滋阴泻火方干预RFRP-3参与光周期改变致性早熟动物模型的神经生物学机制

Idiopathic precocious puberty is the most common type of true precocious puberty. Although the cause is not yet clear, the changing environment factors may play a significant role in the high incidence of precocious puberty. The increase of artificial lighting time is one of the essential factors induced precocious puberty. Previous studies had shown that various causes would increase the level of melatonin as well as its related GnIH releasing, leading to central precocious development in hypothalamus and pituitary. Nourishing "Yin"-removing "Fire" Chinese herbal mixture is commonly used in the treatment of Precocious Puberty in our hospital, reducing the symptoms and signs for syndrome of hyperactivity of fire due to yin deficiency as well as delay sexual characteristics and epiphyseal mature.In our vpreliminary experiments, the animal model of precocious puberty was established by photoperiod extension in order to explore further the mechanism of onset of gonadal axis regulated by nourishing "Yin"-removing "Fire" Chinese herbal mixture. In this study, we are attempted to investigate the effect of Chinese herbal mixture on central neuronal excitability as well as the synthesis and secretion of gonadotropin at the hypothalamus-pituitary level by neurobiological and molecular biological methods.Push-pull perfusion and confocal laser are used to detect the number and distribution of RFRP-3 neurons. Changes of RFRP-3 and GnRH related genes and proteins are measured by immunohistochemistry and western blotting as well as peripheral levels of melatonin and sex hormones. The effects of the serum contained herbs are to investigate in vitro culture GnIH neuron cells for further study. The purpose of the project is to elucidate the mechanism of Chinese herbs on melatonin related synthesis and secretion of GnIH pathway to regulate the arousing of HPG axis.

特发性性早熟是儿童性早熟最常见类型, 环境地理因素改变与其发生明显相关，而延长光照时间可能是诱发的重要因素。既往研究显示：各种原因导致的动物褪黑素水平升高，介导的促性腺激素释放激素抑制素（GnIH）生成增多，在下丘脑及垂体水平抑制中枢性性发育的启动。滋阴泻火方临床可控制性早熟儿童"阴虚火旺"证候，延缓提前的性征发育及骨骺成熟。在预实验初步建立了模拟光照时间延长诱导的性早熟动物模型，本课题拟采用推挽灌流、激光共聚焦神经生物学、分子生物学方法观测药物干预后性早熟大鼠RFRP-3神经元数量和分布的变化以及RFRP-3和GnRH相关基因和蛋白水平的改变，采用含中药血清干预体外培养GnIH神经元细胞，反证滋阴泻火中药的作用，从下丘脑-垂体层面探讨滋阴泻火为主中药对中枢相关神经元兴奋启动及促性腺激素合成和分泌的影响，深入研究中药对褪黑素合成介导的GnIH重要组成-RFRP-3的合成与分泌的影响机制。

青春期发育的原始动力来自体内下丘脑－垂体－性腺轴的激活，但是引起性发育启动的具体机制，目前尚不清楚。青春发育启动可能受多种因素影响,包括遗传因素、营养与肥胖、环境因素（如气候、地理位置、光照）等，这些因素相互整合构成触发信号，激活HPG轴，诱导青春期发育。既往研究认为可能参与调控的因子众多，如Kiss-1、NPY、瘦素、P物质、神经激肽B、RFRP-3等。其中, RFRP-3是迄今为止发现的唯一一个对HPG轴起负调控作用的下丘脑神经肽。但是关于RFRP-3在幼年动物上的研究较少，其是否参与调控性发育的启动值得我们进行研究。. 以SD大鼠作为研究对象，发现 16小时强光组大鼠阴门开启时间和建立两个动情周期的时间提前，该组大鼠性腺发育、血清性激素水平、下丘脑GnRH的表达水平提前达到正常大鼠青春期发育阶段的水平。以16小时、350lux的光照条件建立性发育提前大鼠模型，用滋阴泻火中药干预。发现滋阴泻火中药调节大鼠青春期性发育的作用表现在使下丘脑Kiss-1基因表达下降，RFRP-3基因表达上升，从而降低外周血E2的含量，但对及褪黑素、褪黑素受体作用不明显。. 以SD雌性大鼠为研究对象，采用侧脑室分别注射RF-9下调或RFRP-3上调下丘脑RFRP-3/GPR147信号通路的方法，观察大鼠阴门开启时间，检测大鼠下丘脑性发育相关基因、子宫及卵巢、性激素水平的变化。发现侧脑室注射RFRP-3上调RFRP-3/GPR147信号通路后，大鼠阴门开启时间显著延迟，血清LH、E2水平显著降低，子宫发育成熟度较低，性发育启动明显延迟。这说明RFRP-3/GPR147信号通路可能在延迟性发育启动过程中起着重要的作用。同时，RFRP-3组大鼠下丘脑GnRH及Kiss-1mRNA表达量显著下降。这提示，RFRP-3可能通过抑制Kiss-1-GnRH的表达延迟性发育启动。而注射RF-9下调RFRP-3/GPR147信号通路后，性发育启动时间未提前。下丘脑背内侧核注射慢病毒干扰RFRP-3基因表达，观察雌性大鼠阴门开启时间、性腺发育、血清LH水平的变化。发现大鼠血清LH水平上升，但是阴门开启时间、子宫卵巢的发育水平无显著变化，性发育启动未提前。.