补肾助孕方治疗LPD性不孕症的新机制：垂体GnRH受体后信号通路

The Prescription for Reinforcing Kidney and Promoting Pregnancy is an epirical formula formed under the theoretical guidance of the 'Heart(Brain)-Kidney-Uterus axis' proposed by Xia Guicheng, the renowned specialist of TCM. Previous clinical researches have confirmed its efficacy in the treatment of luteal phase defect (LPD) infertility, which can increase patients' progesterone (P) and luteal hormone (LH) levels during the embryo implantation stage while experimental results suggest that the prescription can improve the corpus luteum's function and endometrial receptivity. Based on the consistency of both Chinese and western medicine's understandings that the LPD infertility is due to the dysfunction of the reproductive axis, we consider that the effective ingredients of the formula can act on the pituitary directly. Therefore, we are proposing a hypothesis about a new mechanism that the prescription can regulate the expression of GTH through the signaling pathway via GnRH receptor to adjust the function of the reproductive axis. In this project, we will study the rat LPD model with Mifepristone and the pituitary cell GnRH receptor antagonistic model with Cetrorelix，and adopt cellular and molecular biology techniques such as qPCR、Western Blot、ELISA and Immunofluorescence laser scanning confocal microscope（IF/LSCM）. For the first time, on the basis of the PKC-MAPK、Ca2+-CAM and cAMP-PKA signaling pathway via GnRH receptor, this project will innovatively explore the new pregnancy-promoting mechanism of the Prescription for Reinforcing Kidney and Promoting Pregnancy in regulating the expression and secretion of GTH and the kidney-tonifying methods in curing LPD infertility. Thus, we will discover new thoughts and methods for the research of treating LPD infertility with Chinese medicine.

补肾助孕方是基于名老中医夏桂成教授提出的"心(脑)-肾-子宫轴"理论指导下形成的经验方剂。临床研究已证实其为治疗黄体功能不全（LPD）性不孕症的有效方药，并能提高患者胚胎着床期P、LH等水平；实验结果提示其改善卵巢黄体功能及子宫内膜容受性。基于中西医对LPD性不孕为生殖轴功能失调认识上的一致性，我们认为补肾助孕方能够作用于垂体，提出"通过GnRH受体后信号通路调控垂体GTH表达进而调节生殖轴功能"的作用新机制假说。本项目以米非司酮构建LPD大鼠模型、西曲瑞克构建GnRH受体拮抗细胞模型，采用qPCR、Western Blot、ELISA、免疫荧光共聚焦扫描等细胞分子生物学技术，首次从GnRH受体后PKC-MAPK、Ca2+-CAM、cAMP-PKA信号通路，开创性研究补肾助孕方对垂体GTH表达分泌的调控及补肾法治疗LPD的助孕新机制，为中药治疗LPD不孕症的研究提供新思路、新方法。

补肾助孕方是基于名老中医夏桂成教授提出的"心-肾-子宫轴"理论指导下形成的经验方剂。临床研究已证实其为治疗黄体功能不全（LPD）性不孕症的有效方药，并能提高患者胚胎着床期P、LH等水平；实验结果提示其改善卵巢黄体功能及子宫内膜容受性。基于中西医对LPD性不孕为生殖轴功能失调认识上的一致性，本项目提出提出"补肾助孕方可以作用于垂体，通过GnRH受体后信号通路调控垂体GTH表达进而调节生殖轴功能"的科学假说。课题组设计了相应的实验方案，通过米非司酮构建LPD大鼠模型、西曲瑞克构建GnRH受体拮抗细胞模型，采用qPCR、Western Blot、ELISA、免疫荧光共聚焦扫描等细胞分子生物学技术，对该假说进行探索和验证。本项目在执行年限内完成全部研究工作。研究结果表明：1、补肾助孕方能够通过调节生殖轴上游垂体的功能，调控FSH，LH表达水平，进而调节生殖轴下游卵巢、子宫功能，达到治疗LPD性不孕症的目的；2、通过体内、外实验发现，补肾助孕方（水煎液或含药脑脊液）既具有显著改善垂体GnRHR表达的作用，还对GnRH受体后PKC-MAPK、Ca2+-CAM、cAMP-PKA三条主要信号通路中关键节点分子具有调节作用，进而影响其信号转导功能；3、综合本项目体内、外试验结论与前期相关研究，补肾助孕方能够调整HPO轴整体功能，证明中医“心（脑）-肾-子宫轴”理论与现代医学HPO轴的内容基本一致，并且说明补肾助孕方可以通过以上机制有效治疗黄体功能不全性不孕症。本研究首次从GnRH受体后PKC-MAPK、Ca2+-CAM、cAMP-PKA信号通路，对补肾助孕方调控垂体GTH表达分泌及补肾法治疗LPD进行了具有一定开创性的研究，探索了中医药助孕的新机制，为中药治疗LPD不孕症的研究提供新思路、新方法。