谷氨酸受体负向变构剂组合对蛛网膜下腔出血后脑损伤的保护机理研究

Subarachnoid hemorrhage (SAH) complicated with brain injury lead to death or permanent neurological disability, but lack of satisfied prevention and control measures. Rapid increase in cerebral glutamate level occurs after subarachnoid hemorrhage. Excessive stimulation of glutamate receptors, such as NMDA and mGluR1/5 receptors, play a key role in brain injury. Allosteric modulators, which interact with allosteric binding sites topologically distincr from the ligand binding site, display varied subunit-selectivity and have therapeutic advantages over the competitive inhibitors. Very recently, multiple classes of negative allosteric modulators (NAM) of NMDA and mGluR1/5 receptors have been identified, these NAMs have not found application in SAH research. Thus, the studies outlined in this application aim at using NAM combination of NMDA and mGluR1/5 receptors to control high concentration glutamate-induced excessive stimulation of glutamate receptors. In this proposal, we study that NAM combination protects cerebellar granule neurons from high concentration glutamate-induced apoptosis, and define the molecular mechanisms that NAM combination control the regulation of NMDA and mGluR1/5 receptors in the siganling pathways. As such, this will uncover the internal relation among cell apoptosis. Furthermore, the proposed studies will focus on testing the effects of NAM combination on pathophysiological process of SAH. The improvement of NAM combination on functional outcome of SAH will be evaluated. The effects on SAH-elated cerebral injury will be investigated. The proposed study will explore the physiological effect and molecular mechanism of the protective effects of NAM combination on cerebral injury after SAH, also evaluate and prove the clinical potential of NAM combination against SAH.

蛛网膜下腔出血(SAH)后脑损伤常致死亡或神经功能残障，但对此缺乏满意防治措施。SAH后脑内谷氨酸浓度急剧升高，过量谷氨酸引起的受体(如NMDA和mGluR1/5受体）过度激活在脑损伤中起关键作用。选择性结合受体变构位点的变构剂能避免配体干扰，且能区分不同亚型的受体，而具有良好的临床应用前景。近年新发现数种NMDA和mGluR1/5受体的负向变构剂(NAM)，尚无应用于SAH研究。本项目拟采用NAM组合控制SAH后高浓度谷氨酸引起的受体过度激活。此课题将检测和分析NAM对高浓度谷氨酸诱导的体外小鼠神经元凋亡模型的保护作用及作用机理，并筛选出最佳的NAM组合。然后检测NAM组合对SAH动物模型病理生理过程的影响，评价NAM组合对SAH功能性预后的改善作用，探讨NAM组合对SAH后脑损伤保护作用的机制。本项目拟阐明以谷氨酸受体为靶点的NAM组合对SAH后脑损伤的保护机理，并评价其临床潜力。

谷氨酸兴奋性毒性及级联的氧化应激损伤、神经元凋亡在蛛网膜下腔出血(SAH)后脑损伤中起着关键作用。本项目主要研究了N-甲基-D-天冬氨酸(NMDA)受体、代谢型谷氨酸受体1/5(mGluR1/5)的负向变构剂对抗SAH后高浓度谷氨酸诱导的神经细胞钙超载和凋亡的效应，并探讨其对SAH后脑损伤的影响。研究结果显示，在SAH后高浓度谷氨酸诱导的原代皮层神经元、海马神经元、小脑颗粒神经细胞钙超载及凋亡模型上，研究筛选出了NR2B型NMDA受体的负向变构剂Ifenprodil和mGluR1受体的负向变构剂JNJ16259685均对抗谷氨酸诱导的神经细胞钙超载及凋亡，两者组合呈现叠加效应，进而抑制大鼠SAH模型后神经功能缺损、血脑屏障破坏、皮层区细胞凋亡；mGluR5受体的选择性激动剂CHPG或正向变构剂VU0360172能减缓大鼠SAH模型后小胶质细胞过度激活和神经元凋亡。此外，研究发现纳米姜黄素减少SAH后谷氨酸的过度释放及氧化应激，临床药物肌肽或半胱氨减轻SAH后氧化应激和神经元凋亡而改善功能性预后。项目研究结果发表SCI论文6篇，为深入理解SAH后脑损伤发展和治疗SAH提供有价值的基础实验数据。