Yi Jung-Sun, Kim Yang-Hee, Koh Jae-Young
National Creative Research Initiative Center for the Study of CNS Zinc, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
Exp Neurol. 2004 Oct;189(2):354-60. doi: 10.1016/j.expneurol.2004.05.032.
In addition to its thrombolytic effect, human recombinant tissue plasminogen activator (tPA) may have parenchymal effects such as protease-dependent neurotoxic and protease-independent neuroprotective effects. The purpose of this study was to examine parenchymal effects of tPA and its non-protease mutant S478A-tPA in permanent focal cerebral ischemia in rats. However, before doing in vivo experiments, effects of tPA and S478A-tPA on zinc or NMDA toxicity were first studied in cortical cultures. Like tPA, which has protease-independent cytoprotective effects, the non-protease mutant S478A-tPA blocked zinc toxicity in cortical cell cultures, but did not affect calcium-mediated NMDA toxicity. Then, effects of tPA and S478A-tPA on infarcts induced by permanent occlusion of middle cerebral artery (MCA) were investigated. tPA and S478A-tPA were administered into the cerebral ventricle 15 min or 1 h after MCA occlusion. Both tPA and its non-protease mutant S478A-tPA, when given 15 min after ischemia, substantially reduced infarcts and ameliorated motor deficits in the MCA occlusion model of focal cerebral ischemia. However, when administered 1 h after MCA occlusion, neither showed protective effects. The protective effects of tPA or S478A-tPA remained unchanged at 7 days after MCA occlusion. Indicating that the native protein conformation is necessary for the protective effect of tPA and S478A-tPA, heat-denatured tPA did not exhibit any protective effect. Since S478A-tPA lacks protease activity, which has been implicated in causing cerebral hemorrhage or aggravating excitotoxicity, its parenchymal neuroprotective effect may be useful in treatment of ischemic stroke.
除了具有溶栓作用外，重组人组织型纤溶酶原激活剂（tPA）可能还具有实质效应，如蛋白酶依赖性神经毒性和蛋白酶非依赖性神经保护作用。本研究的目的是检测tPA及其非蛋白酶突变体S478A-tPA在大鼠永久性局灶性脑缺血中的实质效应。然而，在进行体内实验之前，首先在皮质培养物中研究了tPA和S478A-tPA对锌或N-甲基-D-天冬氨酸（NMDA）毒性的影响。与具有蛋白酶非依赖性细胞保护作用的tPA一样，非蛋白酶突变体S478A-tPA在皮质细胞培养物中可阻断锌毒性，但不影响钙介导的NMDA毒性。然后，研究了tPA和S478A-tPA对大脑中动脉（MCA）永久性闭塞所致梗死的影响。在MCA闭塞后15分钟或1小时将tPA和S478A-tPA注入脑室。在局灶性脑缺血的MCA闭塞模型中，当在缺血后15分钟给予tPA及其非蛋白酶突变体S478A-tPA时，均可显著减少梗死灶并改善运动功能缺损。然而，在MCA闭塞后1小时给予时，两者均未显示出保护作用。在MCA闭塞7天后，tPA或S478A-tPA的保护作用保持不变。这表明天然蛋白质构象对于tPA和S478A-tPA的保护作用是必需的，热变性tPA未表现出任何保护作用。由于S478A-tPA缺乏蛋白酶活性，而蛋白酶活性被认为与脑出血或加重兴奋性毒性有关，其在实质内的神经保护作用可能对缺血性中风的治疗有用。
