基于细胞焦亡及外泌体介导的子宫内膜干细胞活性探讨补肾活血中药改善子宫内膜容受性的机制研究

Low endometrial receptivity is one of the key factors of embryo implantation disorder, which endangers human reproductive health. Previous studies have confirmed that kidney-tonifying and blood-activating herbal compounds can improve endometrial receptivity, and can act on endometrium through multiple pathways. Based on this, we hypothesize that the pyroptosis of endometrial cells and the cell differentiation of Endometrium derived mesenchymal stem cells (EnMSCs) are involved in regulating endometrial receptivity, which may be the pathway and target of kidney-notifying and blood-activating formula improving endometrial receptivity. Indomethacin was used to establish blastocyst implantation disorder model of mouse. The number of blastocyst implantation was observed after intervention of kidney-tonifying and pregnancy promoting formula. The changes of endometrial pinocytes were observed by electron microscopy. The effects of indomethacin on the expression of caspase-1, IL-1beta, IL-18 and GSDMD in endometrial cells were studied by immunohistochemistry and Real-time PCR. Flow cytometry and transmission electron microscopy were used to observe the morphology of EnMSCs-derived exosomes and the effect of the formula on microRNA21. From the perspective of cell exosomes and cell differentiation, the regulation mechanism of endometrial receptivity was explained and the intervention mechanism and target of kidney-tonifying and blood-activating formula were revealed. This study will provide basis for clinical treatment of infertility and enriched the traditional Chinese medicine theory of ‘system of Sanjiao and Moyuan’ from the molecular biological level.

子宫内膜容受性低下是导致胚胎着床障碍的关键因素，危害人类生殖健康。前期研究证实，补肾活血中药能很好地改善子宫内膜容受性，并且能通过多通路作用于子宫内膜。基于此，我们提出假说：子宫内膜细胞的焦亡及内膜间充质干细胞（EnMSCs）的分化参与调控子宫内膜容受性，并且可能是中药改善内膜容受性的作用途径和靶点。拟采用吲哚美辛制备胚泡着床障碍小鼠模型，运用补肾促孕方干预，观察小鼠胚泡着床数目；应用电镜观察子宫内膜胞饮突的变化；采用免疫组化、Real-time PCR等方法研究该方对内膜细胞焦亡相关因子caspase-1、IL-1β、IL-18及GSDMD表达的影响；对小鼠EnMSCs进行诱导分化，利用流式细胞仪及透射电镜等技术观察该方对EnMSCs源性外泌体形态及其miR-21的干预作用。从细胞焦亡及干细胞分化的角度阐释中药干预子宫内膜容受性的机制，为不孕症的治疗提供依据，同时丰富“三焦膜系”理论。

不孕症发病率逐年升高，寻求辅助生殖技术帮助的女性日益增多，但其妊娠率仍较低，子宫内膜容受性低下是其主要影响因素。前期研究证实，补肾活血中药能很好地改善子宫内膜容受性，并且能通过多通路作用于子宫内膜。本项目在前期研究基础之上，基于三焦膜系理论，进一步研究补肾活血中药方改善内膜容受性的效应及机制：理论研究表明，肾膜萎弱、胞膜失疏导致的肾-天癸-冲任-胞宫生殖轴功能失调是子宫内膜容受性低下的病机；温补肾膜、疏利胞膜是治疗子宫内膜容受性低下的治则。动物实验方面，本项目成功制备了小鼠胚胎着床障碍模型；观察了补肾活血中药方对胚胎着床障碍小鼠子宫内膜厚度及腺体数目，子宫内膜胞饮突表达，子宫内膜容受性相关标志物LIF、ανβ3、ER、PR表达，血清E2、PG水平以及胚胎着床数目的影响，评价了补肾活血中药方改善子宫内膜容受性的药效；分析了补肾活血中药方对胚胎着床障碍小鼠子宫内膜细胞DNA损伤，BDNF/TrkB通路以及细胞焦亡相关蛋白表达的影响，发现其作用机制可能与激活BDNF/TrkB通路以及减少细胞焦亡有关。细胞实验方面，分析了补肾活血中药方对子宫内膜间充质干细胞（MSCs）活性的影响，基于细胞焦亡研究了本方促进MSCs活性的机制，基于MSCs及其外泌体活性研究本方对小鼠子宫内膜上皮细胞活性的影响，发现本方能够通过MSCs及其外泌体增加子宫内膜上皮细胞的活性，其作用机制可能是增加MSCs来源的外泌体miR-21活性以对抗MSCs焦亡。本项目从细胞焦亡及干细胞活性的角度揭示中药改善子宫内膜容受性的效应机制，为临床反复着床失败的中医辨治提供实验依据，同时从生殖的角度初步阐释了中医三焦膜系理论的科学内涵。