芎芪合剂对于调控NMDA受体-调节神经突触在脑缺血-再灌注损伤中的机制研究

The use of recombinant tissue plasminogen activator (t-PA) as an intravenous thrombolytic agent within the first three hours of stroke onset is the only therapy approved by the Food and Drug Administration for acute ischemic stroke in United States. However, revascularization of the occlusive cerebral artery will inevitably aggravate the ischemia-reperfusion injury. Therefore, alleviating the ischemia-reperfusion injury and extending the time window of thrombolytic therapy are clinical central issues. Calcium influx through NMDA receptors (NMDARs) plays key role in ischemia-reperfusion injury. And, controlling the calcium influx via inhibiting NMDARs’ activation is a promising way to alleviate ischemia-reperfusion injury, lengthen time window and ameliorate neurological deficits. In this study, we will administrate the combination of astragalus membranaceus and ligustrazine on the C57BL/6 mice, which have been induced permanent MCAO, and, on the primary neurons of SD rats, which have been induced hypoxia. Then, the quantitative detection of NR2A, NR2B, PSD-95, SYN and GAP-43 expressions in the concerned cerebral areas of the mice, as well as mitochondria membrane potential assessment and Ca2+ influx analysis of the primary neurons will be used to evaluate the effects of the combination of astragalus membranaceus and ligustrazine on NMDARs activating pathway after cerebral ischemia and reperfusion induction. The effects of the combination of astragalus membranaceus and ligustrazine on neuronal plasticity will be analyzed as well.

在急性脑缺血3小时内利用纤维蛋白溶酶原激活剂(rt-PA)进行静脉溶栓是目前唯一被美国FDA批准的超急期临床治疗方法，然而，对脑动脉闭塞所导致的缺血进行溶栓将不可避免地加重脑组织的缺血-再灌注损伤，如何减少再灌注损伤、延长时间窗是目前临床所关注的焦点。NMDA受体介导的Ca2+内流损伤是脑缺血-再灌注损伤的关键损伤机制，抑制该途径的激活，有望显著减轻再灌注损伤、延长时间窗、改善神经功能。在本研究中，我们拟将补阳还五汤有效成分芎芪合剂作用于临时性MCAO小鼠模型及缺氧处理的SD大鼠原代神经元，通过对缺血区域的NR2A、NR2B、PSD-95及SYN、GAP-43表达情况定量检测，以及神经元的线粒体功能检测、钙离子内流测定等，了解芎芪合剂对于脑缺血-再灌注所引起的NMDA受体介导的Ca2+内流损伤途径的作用效果及其影响机制；同时，探讨其对于神经突触可塑性的影响以了解其对于神经功能康复的作用。

急性脑梗死后溶栓或取栓治疗出现的脑缺血-再灌注损伤目前尚无理想治疗药物，中医认为其主要病机为气虚血瘀,针对这一病机我们前期应用具有益气活血效用的黄芪联合川芎嗪注射液组合的“芎芪合剂”，发现其可通过大鼠血脑屏障，减少急性脑梗死大鼠溶栓后血管紧密连接的破坏和出血转化的发生，但其抗脑缺血再灌注损伤的作用机制并不十分清楚。因此，本项目从芎芪合剂对于脑缺血-再灌注所引起的NMDA受体介导的Ca2+内流损伤途径的干预作用及其对于神经突触可塑性的影响两方面以了解其保护脑缺血再灌注损伤的功效。通过构建临时性大脑中动脉栓塞/再复灌小鼠模型及SD大鼠原代皮层神经元氧糖剥夺/复氧复糖模型，从体内外两方面证实芎芪合剂能通过抑制NMDA受体NR2亚基活性，从而抑制神经元细胞Ca2+超载，维持神经元线粒体膜电位稳态，减少线粒体损伤，上调抗凋亡因子Bcl-2表达，下调促凋亡因子Caspase3等表达，减少神经元凋亡；同时上调缺血区脑组织突触调节蛋白SYN与GAP-43表达，减少MDA含量，提升脑组织SOD活力，减少氧化应激损伤，保护脑缺血再灌注损伤小鼠神经功能。研究证实芎芪合剂调控NMDA受体介导Ca2+内流损伤途径防治脑缺血再灌注损伤的作用机制，并为其用于急性脑梗死临床治疗提供研究依据，在急性脑梗死的中医特色治疗方面具有重要临床意义。