低温对新生大鼠缺氧缺血后脑组织硝基酪氨酸形成及细胞骨架蛋白降解影响

Effects of hypothermia on nitrotyrosine formation and cytoskeleton protein degradation in post hypoxia- ischemia neonatal rats brain

目的 探讨低温对新生大鼠脑缺氧缺血(HI)后硝基酪氨酸形成及细胞骨架蛋白(微管相关蛋白-2和胞衬蛋白)降解影响。方法 结扎7日龄Wistar大鼠左侧颈总动脉后吸入7.8％氧气60 min制成HI模型,随机分成低温组(30℃,n=18)和常温组(36℃,n=18),均予维持恒定目标温度10 h。每组8只,在HI后24 h处死,脑组织匀浆用于Western蛋白印迹检查,余每组10只在HI后72 h处死,进行硝基酪氨酸免疫组织化学染色。结果 正常脑组织有少量硝基酪氨酸形成,在HI后24 h硝基酪氨酸生成明显增加,低温干预后硝基酪氨酸生成减少,HI后72 h皮层及海马齿状回的硝基酪氨酸的阳性细胞数低温组明显低于常温组(P<0.02,P<0.01)。微管相关蛋白-2的相对分子质量820、70条带在低温组降解明显低于常温组,胞衬蛋白在HI后降解为相对分子质量150、120两条带,低温组胞衬蛋白降解明显减少。结论 低温能抑制硝基酪氨酸生成和细胞骨架蛋白的降解,对缺氧缺血性脑损伤的保护作用可能与抑制一氧化氮合酶、钙离子蛋白酶和半胱天冬酶的活性有关。

Objective To study the effects of hypothermia on nitrotyrosine formation and degradation of cytoskeleton protein, mi-crotubule associated protein 2(MAP-2) and fodrin in cerebral hypoxia-ischemia neonatal rats. Methods Seven - day - old Wistar rats were subjected to left carotid artery ligation and hypoxia for 60 min. Immediately, at the end of hypoxia, animals were maintained either at 36 ℃ or 30 ℃ for 10 hours at random. Nitrotyrosine formation and degradation of MAP-2 and fodrin were measured at 24 hours post-HI (n = 8 for each group) by Western blots. Immunoreactivity of nitrotyrosine was detected in cortex and dentate gyrus at 72 hours post-HI by immnohistochemistry. Results Nitrotyrosine formation could be detected in normal brain and large amounts of nitrotyrosine was produced at 24 hours post-HI. Hypothermia inhibited nitortyrosine formation and reduced the number of nitrotyrosine positive cells in cortex and dentate gyrus of hippocampus(P<0.02, P<0.01). The degradation of MAP-2 and fodrin was inhibited by hypothermia. Conclusions Hypothermiacan reduces nitrotyrosine formation and cytoskeleton degradation. It indicates that hypothermia plays a protective role on brain injury probably by inhibiting the activation of nitric oxide synthase, calpain and caspase.