补肾助孕方调控下丘脑KISS-1/GPR54系统治疗LPD性不孕症的机制研究

The Prescription for Reinforcing Kidney and Promoting Pregnancy (Bu Shen Zhu Yun Fang, BSZYF) is a rich experienced formula under the theory of “Heart(Brain)-Kidney-Uterus axis” proposed by Professor Xia Guicheng, the “National TCM Master”. Previous clinical and experimental studies have confirmed its efficacy in the treatment of luteal phase defect (LPD) by improving the function of pituitary, ovary and uterus. Based on the understanding that the LPD is due to the dysfunction of reproductive axis, and KISS-1/GPR54 neurons system in hypothalamus is the major regulator of GnRH secretion, we are proposing the hypothesis that the BSZYF could improve the pulse mode of GnRH secretion by targeting KISS-1/GPR54 neurons system to adjust the function of the reproductive axis..In this project, we will establish rat LPD model with mifepristone and the anti-estrogen hypothalamus cell model with Tamoxifen, PHTPP and/or G-15 on GT1-7 cell line. It is the first time in China that GnRH will be detected by Fast-scan Cyclic Voltammetry. This project will innovatively explore the multi-level mechanisms of BSZYF in regulating the expression of KISS-1/GPR54 neurons system and support the methods of kidney-tonifying in treating LPD theoretically.

补肾助孕方是国医大师夏桂成教授依据 “心（脑）-肾-子宫轴”理论形成的经验方，临床及实验研究均表明可通过改善生殖轴中垂体、卵巢、子宫的功能，有效治疗黄体功能不全（LPD）性不孕症。中西医均认为生殖轴功能失调是引起LPD性不孕的重要因素，又因下丘脑KISS-1/GPR54系统是调控GnRH分泌必经环节，前期研究已表明，补肾助孕方可改善血清GnRH水平，据此我们提出科学假说：补肾助孕方调节生殖轴功能可能是通过调控KISS-1/GPR54神经元系统，干预GnRH脉冲模式，或者影响雌激素水平，反馈作用于该神经元系统，进而调节生殖轴功能。本项目以LPD大鼠整体动物模型与雌激素竞争性拮抗神经元细胞模型进行实验，采用快速扫描循环伏安技术实时监测GnRH脉冲式分泌，多层次研究补肾助孕方对下丘脑KISS-1/GPR54神经元系统以及GnRH分泌的调控作用，为补肾法治疗LPD提供理论依据。

补肾助孕方是国医大师夏桂成教授依据“心（脑）-肾-子宫轴”理论形成的经验方，临床及实验研究均表明其可通过改善生殖轴中垂体、卵巢、子宫的功能，有效治疗黄体功能不全（LPD）性不孕症。基于生殖轴功能失调是引起LPD性不孕的重要因素，且下丘脑KISS-1/GPR54系统是调控GnRH分泌必经环节，本项目提出科学假说：补肾助孕方可通过调控 KISS-1/GPR54神经元系统，干预GnRH脉冲模式，或者影响雌激素水平，反馈作用于 KISS-1/GPR54神经元系统，进而调节生殖轴功能。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、雌激素受体拮抗剂构建雌激素竞争性拮抗神经元细胞模型，采用快速扫描循环伏安法监测 GnRH脉冲，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KISS/GPR54神经元系统，调控GnRH脉冲分泌，进而调节生殖轴功能，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）不仅能显著改善下丘脑KISS/GPR54神经元系统，还对GPR54受体后cAMP-MAPK、Ca2+-PKC信号转导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，进一步阐述了中医学“心（脑）”对月经周期节律、孕育具有调控作用，也为中医“心（脑）-肾-子宫轴”理论与现代医学生殖内分泌HPO轴功能在内涵的一致性上提供了科学依据。本研究着眼于补肾中药对下丘脑的调节作用，立足于KISS-1/GPR54神经元系统，通过在体动物实验及离体细胞实验初步阐明了补肾助孕方影响GnRH脉冲分泌、调控HPO轴、治疗LPD性不孕症的作用机制，为中药治疗LPD不孕症的研究提供新思路、新方法、新技术。