星形胶质细胞ndrg2在雌激素受体β脑缺血保护效应中的作用及机制研究

The incidence and mortality of stroke in postmenopausal women are significantly increased and higher than those of men of the same age, and the postmenopausal women has become the focus of stroke prevention and treatment. Estrogen has neuroprotective effects against stroke, but its carcinogenic effects greatly restricts its clinical application. The estrogen receptor-ERβ is not expressed in female reproductive organs, and the activation of ERβ, which has no carcinogenic effects, is expected to be a new strategy for the prevention and treatment of stroke in postmenopausal women. In previous studies, the applicant found that the activation of ERβ exerts neuroprotective effects against cerebral ischemia by activating astrocytes; ndrg2 is specifically expressed in astrocytes, and could regulate astrocyte activation, and plays an important role in the neuroprotection against cerebral ischemia, and is regulated by estrogen. In the pre-experiments, the applicant found that the activation of ERβ stimulated the expression of ndrg2; and the neuroprotective effects of ERβ against cerebral ischemia disappeared in ndrg2-/- mice, suggesting that ndrg2 might be the key molecule mediating the neuroprotective effects of ERβ, but the underlying mechanism remains unclear. By using the ndrg2-/- mice and ndrg2 overexpression lentivirus, constructing cerebral ischemia model both in vivo and in vitro, and using iTRAQ mass spectrometry analysis and other morphology, behaviourology, proteomics and molecular biology technologies, this program aims to verify the role and mechanism of ndrg2 in the neuroprotective effects of ERβ against cerebral ischemia, which may provide a new molecular target for the prevention and treatment of stroke in postmenopausal women.

绝经女性脑卒中发病率和死亡率显著增加且高于同年龄段男性，已成为脑卒中防治的重点人群。雌激素具有脑卒中保护作用，但致癌作用限制其临床应用。雌激素受体ERβ不表达于女性生殖器官，激动ERβ（无致癌作用）有望成为防治绝经女性脑卒中的新策略。申请人前期发现，激动ERβ通过激活星形胶质细胞减轻脑缺血损伤；发现ndrg2特异性表达于星形胶质细胞，调控星形胶质细胞激活，在脑缺血保护中发挥重要作用，受雌激素调控。预实验发现，激动ERβ可促进ndrg2表达；ERβ脑缺血保护作用在ndrg2-/-小鼠消失，提示ndrg2可能是介导ERβ脑缺血保护作用的关键分子，但机制还不清楚。本课题拟利用ndrg2-/-小鼠和ndrg2过表达慢病毒，构建在体离体脑缺血模型，采用iTRAQ质谱分析等形态学、行为学、蛋白质组学、分子生物学技术，阐明ndrg2在ERβ脑缺血保护中的作用及机制，为防治绝经女性脑卒中提供新的分子靶点。

脑卒中已成为威胁我国国民健康的主要杀手。绝经女性脑卒中发病率和死亡率显著增加且高于同年龄段男性，是脑卒中防治的重点人群。雌激素具有脑卒中保护作用，但致癌作用限制其临床应用。雌激素受体ERβ不表达于女性生殖器官，激动ERβ（无致癌作用）有望成为防治绝经女性脑卒中的新策略，但分子机制尚不清楚。负责人前期发现Ndrg2在脑缺血损伤中发挥重要作用，其脑内表达受雌激素调控，可能是ERβ的靶基因。因此，我们推测Ndrg2可能是介导ERβ脑缺血保护作用的关键分子，但确切作用和机制尚需研究证实。我们利用Ndrg2敲除小鼠和原代星形胶质细胞，构建去卵巢（OVX）、雌激素替代、ERβ激动剂（DPN）替代治疗模型、大脑中动脉阻塞脑缺血和细胞氧糖剥夺损伤模型，运用行为学、形态学和分子生物学等方法，主要研究：①Ndrg2在脑缺血损伤中的确切作用及分子机制；②Ndrg2是否是ERβ的靶基因；③Ndrg2在ERβ脑缺血保护中的作用及机制。主要研究结果：①Ndrg2广泛表达于脑内星形胶质细胞，可以作为新的星形胶质细胞分子标志物；②Ndrg2通过调控Rho-GTPase信号通路来调节星形胶质细胞形态（扁平化与星形化）；③Ndrg2通过稳定Na+-K+-ATPaseβ1亚基减轻星形胶质细胞毒性水肿介导的脑缺血水肿损伤，发挥脑缺血保护效应；④激动ERβ的替代治疗可通过抑制小胶质细胞和星形胶质细胞炎性激活以及NF-κB炎症通路，减轻脑缺血再灌注损伤；⑤雌激素和ERβ能够显著上调Ndrg2表达，证实Ndrg2是ERβ靶基因；⑥Ndrg2是介导雌激素和ERβ脑缺血保护效应的关键分子。这些结果阐明了激动ERβ防治绝经动物脑缺血损伤的作用和分子机制，为防治绝经女性脑卒中提供新的治疗方向、研究思路和分子靶点，具有重要的理论和临床指导意义。