PI3K/Akt信号通路在经皮穴位电刺激治疗卵巢早衰中的机制研究

Premature ovarian failure (POF) is the hot spot in the field of gynecological and reproductive endocrinology research. The patients with POF have some diseases including osteoporosis, cardiovascular diseases, infertility, which affect the women's quality of life. In the previous study, we found that transcutaneous electrical acupoint stimulation (TEAS) improve the POF ovarian function. Our preliminary experiment results also showed that phosphatidylinositol-3-OH kinase (PI3 kinase) stimulator improved the activation of primordial follicles of human ovarian cortex and TEAS increase phosphorylation of protein kinase B (AKt) of the signal pathway, which suggested that mechanism of TEAS improving POF ovarian function was associated with PI3K/AKt signal pathway. Therefore, we hypothesized that TEAS activate primordial follicles and inhibit granulocyte cell apoptosis through the activation of PI3K/Akt signal pathway. The present study is to investigate the effect of TEAS on ovarian function and morphology indicators in POF mice, and PI3K/AKt signal pathway in vitro. To further investigate the mechanism by which TEAS improving ovarian function in POF mice, we used the activation of primordial follicle technology, as well as co-cultured with granulocytes in vitro. As the mechanism of TEAS improving POF ovarian function has not been publicized, our present study will reveal that the molecular mechanism of TEAS involved in the treatment of POF, and lead to potential therapeutic target for the treatment of POF ovarian function.

卵巢早衰（POF）是妇科生殖内分泌领域的研究热点，其对女性生育力和健康可造成严重影响。前期发现：体内实验发现经皮穴位电刺激（transcutaneous electrical acupoint stimulation, TEAS）可降低POF患者促性腺激素水平，改善卵巢功能；体外研究发现PI3K激动剂能够激活卵巢始基卵泡，TEAS可上调PI3K/Akt信号通路AKt及P-AKt水平，提示TEAS改善POF卵巢功能可能与PI3K/Akt信号通路有关。我们假设：TEAS通过PI3K/Akt信号通路激活POF小鼠始基卵泡，抑制颗粒细胞凋亡，改善卵巢功能。本研究拟：在体观察TEAS处理POF小鼠后卵巢形态学及功能指标的变化情况；以PI3K/Akt信号通路为切入点，深入分析TEAS是通过激活始基卵泡、抑制颗粒细胞凋亡，改善POF小鼠卵巢功能的分子机制，为TEAS治疗POF提供了理论依据。

经皮穴位电刺激（TEAS）已被广泛应用于卵巢功能减退及早发性卵巢功能不全（POI）治疗，但具体机制尚不明确。本研究选取60只8周龄SPF级C57BL6雌性小鼠，随机分为6组，每组10只，分别为：① 对照组+模拟TEAS治疗（control-），② 对照组+TEAS治疗（control+），③ 实验组放疗后2天+模拟TEAS治疗（IR 2D-），④ 实验组放疗后2天+TEAS治疗（IR 2D+），⑤ 实验组放疗后1周+模拟TEAS治疗（IR 1W-），⑥ 实验组放疗后1周+TEAS治疗（IR 1W+）。观察治疗前后小鼠动情周期，检测小鼠血清抗苗氏勒管激素（AMH）水平，观察卵巢组织形态学并计数各级卵泡数。检测卵巢组织PTEN、Akt、FOXO3a、GSK3β、ERK1/2磷酸化蛋白和总蛋白的表达含量，及FOXO3a和p-FOXO3a在原始卵泡中的表达及定位。测定卵巢组织超氧化物歧化酶2（SOD2），谷胱甘肽过氧化物酶（GSH-Px）以及丙二醛（MDA）水平变化。全身单次4 Gy X线辐照可造成小鼠动情周期紊乱，AMH水平下降，对卵巢形态学及各级卵泡数量产生明显的损伤作用。与IR 2D-相比， IR 2D+组小鼠动情周期恢复正常，AMH水平明显升高，原始卵泡损失数量明显减少，但初级卵泡、次级卵泡及窦卵泡的数量变化不明显。IR 1W+组无上述改变。IR 2D-组与对照组相比，小鼠卵巢组织PTEN，Akt，ERK，GSK3β及 FOXO3a磷酸化蛋白相对表达水平均显著增加。同时 MDA含量升高，SOD2及GSH-Px含量降低。IR 2D+组PTEN，Akt，ERK，GSK3β及 FOXO3a磷酸化蛋白水平显著降低。通过免疫荧光检测发现，IR 2D-组FOXO3a磷酸化蛋白在原始卵泡中表达明显增加，而予以TEAS治疗后可显著抑制FOXO3a磷酸化水平及由细胞核向胞质转运效应。并且TEAS治疗可显著抑制卵巢组织的MDA含量，升高SOD2及GSH-Px活性。全身单次4Gy X线辐照可造成小鼠动情周期紊乱，AMH水平降低，且对卵巢形态学及各级卵泡计数产生显著的损伤作用。辐照后2天即予以TEAS治疗通过拮抗PTEN/PI3K/FOXO3a信号通路激活，降低FOXO3a磷酸化水平，进而抑原始卵泡过度激活，以及增强卵巢组织抗氧化能力，可能是TEAS保护放疗小鼠卵巢功能的机制之一。