GSS脑保护作用机制研究：基于GluN2B型NMDA受体的调控

NMDA (N-methyl-d-aspartate) receptors are key molecules involved in excitotoxicity following cerebral ischemia/reperfusion injury. However, NMDA recptor antagonists show prominent side effects in clinical trials. Finding new drugs targeting NMDA receptors is of great significance. Genistein sodium sulfonate(GSS) is novel water soluble substances modified from genistein. Our preliminary data indicate that the neuroprotective effects of GSS against cerebral ischemia is significantly improved compared with genistein and the effective dose was greatly decreased. Thus GSS is a promising drug for cerebral ischemia. Activtion of ERK1/2 signaling pathway and decreased protein level of GluN2B subunit of NMDA receptors might contribute to the neuroprotective effets of GSS. As genistein can activate the membrance estrogen receptor,we speculate that GSS might also have the abitity to activate membrance estrogen receptor and regulate the level and activation of extrasynaptic GluN2B-containing NMDA receptors, which are closely related with cell injury, thus protect against cerebral ischemia/reperfusion injury. In the present project, we will further investigate the neuroprotective effects of GSS through in vitro and in vivo studies and educidate whether GSS can regulate the protein level and activation of GluN2B-containing NMDA receptors throuth activating GPR30,using molecular biological, electrophysiological, morphological, pharmacological and behavioral methods. The present project will be helpful for our understanding of the neuroprotective role of GSS and its underlying mechanisms and will provide a new clue for the clinical preventive medication and treatment of ischemic cerebrovascular disease.

NMDA受体是兴奋性毒性损伤的关键分子，但其拮抗剂在临床试验中显示明显的副作用，因而寻找新的NMDA受体靶向药物异常重要。染料木素磺酸钠(GSS)是对染料木素进行结构修饰的强水溶性物质。前期结果表明，与染料木素相比，GSS脑保护作用明显提高，有效保护剂量显著降低，其机制可能与ERK1/2信号通路激活、GluN2B蛋白水平下调有关。结合染料木素能激活雌激素膜受体GPR30的特性，我们推测GSS很可能通过GPR30调控与细胞损伤信号密切相关的突触外GluN2B型NMDA受体功能而发挥对脑缺血再灌注损伤的保护作用。本项目拟通过分子生物学、电生理学、形态学和药理学等方法，分别从离体和在体水平明确GSS在脑缺血再灌注损伤中保护作用，并对GSS是否通过激活GPR30进而调控GluN2B型NMDA受体功能进行深入研究。该项目有助于深入了解GSS的脑保护作用及机制，为缺血性脑病的预防和治疗提供新线索。

NMDA 受体是兴奋性毒性损伤的关键分子，但其拮抗剂在临床试验中显示明显的副作用，因而寻找新的 NMDA 受体靶向药物异常重要。GSS 的脑保护作用可能与 ERK1/2 信号通路激活、GluN2B 蛋白水平下调有关。结合染料木素能激活雌激素膜受体 GPR30 的特性，我们推测 GSS 很可能通过 GPR30 调控与细胞损伤信号密切相关的突触外GluN2B型NMDA受体功能而发挥对脑缺血再灌注损伤的保护作用。本项目采用大鼠大脑中动脉栓塞动物模型、谷氨酸损伤PC12细胞及原代培养的皮层神经元细胞模型，分别从离体和在体水平探讨GSS减轻脑损伤的机制。. 结果：（1）GSS降低MCAO大鼠神经功能评分、脑梗死体积、血清LDH及CK活性，提高脑组织LDH、tNOS、cNOS、T-AOC和抗氧化酶活性，升高NO含量，降低iNOS活性和MDA含量。（2）GSS升高脑组织钠钾及钙镁ATPase活性。（3）GSS降低MCAO大鼠皮层半损伤区Bax、Caspase-3 及Caspase-9蛋白和mRNA表达，升高Bcl-2蛋白和mRNA表达。GSS减少谷氨酸诱导的原代培养的皮层神经元凋亡细胞，降低Bax及Caspase-3表达降低，升高Bcl-2表达。（4）GSS抑制谷氨酸诱导的PC12细胞活性降低。提高谷氨酸诱导的皮层神经元活性，降低LDH释放率。在离体和整体水平，升高PSD95、NMDA受体亚单位及DAPK1的mRNA和蛋白表达，升高GluN2B mRNA表达，但降低GluN2B蛋白及磷酸化，升高p-DAPK1及p-ERK水平。（5）给予ERK抑制剂U0126后，取消了GSS对谷氨酸诱导的神经元LDH释放率升高的抑制作用；GSS升高皮层半损伤区PSD95、NMDA受体亚单位及DAPK1 mRNA表达的作用被U0126逆转。. 结论：GSS对脑缺血再灌注损伤有较好的保护作用，其作用机制与提高能量代谢，抑制氧化应激、细胞凋亡及兴奋性毒性损伤有关。DAPK1/ERK信号通路参与GSS的脑保护机制。