枸杞子新成分LyA选择性拮抗GluN2B/Ca2+/CaMKII通路减轻缺血性脑损伤的机制研究

It is evidenced that Ca2+ influx and overload activated the apoptotic pathway to induce neuron injury during ischemic stroke, which is mediated by overactivation of GluN2B, a submit of NMDAR (glutamate receptor). Selective antagonist for GluN2B is one of the important strategies for ischemic stroke treatment, however, there is no safe and effective antagonists for clinical therapy at present. It is the first time that we isolated and identified a new active ingredients LyA from the fruits of Lycium barbarum, and found the new compound LyA significantly attenuated cerebral ischemia/reperfusion injury in middle cerebral artery occlusion (MCAO) rats and reduced the infarct size. The result of molecular docking and preliminary experiment showed that LyA combined well with the key residues of the extracellular N-terminal (NTD) in the binding domain of allosteric regulation of GluN2B subunit, and suppressed calcium influx mediated by GluN2B subunit. These data suggested that LyA might exert brain protection by selectively antagonizing GluN2B subunit to reduce calcium influx and then inhibiting the downstream apoptotic pathways. This project firstly proposes that LyA combines with GluN2B subunit to exert its neuroprotective effects by inhibiting the downstream Ca2+/CaMKII pathway, and we will combine research methods including electrophysiology, molecular biology and radiolabeled technology and as well as pharmacology to investigate the combination of LyA and GluN2B subunit, the pharmacodynamics and the related molecular mechanisms. The research results will further explore the clinical application of LyA in ischemic stroke and provide evidence for new potential targets in ischemic stroke therapy.

缺血性脑卒中时，谷氨酸受体NMDAR亚单位GluN2B过度激活引发Ca2+内流超载，激活凋亡通路造成神经元损伤。选择性拮抗GluN2B是治疗脑卒中的重要措施之一，但临床尚无安全有效的该类药物。申请者前期从枸杞子中分离得到新活性成分LyA，发现其显著降低大鼠脑中动脉栓塞（MCAO）的缺血再灌注损伤，减少脑梗死面积；分子对接及预实验结果显示，LyA与GluN2B胞外段N-末端（NTD）变构调节区的关键氨基酸结合，显著抑制GluN2B介导的Ca2+内流。提示LyA可能通过选择性拮抗GluN2B抑制Ca2+内流，从而抑制下游凋亡通路发挥脑保护作用。本项目拟在前期工作基础上，结合电生理学、分子生物学、放射性同位素标记、药理学等技术，深入研究LyA与GluN2B的结合及结合区域，验证LyA抗脑缺血损伤的药效，阐明其对下游Ca2+/CaMKII通路的影响，为LyA治疗缺血性脑卒中的临床应用提供理论依据。

缺血性脑卒中的发病机制主要包括兴奋性毒性、钙离子超载、各种酶的激活、氧自由基的形成、再灌注后产生的炎症反应等，最终启动细胞凋亡程序引起细胞死亡，它们发生在不同时期，却又相互联系。N-甲基-D-天冬氨酸受体（N-methyl-D-aspartate receptor, NMDAR）是谷氨酸离子型受体中最主要的一类受体，其介导的兴奋性毒性，即细胞内Ca2+超载导致的神经元结构和功能受到破坏，是其它发病机制的触发者。LyA是从枸杞子中提取得到的天然抗氧化成分，前期已证实其具有神经保护作用。本课题通过体内外模型，即体内公认的MCAO脑缺血动物模型，以及体外OGD脑缺血细胞模型和NMDA诱导的神经细胞模型进行LyA抗脑损伤的研究。结果显示，在MCAO模型中，LyA能显著改善再灌注48小时后大鼠的神经学评分，减少梗死体积，减轻氧化应激损伤。同时，LyA显著增加缺血脑皮层中Nrf2和HO-1的转录表达；体外OGD模型实验结果表明，LyA可保护分化的SH-SY5Y细胞免受OGD诱导的损伤，采用细胞转染技术证实了LyA是通过PKCε/Nrf2/HO-1通路缓解氧化应激损伤和神经凋亡进而发挥脑保护作用；体外NMDA诱导神经损伤模型实验结果发现，LyA能显著抑制NMDA诱导的细胞凋亡，减少乳酸脱氢酶(LDH)的释放。同时，LyA可显著逆转NMDA诱导的细胞内钙离子增加及ROS的生成。western blot结果表明，NMDA能抑制p-NR2B、p-JNK、p-p38水平的升高。综上实验结果，可以看出LyA最终通过氧化应激PKCε/Nrf2/HO-1通路和GluN2B/Ca2+/MAPKs通路发挥神经保护作用。本项目首次发现了枸杞子成分LyA的脑保护作用，并通过多靶点揭示其分子机制，本课题的顺利完成，不仅丰富了枸杞子神经保护作用的药效物质及作用机制，同时为后续相关制剂的开发奠定基础。