin和PI3K/AKT信号通路关键蛋白表达水平下降，提示MENK促进炎性反应时BESC细胞增殖，其机制可能是通过调控细胞周期、增殖相关因子以及Wnt/β-catenin和PI3K/AKT通路发挥作用。.（2）非选择性阿片受体拮抗剂纳洛酮（NAL）和δ阿片受体拮抗剂ICI 154129能够不同程度的逆转炎性反应时MENK对BESC细胞的促增殖作用，对CTGF和TGFβ1基因的上调作用以及对Wnt/β-catenin和PI3K/AKT通路的抑制作用；μ受体拮抗剂CTAP能够逆转炎性反应时MENK对CTGF基因的上调作用和对PI3K/AKT通路抑制作用，提示μ阿片受体和δ阿片受体均参与了炎性反应条件下MENK对BESC细胞增殖的调控。.（3）大肠杆菌LPS可促进奶牛子宫内膜上皮细胞（BEEC）细胞周期进程和增殖相关基因表达，激活Wnt/β-catenin和PI3K/AKT通路。MENK能够抑制LPS诱导的细胞周期进程，增殖相关基因表达，以及Wnt/β-catenin和PI3K/AKT通路活化，提示MENK能够抑制炎性反应条件下的BEEC细胞增殖，潜在调控机制包括增殖相关因子以及Wnt/β-catenin和PI3K/AKT通路。.（4）阿片受体拮抗剂NAL和ICI154129均能逆转炎性反应条件下MENK对BEEC细胞增殖的抑制作用；μ受体拮抗剂CTAP对MENK的抑制增殖效应无显著影响。这些结果提示δ阿片受体参与炎性反应条件下MENK对BEEC细胞增殖的调控。.（5）产后山羊子宫灌注大肠杆菌可造成子宫的急性感染，孕酮能够降低大肠杆菌感染所致的山羊理化指标升高和子宫内膜结构损伤，对子宫内膜组织中的增殖相关基因EGFR、VEGF和TGFβ的表达影响不显著；山羊产后急性子宫感染可诱导子宫内膜组织中前脑啡肽原（PENK）基因表达，子宫液中MENK水平无显著变化；孕酮可诱导子宫内膜组织PENK基因表达，对子宫液MENK水平无显著影响。