基于RFRP-3-kisspeptin调控GnRH/LH脉冲模式探讨加味当归补血汤防治生殖衰老的作用机制

Follicular exhaustion is the cause of premature ovarian insufficiency (POI), which with little effective treatment so far, affects women’s reproductive health seriously. Recent studies have revealed that neuroendocrine changes gradually accelerate follicular depletion. Decreased hypothalamus GnRH pulse frequency leads to the delayed and deficiency of LH pulse, which is the main endocrine characteristic of female early reproductive aging. Our group has been dedicated to the research on the prevention and treatment of female reproductive aging with traditional Chinese medicine for many years. Previous studies showed that the dynamic expression of kisspeptin/ GPR54 neurons in the hypothalamus of mice at different ages affected the release of LH pulses, in addition, we also used the rat POI model, detected the dynamic expression of RFRP-3 and Kisspeptin were to be associated with ovarian decline for the first time. The "Jia Wei Dang Gui Bu Xue decoction" created by our group has shown the effect of delaying reproductive aging in previous clinical and basic studies. In this project, castrated rat models of different ages and intracerebroventricular injection (ICV) of RFRP-3 were used to observe the time-phase expression of RFRP-3-Kisspeptin/GPR54 in reproductive aging and the effect on GnRH/LH pulse frequency after TCM intervention. The implementation of this project will elucidate the central nervous mechanism of Jia Wei Dang Gui Bu Xue decoction on delay reproductive aging, which has great theoretical and application value.

卵巢早衰严重影响女性生殖健康，其病身是卵泡耗竭，尚缺乏有效治疗手段。最近研究揭示神经内分泌改变会渐进性的加速卵泡耗竭，下丘脑GnRH脉冲频率减弱导致LH脉冲波的延迟和缺失是女性早期生殖衰老的主要内分泌特征。本课题组多年来致力于中医药防治女性生殖衰老的研究，前期研究显示不同年龄阶段小鼠下丘脑内kisspeptin/GPR54等神经元的动态表达影响LH脉冲释放，此外，我们首次利用大鼠早衰模型，检测到RFRP-3、Kisspeptin动态表达与卵巢衰退有关。课题组创制的“加味当归补血汤”，在既往临床与基础研究中显示出延缓生殖衰老的作用。本项目采用不同年龄去势大鼠模型，通过侧脑室微量注射RFRP-3，观察中药干预后RFRP-3、Kisspeptin/GPR54在生殖衰老中的时相表达及对GnRH/LH脉冲频率的影响，阐明加味当归补血汤防治生殖衰老的中枢神经机制，有重大理论和应用价值。

卵巢早衰严重影响女性生殖健康，其病身是卵泡耗竭，尚缺乏有效治疗手段。最近研究揭示神经内分泌改变会渐进性的加速卵泡耗竭，下丘脑GnRH脉冲频率减弱导致LH脉冲波的延迟和缺失是女性早期生殖衰老的主要内分泌特征。LH释放机制的年龄相关变化反映了促性腺激素释放激素神经元兴奋性和抑制性传入输入对雌二醇正反馈条件的反应性降低。项目首先利用6月龄及18月龄大鼠，构建去势模型，通过注射雌激素诱导LH峰，结果显示：老年去势大鼠对雌激素反馈及LH的产生及释放能力均降低，雌性大鼠的生殖衰老表现在下丘脑RFRP-3、Kispeptin等神经内分泌因子及垂体分泌功能减退。RFRP-3是已经发现的唯一抑制HPO轴的神经肽，现已明确RFRP-3在下丘脑kisspeptin/GPR54/GnRH系统水平上发挥作用，但对于其在 HPO轴调控中的作用机制尚不完全清楚。接着项目通过对青老年大鼠侧脑室注射RFRP-3，检测GnRH/LH的释放情况，RFRP-3降低了LH脉冲的频率，RFRP-3可通过中枢水平抑制下丘脑Kispeptin或者GnRH的表达，还能够直接作用于脑垂体，抑制促黄体生成素LH的分泌，进而影响生殖。加味当归补血汤并不影响LH脉冲释放，且一定程度上增加了LH总体释放浓度（尤其是青年大鼠），LH的释放增加与加味当归补血汤增加GnRH脉冲释放量有关。此外，加味当归补血汤能一定程度减少RFRP-3对GnRH的释放抑制，增加去势大鼠的GnRH脉冲释放。基于前面的研究结果，项目最后探究了加味当归补血汤药对自然衰老大鼠的药效实验及机制，通过对卵巢病理观察、卵泡病理计数、血清性激素、HPO轴神经内分泌指标的检测。结果显示加味当归补血汤能显著增加自然衰老大鼠原始卵泡、初级卵泡数量，降低了FSH及E2水平，增加AMH水平。加味当归补血汤防治生殖衰老的保护作用可能通过抑制RFRP-3表达，促进Kisspeptin的表达，进而增加GnRH的活性及释放有关。本项目密切关注中药通过下丘脑RFRP3-kisspeptin的影响改善GnRH/LH释放，将中药在女性的生殖衰老中的机制研究提升到整个HPO轴，为进一步阐明中药防治生殖衰老的中枢神经调控机制奠定基础，提供研究思路参考。