Akt/AMPK/mTOR信号通路调控子宫内膜自噬水平和巨噬细胞极化在二硫化碳致胚胎植入障碍中的作用机制

Carbon disulfide (CS2) has obvious reproductive toxicity, but the mechanism remains unclear. In the previous study, we established an animal model of the embryo implantation defects caused by exposure to CS2 during embryo implantation, and found that the expression of adhesion molecules mRNA and protein in uterine tissue was reduced and DNA damage was obvious. Based on our previous results and the recent discovery that the Akt/AMPK/mTOR signaling pathways regulate the processes of autophagy and immune tolerance, we intend to observe the mRNA and protein expression of proteins related to signaling pathways, the levels of endometrial autophagy, and the typing and functional changes of macrophage in uterine tissue of mouse exposed to CS2 at different time and sacrificed at different endpoints during embryo implantation. And meanwhile we plan to analyze the relationship between these three factors and the embryo implantation defects, screen and locate the key regulatory proteins in the signaling pathways that disrupt the process of embryo implantation after CS2 exposure. Moreover, amino acids will be added in the daily diet and the variation in expression of the proteins in signaling pathways will be observed in order to further confirm that the signaling pathways play an important role in the mechanism of the embryo implantation defects caused by CS2 and to evaluate the intervention effect of amino acids in mouse exposed to CS2. Therefor, the mechanism of embryo implantation defects will be revealed in different levels, which may provide the scientific basis and research methods for protecting the occupational employees exposed to environmental hazardous Substances from reproductive toxicity.

二硫化碳（CS2）具有明显的生殖毒性，但其毒作用机制尚不明确。前期研究工作建立了CS2致小鼠胚胎植入障碍动物模型，发现植入期小鼠子宫组织粘附分子的蛋白、mRNA表达水平下降及其基因损伤。本研究拟以课题组前期研究发现为切入点，基于近期研究发现的Akt/AMPK/mTOR信号通路对生殖功能的调控作用，观察不同CS2染毒时间及不同胚胎植入时间断面上该信号通路分子蛋白及其mRNA表达水平、子宫内膜细胞自噬水平和子宫内膜巨噬细胞分型及功能变化，分析三者变化规律与胚胎植入障碍关系，筛选信号通路中CS2干扰胚胎植入的关键调控分子；增补氨基酸后观察该通路关键调控因子的变化规律，进一步验证该信号通路在CS2致胚胎植入障碍的毒作用机制，并评价其对CS2致胚胎植入障碍的干预效果。从蛋白质和基因、细胞和亚细胞以及整体水平不同层面进一步揭示胚胎植入障碍机制，为有效预防和控制接触CS2作业女工胚胎损伤提供理论依据。

背景 课题组前期研究发现二硫化碳（carbon disulfide, CS2）可导致胚胎丢失，但其机制尚不明确。AKT/AMPK/mTOR信号通路、细胞自噬和巨噬细胞极化均与胚胎植入过程相关。主要研究内容 检测围植入期不同时间断面孕鼠暴露于CS2后子宫内膜组织AKT/AMPK/mTOR通路分子表达水平、自噬水平、M1/M2巨噬细胞极化方向、炎性因子和免疫耐受细胞因子及生长因子表达水平，体外培养小鼠腹腔巨噬细胞探讨CS2对巨噬细胞极化影响，膳食增补N-氨甲酰谷氨酸（N-carbamyl glutamate, NCG）以验证mTOR信号通路在CS2致胚胎植入障碍中的作用。重要结果 ①不同时间点染毒CS2后的第一个观察终点孕鼠子宫组织pAKT蛋白表达量明显降低和pAMPK蛋白表达量明显升高，植入窗口关键期mTOR和p-mTOR蛋白及其mRNA表达水平明显降低。②植入窗口期，CS2可上调P62并下调LC3B蛋白和mRNA表达，子宫自噬小体数量明显减少。③CS2染毒后孕鼠子宫内膜中免疫炎性因子IL-6和IL-12的蛋白和mRNA表达水平明显升高，免疫耐受因子TGF-β和IL-10 mRNA表达水平明显下降，生长因子VEGF蛋白和mRNA表达水平明显降低。④GD3染毒后第一个观察终点（GD4）子宫内膜中M1型巨噬细胞比例明显升高，胚胎植入窗口（GD5）M2型巨噬细胞明显增加，植入晚期（GD6、GD7）M1型巨噬细胞再次明显升高。⑤CS2致体外培养巨噬细胞NOS2 蛋白质和mRNA以及IL-6 mRNA表达水平明显下降，ARG1蛋白和mRNA以及CHI3L3 mRNA表达水平明显上升。⑥膳食增补NCG后明显增加孕鼠子宫组织mTOR信号通路关键蛋白表达水平和胚胎植入数目，逆转自噬相关蛋白LC3B和P62变化。⑦CS2降低卵巢黄体功能，改变卵巢组织COX-2和Hif1-αmRNA表达水平，并降低子宫蜕膜化标志性因子IL-11的蛋白和mRNA表达水平。结论 本项目揭示CS2可抑制AKT/AMPK信号通路和自噬，扰乱巨噬细胞极化平衡，导致植入关键窗口期mTOR、免疫因子、生长因子和蜕膜化相关因子表达水平改变，最终引起胚胎植入障碍。意义 本课题研究进一步揭示CS2致胚胎损伤机制，为预防和控制CS2作业女工胚胎损伤提供科学依据。