GPR30激动剂和脑源雌激素协同降低缺血脑损伤的新机制

Our recent studies have shown that aromatase (Aro), the key enzyme to synthetize 17β-estradiol exhibits high expression in the injured brain, and the brain-derived estradiol (BDE2) triggers innate immunity response to protect against secondary neuronal apoptosis. Translationally, an important goal would be to identify mechanisms and potential therapies that can induce BDE2 in the brain so as to provide endogenous CNS protection from secondary apoptosis that occurs in neurodegenerative disorders. In pursuance of this goal, we are going to focus on the membrane estrogen receptor GPR30 that might be an important novel regulator of BDE2 in the brain, and examine the potential synergistic action between GPR30 specific agonist G1 and BDE2 to attenuate inflammatory injury following global cerebral ischemia (GCI) in surgical menopausal rats. The aims of our study would thus test the following hypotheses: 1)G1 replacement upregulates Aro protein expression and activity in the hippocampus through a GPR30-mediated mechanism, which decreases the secondary apoptotic neuron; 2)Synergistic action between G1 and BDE2 targets GPR30 activation to attenuate astrogliosis and inflammasome formation, as well as enhance anti-inflammatory cytokines; 3)Synergism between G1 and BDE2 also promotes microglia polarization from M1 to M2 phenotype, which favors neuronal repair following GCI; and 4)The AKT/PTEN-NFkB rapid signaling pathway induced by GPR30 is an important mechanism to provide neuroprotective and repair roles for synergism between G1 and BDE2 following GCI. If successful, the proposed studies could significantly advance the field by revealing novel functions of GPR30 and BDE2 against cerebral ischemic injury, which could have important translational significance.

课题组最近研究发现，脑源17β-雌二醇 (BDE2) 合成的关键酶Aromatase (Aro) 在损伤的大脑高表达，且BDE2 可启动天然免疫反应，降低神经元继发性凋亡。因此，揭示BDE2的作用机制、探索诱导BDE2的有效策略对治疗神经退行性疾病至关重要。本研究采用手术绝经大鼠全脑缺血模型，以雌激素膜受体GPR30为切入点，探索其特异激动剂G1上调Aro/BDE2、并协同作用降低神经炎症的分子机制。研究验证以下假设：1)G1通过GPR30上调海马Aro表达及活性，降低神经元迟发性凋亡；2)G1与BDE2协同作用于GPR30降低星形胶质细胞增殖及炎性小体形成，促进抗炎因子表达；3)G1协同BDE2促进小胶质细胞M2表型极化，促进神经元修复；4)AKT/PTEN信号通路是BDE2与G1协同作用降低脑缺血损伤的重要机制。此研究旨在揭示GPR30和BDE2抗脑缺血损伤的新功能，具有医学转化价值。

心脏骤停仍是当下世界面临的重大公共卫生问题，认知障碍是幸存者最严重的后遗症，目前尚无有效治疗措施。过去20年，雌激素替代治疗一直是基础与临床脑血管病防治研究的热点。最近，脑源雌激素(BDE2)作为内源性神经递质逐渐被研究者关注。课题组及他人前期研究发现，脑损伤如脑外伤、缺血性卒中、全脑缺血均可诱导BDE2合成，而BDE2合成关键酶Aromatase特异抑制剂、miRNA、基因敲除加重脑损伤。提示，BDE2具有损伤依赖性。可见，诱导BDE2可能成为修复已受损神经元的新思路。雌激素膜受体GPR30可通过基因和非基因通路介导促生存信号，其特异激动剂G1不但具有雌激素活性，又无雌激素依赖的副作用（如雌激素依赖的恶性肿瘤）；因此，G1较外源性雌激素更具有潜在的临床应用前景。.本研究切除SD雌性大鼠双侧卵巢以模拟手术绝经女性雌激素剥夺，建立全脑缺血动物模型模拟心脏骤停所致的海马CA1区神经元损伤和认知功能障碍，采用Cre-loxP基因重组技术制备前脑神经元Aromatase条件敲除大鼠，小干扰RNA敲低技术、免疫荧光染色、TUNEL形态学和Western Blot、ELISA分子生物学等实验技术，以及水迷宫、巴恩斯迷宫、旷场、新事物识别等功能行为学实验技术，系统研究G1与BDE2协同降低缺血再灌注后海马神经元损伤、改善认知功能、促进缺血后神经功能修复的作用及分子机制。研究证实：1) G1可通过降低小胶质细胞NLRP3炎性小体活性，上调神经元IL1RA蛋白表达，从而增加神经元的自我防御能力，减少神经元延迟性凋亡；2) 脑缺血再灌注可增加星形胶质细胞Aromatase-BDE2内源性保护信号通路，降低再灌注早期炎症损伤，此过程是GPR30依赖的；3）G1与BDE2协同作用于GPR30，促进缺血再灌注早期小胶质细胞M1型向M2型极化，从而阻止神经元延迟性死亡； 3) G1可促进内源性神经干细胞增殖和分化，保护新生神经元，具有长期神经保护作用，其分子机制与星形胶质细胞Aromatase-STAT3信号通路密切相关。本研究首次揭示通过星形胶质细胞、小胶质细胞和神经元的交互作用，G1与BDE2协同降低脑缺血神经元损伤、促进神经功能修复的作用机制。结果说明GPR30受体可作为诱导内源性神经保护剂BDE2的潜在靶点。.