不同时机电针预处理调控PI3K/Akt通路保护早发性卵巢功能不全模型大鼠卵巢功能的时效差异及机制

Female primordial germ cells physiologically decreased at the beginning of birth and depleted at menopause, when the female ovarian function will permanently lost. During the nature process of premature ovarian insufficiency, the ovarian function will begin to decline or deplete earlier than normal. The prevention and early treatment revealed a more promising method for premature ovarian insufficiency than the treatment when the disease appeared, however, the optimal time of prevention and early treatment is unclear. In our previous study, we have found that continuous electroacupuncture pretreatment before model establishment and during model process could provide some protective effect on ovarian function, and one of the possible mechanism accounting for the protective effect is that it could up-regulate Bcl-2 protein expression, and down-regulated Bax protein expression in granulosa cell in ovarian. In this study, by using ELISA、RT-PCR、Western blot, TUNEL detection, light microscopy and other experimental technique, we planned to evaluated the different effects and mechanisms of electroacupuncture pretreatment at different time-point on protecting ovarian function in premature ovarian insufficiency rat via apoptosis-related factors mediated PI3K/Akt pathway. This study will preliminarily reveal the scientific connotation of different effects of electroacupuncture pretreatment at different time-point on protecting ovarian function from the perspective of biological mechanisms and molecular basis. Meanwhile, this study will also provide some ideas of prevention and treatment on premature ovarian insufficiency.

女性原始生殖细胞的生理性损耗自出生后即已开始，直至绝经期生殖细胞耗竭、卵巢功能永久丧失。早发性卵巢功能不全自然病程中，随病情进展，疾病患者卵巢功能较正常女性会提前过渡至低下或耗竭状态。早发性卵巢功能不全的预防和早期治疗比疾病形成后的治疗会更有意义，但预防和早期治疗的最佳时机尚不明确，我们前期研究发现，在造模前和造模中进行电针预处理可能通过调控卵巢颗粒细胞凋亡相关因子Bcl-2蛋白和Bax蛋白的表达，对早发性卵巢功能不全大鼠卵巢具有一定保护作用。我们拟采用ELISA、RT-PCR、Western blot、TUNEL法、光镜等技术，围绕PI3K/Akt通路介导的凋亡相关因子，探索不同时机电针预处理保护早发性卵巢功能不全大鼠卵巢功能的时效差异和机制。本项目将从生物学机制和分子基础角度，初步揭示不同时机电针预处理卵巢保护时效差异的科学内涵，同时也为针灸防治早发性卵巢功能不全方案的优化提供思路。

卵巢功能不全至卵巢衰竭一般是渐进过程，使用保护性措施阻止或延缓卵巢功能不全进程，对保护女性生育能力具有重要意义。本研究通过在POI大鼠造模前、造模中、造模后三个不同时机进行电针预处理，并在造模前使用电针、促性腺激素释放激素激活剂(Gonadotropin-releasing hormone agonist，GnRHa)与电针联合GnRHa预处理，观察比较各组大鼠模型成功后卵巢湿重、卵泡形态、各级卵泡数量、性激素水平、颗粒细胞凋亡情况、PI3k/Akt信号通路下游分子的表达情况，阐释了不同时机电针预处理通过调控PI3K/Akt信号通路保护卵巢功能的时效差异及生物学机制，以及电针联合GnRHa预处理保护卵巢功能的中西医协同效应和相应机制。本研究发现，不同时机电针预处理保护卵巢功能机制与电针介导PI3K-Akt-mTOR通路不同程度上调Akt、mTOR，以及不同程度上调Bcl-2和下调Bax有关，特别是造模前（电针I组）与造模中（电针II组）的电针干预上调效应较造模后电针（电针III组）干预更明显。另外，相较造模前电针预处理或皮下注射GnRHa预处理，造模前电针联合皮下注射GnRHa可通过调控下丘脑-垂体-卵巢轴（HPO轴）更好抑制卵巢组织窦前卵泡过度招募与激活，更好地保存卵巢窦前卵泡，进而保护卵巢功能。本实验发现在未发病与发病早期使用电针预处理可更好保护卵巢储备功能，本研究提示针对存在卵巢损伤风险、且有生育力保护需求的女性，可使用电针预处理或电针联合皮下注射GnRHa保护卵巢功能，且电针预处理的时机越早越好。

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