阻断TRPV4受体对脑缺血再灌注损伤的作用及其分子机制

Intracellular calcium overload and brain edema are important mechanisms underlying the cerebral ischemia-reperfusion injury. As its permeability to calcium ion, activation of TRPV4 receptor by the cell swelling etc. during the acute cerebral stoke, can lead to the increase in intracellular free calcium concentration ([Ca2+]i). Activation of TRPV4 receptor mediates astrocytic cell death, leading to damage of blood-brain barrier. Our previous study found that activation of TRPV4 receptor enhanced NMDA-induced current and could induce hippocampal pyramidal neuron death. Based on our previous result that intracerebroventricular injection of TRPV4 antagonist markedly decreased the volumes of cerebral infarction after 24h reperfusion in middle cerebral artery occlusion model mice, the present project will explore the molecular mechanisms underlying the neuroprotection of blocking TRPV4 receptor on ischemia-reperfusion injury. We will study, firstly, the key time window of the neuroprotection of blocking TRPV4 receptor on cerebral ischemia-reperfusion injury; secondly, whether blocking TRPV4 receptor exerts the neuroprotection through multiple mechanisms including (1) decreasing [Ca2+]i to inhibit the production of free radicals and nitric oxide or to downregulate p38MAPK signaling pathway, (2) reducing the permeability of the blood-brain barrier to prevent the vasogenic brain edema, (3) inhibiting glutamate excitotoxicity. The results are expected to provide the theoretical and experimental basis for new strategy of clinical treatment of cerebral ischemia injury.

胞内钙离子（[Ca2+]i）超载和脑水肿是导致和加重脑缺血再灌注神经元死亡的重要因素。对Ca2+具有通透性的TRPV4受体可被急性脑卒中时细胞水肿等因素激活，使[Ca2+]i升高。TRPV4受体激活后能损伤星形胶质细胞，破坏血脑屏障。申请者前期研究发现激活TRPV4受体能增强NMDA电流，诱导海马神经元死亡。基于TRPV4受体阻断剂能减少缺血再灌注后脑梗死体积的预实验结果，本课题将探讨阻断TRPV4受体保护脑缺血再灌注损伤的分子机制。本课题首先确定阻断TRPV4受体保护脑缺血再灌注损伤的时间窗；并阐明阻断TRPV4受体是否通过（1）抑制[Ca2+]i升高，进而减少自由基和一氧化氮的生成或下调p38MAPK信号通路；（2）降低血脑屏障的通透性，阻止脑水肿的发生；（3）降低谷氨酸的兴奋毒作用等多靶点的调控机制对脑缺血再灌注损伤起保护作用，为开辟缺血性脑损伤临床治疗的新途径提供理论和实验依据。

瞬时感受器电位香草酸受体4（TRPV4R）可被急性脑卒中时细胞水肿等因素激活，导致[Ca2+]i升高。本课题主要研究阻断TRPV4受体对脑缺血再灌注损伤的作用及其分子机制。研究结果如下：（1）大脑中动脉栓塞（MCAO）小鼠再灌注4h至48h TRPV4R蛋白水平升高；侧脑室注射（icv.）TRPV4R特异性阻断剂HC-067047能减小MCAO再灌注24h至48h 脑梗死体积；TRPV4R激动剂GSK1016790A（icv.）能剂量依赖性地导致海马神经元死亡，表明过度激活TRPV4R具有神经毒性作用，阻断该受体对脑缺血再灌注损伤具有保护作用。（2）GSK1016790A能导致海马CA1区神经元凋亡， HC-067047能抑制MCAO再灌注48h海马CA1区神经元凋亡并能阻断MCAO再灌注48h海马组织p-Akt蛋白水平降低， p-p38MAPK和caspase-3剪切体蛋白水平增加，Bcl-2/Bax蛋白比值降低；GSK1016790A能模拟MCAO时上述蛋白的改变；激活PI3K或 阻断p38 MAPK能抑制GSK101690A所致的海马神经元凋亡及对凋亡相关蛋白的调节作用。（3）GSK1016790A能增加海马组织丙二醛和一氧化氮的水平，抑制过氧化氢酶和谷胱甘肽过氧化物酶的活性，增强神经元型一氧化氮合酶（nNOS）的蛋白水平和活性；给予氧化物清除剂Trolox和nNOS抑制剂能阻断GSK1016790A所致的海马神经元损伤。（4）HC-067047能降低MCAO再灌注48h脑组织的含水量和伊文思蓝的溢出率，并阻断MCAO再灌注48h海马基质金属蛋白酶-9（MMP-9）的蛋白水平和活性升高，紧密连接蛋白ZO-1和occludin蛋白水平下降，GSK1016790A能模拟MCAO小鼠的上述改变；给予MMP-9阻断剂能抑制GSK1016790A降低紧密连接蛋白表达的作用。（5）GSK1016790A能增强海马CA1区锥体神经元NMDA介导的电流，阻断NR2B亚基或CaMKII可阻断此作用；GSK1016790A能增强海马脑片兴奋性突触后电位、降低双脉冲易化；GSK1016790A能增强AMPA介导的电流，阻断PKA或PKC可以有效阻断此作用。因此，阻断TRPV4R能通过多靶点的调控机制对脑缺血再灌注损伤起保护作用，为临床治疗缺血性脑损伤提供了新的方向和治疗靶点。