Knoblach Susan M, Alroy Daniel A, Nikolaeva Maria, Cernak Ibolja, Stoica Bogdan A, Faden Alan I
Department of Neuroscience, Georgetown University Medical Center, Washington, DC 20057, USA.
J Cereb Blood Flow Metab. 2004 Oct;24(10):1119-32. doi: 10.1097/01.WCB.0000138664.17682.32.
In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 microM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated alpha-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated alpha-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated alpha-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.
在旨在评估创伤性脑损伤治疗窗的研究中，半胱天冬酶3抑制剂z-DEVD-fmk在损伤后1小时给药时可改善神经功能并减小损伤体积，但在损伤后4、8或24小时给药则无此效果。此外，在损伤后1至72小时，未治疗的损伤皮质中半胱天冬酶3及其底物聚（ADP-核糖）聚合酶（PARP）均未被切割。损伤后6至24小时，很少有皮质神经元表达活性半胱天冬酶3或TUNEL阳性，且TUNEL染色主要为I型（坏死型）。尼氏染色显示，撞击后6至24小时，损伤皮质中有广泛的神经元坏死。综合这些数据表明，z-DEVD-fmk可能减少神经元坏死，因此我们使用了由 maitotoxin诱导的坏死性细胞死亡的体外模型来进一步测试这一点，并探索其中潜在的机制。Z-DEVD-fmk（1 nM至100 microM）显著减轻了maitotoxin诱导的神经元细胞死亡，并显著降低了maitotoxin损伤后145 kD钙蛋白酶介导的α-血影蛋白降解产物的表达。maitotoxin损伤后，既未表达120 kD半胱天冬酶介导的α-血影蛋白切割产物，也未表达组织蛋白酶B。在无细胞试验中，z-DEVD-fmk减少了纯化的钙蛋白酶I对酪蛋白的水解。最后，z-DEVD-fmk降低了体内创伤性脑损伤后145 kD钙蛋白酶介导的α-血影蛋白切割片段的表达。这些数据表明，z-DEVD-fmk提供的神经保护作用可能部分反映了对钙蛋白酶相关坏死性细胞死亡的抑制。
