疏肝泻火方治疗CPP的机制探索：对垂体GnRH受体信号转导及反馈系统的影响

Central precocious puberty (CPP) is a kind of pediatric endocrinopathy characterized by prematurely secretion of GnRH and premature development of hypothalamus-pituitary-gonad axis（HPGA）. Based on the theory of Yi and Kui are derived from the same origin, Prof.Liu Yi-Min, a veteran TCM physician, created Shuganxiehuo recipe in TCM. Previous studies have confirmed the efficacy of Shuganxiehuo recipe in the treatment of CPP by improving the function of ovary and uterus. it also regulates the secretion of gonadotropic hormone (GTH) in the pituitary gland.The GnRH receptor signal system and the estrogen feedback mechanism are closely related to the expression of GTH. Therefore, we are proposing the hypothesis that Shuganxiehuo recipe may regulate the whole function of HPG axis through regulating the three major signal transduction pathways and/or E2 feedback system of pituitary GnRHR, and ultimately achieve the effect of CPP treatment.In this project, we will establish rat CPP model with NMA and the anti-GnRHR pituitary cell model with Cetrorelix. From the point of view of PKC-MAPK, Ca2+-CAM and cAMP-PKA signaling pathway after GnRH receptor, From the whole animal experiment to the cell test in vitro, we will study the regulation mechanism of Shuganxiehuo recipe on the function of pituitary through a variety of pathology and molecular biology techniques. we will reveal the pituitary GTH secretion and related feedback regulation effect, further clarify the regulatory mechanism of the side of the HPG axis, and provide a basis for clinical medicine.

中枢性性早熟(CPP)是GnRH过早分泌，导致HPG轴提前启动的儿科疾病。全国名中医刘以敏教授基于“乙癸同源”理论，从肝论治，创制疏肝泻火方。前期研究表明，该方可改善卵巢、子宫功能，影响垂体促性腺激素（GTH）分泌水平。因垂体GnRH受体信号系统以及雌激素反馈机制与GTH表达密切相关，据此提出：疏肝泻火方可能通过影响垂体GnRH受体三大信号转导途径与/或E2反馈系统，进而调节HPG轴整体功能，实现矫治CPP的功效。为证实上述假说，本项目采用NMA构建CPP雌性大鼠模型和西曲瑞克构建垂体GnRH受体拮抗细胞模型，围绕GnRH受体后PKC-MAPK、Ca2+-CAM、cAMP-PKA信号通路以及雌激素反馈机制，运用病理学及分子生物学方法，从体内到体外细胞验证，探讨疏肝泻火方对垂体的调节作用，揭示其对垂体GTH分泌及相关反馈调节的影响，进一步阐明该方对HPG轴的调控机制，为临床用药提供依据。

本项目立足于垂体GnRH受体信号转导系统，围绕GnRH受体后信号通路（PKC-MAPK）以及雌激素反馈机制，运用病理学及分子生物学方法，从体内到体外细胞实验，探讨疏肝泻火方对垂体的调节作用，揭示其对垂体促性腺激素分泌及雌激素反馈调节的影响，进一步阐明该方对HPG轴的调控机制。.1. 用NMA构建雌性CPP大鼠模型，观察疏肝泻火方对模型大鼠HPG轴的影响，对GTH分泌水平的影响及雌激素反馈调节的作用。结果表明，疏肝泻火方可以抑制模型大鼠性器官子宫、卵巢的发育；降低模型大鼠血清FSH、LH、PRL、E2的表达水平、下丘脑中GnRH蛋白表达水平、垂体中GnRH、GnRHR、ERα和GPR30 mRNA和蛋白表达水平以及卵巢中GnRHR蛋白表达水平。提示疏肝泻火方可能通过抑制下丘脑中的GnRH、垂体中的GnRHR表达，调节雌激素受体ERα和GPR30的表达，从而抑制雌激素和FSH、LH的分泌，达到抑制性器官的发育和HPG轴的激活。.2. 用瘦素干预体外大鼠垂体细胞，观察疏肝泻火方对PKC-MAPK信号通路的作用及雌激素受体的影响。结果表明，HY-N2180可部分逆转疏肝泻火方对垂体细胞PKC-MAPK通路的抑制作用，疏肝泻火方可抑制PKC-MAPK通路中关键蛋白PKC、p38MAPK、EPK和JNK的表达水平，缓解瘦素干预的垂体细胞分泌FSH、LH和抑制垂体细胞中GnRHR、ERα和GPR30蛋白的表达。提示疏肝泻火方可能通过抑制垂体细胞中的PKC-MAPK信号通路，降低雌激素受体和促性腺激素的表达，进而缓解CPP。.本项目结果进一步阐明疏肝泻火方对HPG轴的调控机制，为疏肝泻火方治疗CPP提供理论依据。