GPER1介导的雌激素非基因组效应在脑缺血神经元损伤中的保护作用和机制研究

There is still a dispute involved in the clinical application of estrogen because of the undefined mechanisms underlying its neuroprotective effects, although its significant neuroprotective activities have been identified. The simple classification of the neuroprotective mechanisms of estrogen is genomic and non-genomic processes. The identification of G protein－coupled estrogen receptor 1 has introduced an receptor potentially responsible for non-genomic estrogen signaling. Several studies have demonstrated that G protein－coupled estrogen receptor 1 may mediate protective effects of estrogen against ischemic damage in cardiac muscle and hepatocyte. Our previous research had suggested that there is G protein－coupled estrogen receptor 1 in central nervous system by immunostaining. Furthermore,we found that the expression of G protein－coupled estrogen receptor 1 is increased in the rat brain following cerebral ischemia. Thus, we presume that estrogen exerts neuroprotective effects via G protein－coupled estrogen receptor 1. In the study, the hypothesis that G protein－coupled estrogen receptor 1 has a neuroprotective effect that is achieved through the inhibition of ASK1 on PI3K/Akt signaling cascade is investigated by Immunohistochemistry, immunoblotting，gene transfection，water maze，patch clamp and so on in a rat model of global cerebral ischemia and a model of oxygen-glucose deprivation in vitro rat hippocampal neurons. These research results could help us increase understanding of the mechanisms of neuroprotective effects of estrogen and provide a new target for neuroprotection.

雌激素有显著脑保护作用，但其临床应用存在争议，主要原因是其脑保护机制不详。仅研究雌激素脑保护基因组机制是不全面的，因为其脑保护中也存在非基因组机制。G蛋白偶联雌激素受体-1（GPER1）是目前唯一公认的雌激素膜受体，雌激素可通过它启动非基因组生物效应。研究已证实GPER1介导的非基因效应在心肌、肝细胞缺血损伤中有明显的保护作用。我们前期发现GPER1在中枢神经系统表达，缺血损伤后脑GPER1表达增加。据此，我们推测GPER1在脑缺血神经元损伤中有保护作用。本研究拟分别建立大鼠全脑缺血和海马神经元糖氧剥离模型，借助免疫组化、免疫印迹、MTT法、 水迷宫等技术观察GPER1对脑缺血海马神经元的保护作用,并探讨"GPER1 介导雌激素通过PI3K/Akt 通路调节ASK1的活性来抑制脑缺血诱导的神经元凋亡"这一机制。本课题的完成能深化对雌激素脑保护机制的认识，也可为研究神经保护提供新靶点。

由于机制不详，雌激素作为脑保护及应用于临床仍存在争议。G蛋白偶联雌激素受体-1（GPER1）是雌激素膜受体，雌激素可通过它启动非基因组生物效应。本实验旨在证实GPER1对脑缺血海马神经元的保护作用,并验证其信号通路。.我们改良了大鼠全脑缺血模型。直视下阻断大鼠C1-C2横突孔之间的椎动脉可以大大提高模型的成功率、稳定性和可复制性。尼氏染色确定10min是最佳缺血时间，大鼠海马CA1区是最敏感的缺血区。.我们应用免疫组化法对GPER1在大鼠海马区神经元的表达进行了定位，发现GPER1在CA1-3区和齿状回神经元细胞膜上均呈现。在去势的全脑缺血大鼠模型上，缺血前后不同时间点注入不同剂量的G1，通过尼氏染色发现G1对全脑缺血后海马CA1区神经元损伤的保护作用具有时间和浓度依赖性。Morris水迷宫测试证实G1能够减轻全脑缺血造成的认知障碍。我们运用westernblot技术检测了缺血再灌注后各时间点p-Akt、p-ASK1、p-JNK三种蛋白的表达水平，发现激活GPER1可明显提高缺血后1h海马CA1区p-Akt的水平，降低p-ASK1和p-JNK的水平，这种效果可以被LY294002所阻断。.离体实验中，糖氧剥离（OGD）处理后，神经元GPER1的表达较正常组显著升高，24h时表达量达最高峰。OGD 3h后，光镜下细胞形出现固缩，密度下降，MTT观察细胞活性明显下降，而E2和E2-BSA预处理能明显减轻上述损伤。流式细胞仪观察到OGD 3h后，神经元凋亡明显增加，而E2和E2-BSA预处理能明显抑制凋亡。在过表达GPER1的神经元中，E2和E2-BSA对细胞活性和LDH释放率的保护显著增加，而在GPER1低表达的神经元中，E2仍能逆转OGD引起的细胞活性下降和LDH释放率增加，但E2-BSA的保护作用消失。E2和E2-BSA能增加OGD处理神经元Akt磷酸化水平，降低ASK1磷酸化水平，而LY294002可以显著抑制上述效应。通过MTT实验和LDH检测，我们观察到LY294002可以完全逆转E2-BSA的保护作用，部分逆转E2的保护作用.这些结果证明GPER1通过激活PI3K/Akt通路，下调ASK1/JNK活性，能够有效减轻全脑缺血对海马神经元造成的损伤，改善全脑缺血造成的认知功能障碍，预示着GPER1的激活在全脑缺血后有神经保护作用，可能成为脑缺血后药物的潜在靶点。