基于ER与PI3K/Akt信号通路关联性探讨补肾活血汤对免疫性卵巢早衰小鼠模型作用机制的研究

The pathogenesis of premature ovarian failure(POI) is still unknown so far, also the specific therapy and drug are lack.The abnormalities of autoimmune is one of the factors leading to disease has been confirmed. The occurrence and development of POI can take several years, traditional Chinese medicine(TCM) is a safe and effective means of early intervention. We have successfully established the B6AF1 immune mice model of premature ovarian failure, and found the model existence of hormone receptor expression decreased, a large number of follicular atresia in ovarian tissue and serum related factors decreased.In our previous study, we found that Bushen Huoxue decoction can filled lean kidney, promoting blood circulation and removing blood stasis, also can significantly improve hormone receptor expression, has definite clinical effect on POF.But its pharmacological mechanism of action awaits further studies.Based on the above research,we hypothesize that Bushen Huoxue decoction treating autoimmune POF may by regulate ER expression in ovary, influences key components of PI3K/Akt pathway.So that compound can promote the proliferation of granulosa cells and repair damaged ovarian function. In order to verify this hypothesis, our study intended to explore the mechanism of Bushen Huoxue Decoction in the treatment of autoimmune POF from the angle of Estrogen Receptor mediated PI3K/Akt signaling pathway on in vitro and in vivo experiment to provide new scientific evidence for the clinical application of Bushen Huoxue decoction.

卵巢早衰（premature ovarian failure，POF）发病的具体机制未明，自身免疫异常是导致疾病的因素之一已得到证实。本病的发生发展过程可历时数年，中医药是早期干预的安全有效手段。我们已经成功建立B6AF1小鼠免疫性卵巢早衰模型，并发现该模型存在激素受体表达减弱，大量卵泡闭锁，血清相关因子水平下降。本课题组前期研究显示补肾活血汤具有填精益肾、活血化瘀的功效，复方可明显改善激素受体表达，对卵巢早衰具有确切的临床疗效。因此，我们提出复方可调节ER在卵巢中的表达，影响PI3K/Akt信号通路的某一个或几个关键分子的激活，促进颗粒细胞增殖，修复受损的卵巢功能的科学假说。为了验证此假说，本项目拟运用多种生物技术手段，从体内、外角度，来研究补肾活血汤治疗免疫性卵巢早衰的药理机制，为补肾活血汤的临床应用提供新的科学证据。

卵巢早衰（Premature ovarian failure，POF）是当前妇产科领域的疑难问题，激素替代治疗（Hormone replacement therapy，HRT）作为一线方案存在诸多限制，且并不能改善生育力。补肾活血汤（BuShen HuoXue decoction，BSHXD）是我院名老中医经验方，前期实验及临床研究均显示复方疗效满意，但具体分子机制尚不清楚。复方富含黄酮类植物雌激素，雌激素受体是雌激素发挥效应的关键靶点，而PI3K/AKT信号通路能够被ER激活，从而在卵泡发育中起到促进作用。前期研究已发现补肾活血汤可以显著提高POF患者靶器官中ER表达。为了验证这一科学问题，我们分别从动物实验和细胞实验深入探究补肾活血汤治疗POF的效应机制。首先利用生物信息学方法，证实ESR1是核心成员，PI3K/Akt是药物-疾病关键信号通路。采用UPLC-MS/MS 对补肾活血汤（BSHXD）进行鉴定，其所含黄酮、异黄酮、类黄酮等化合物具有类雌激素受体激动剂的作用。选择分子对接结合性能高的单体，利用表面等离子共振技术（Surface plasmon resonance, SPR）进行中药小分子—ESR1蛋白互作检测，证明复方中筛选出的中药活性成分与ESR1靶点有强亲和力，其所具备的类雌激素效应对POF的低雌相关症状具有良好的治疗作用。动物实验结果显示：补肾活血汤能显著改善POF模型小鼠血清性激素水平，促进各级卵泡生长。进一步研究发现，补肾活血汤干预后模型小鼠卵巢组织中ER明显表达，与此同时，卵巢内PI3K/Akt信号通路被激活，通路相关磷酸化蛋白明显增加。进而，我们选择人卵巢颗粒细胞（KGN细胞）进行实验，发现补肾活血汤含药血清干预后，细胞激素分泌水平上升，AKTmRNA、mTORmRNA相对表达量增加，增强了cyclinD2、PCNA、survivin蛋白的表达，下调Cleaved-caspase3蛋白表达。应用雌激素受体抑制剂后，相关作用被削弱。补肾活血汤含药血清体外培养人卵巢颗粒细胞卵巢颗粒细胞系，可显著减少细胞凋亡，恢复细胞周期。本项目的研究成果为补肾活血汤治疗卵巢早衰的临床转化提供了可靠的实验依据，同时为基于调节ER、PI3K/AKT信号通路活性为靶点的POF治疗药物的相关研发提供了新的策略。