亚低温对脑损伤大鼠海马神经元自噬相关蛋白表达的影响  

Effect of mild hypothermia on expression of autophagy-related proteins in hippocampus after traumatic brain injury in rats

作　　者：张坤[1] 张宏义[1] 王东春[1] 孙立倩[1] 祁大勇[1] 周洪霞[2] 

机构地区：[1]唐山市工人医院神经外科,063000 [2]河北联合大学基础医学院

出　　处：《中华创伤杂志》2016年第2期166-170,共5页Chinese Journal of Trauma

基　　金：唐山市科学技术研究与发展计划（12140209A-31）

摘　　要：目的探讨亚低温治疗对大鼠创伤性脑损伤（TBI）后海马区微管相关蛋白1轻链3（LC3）和Beclin-1表达的影响。方法将60只成年健康雄性sD大鼠按随机数字表法分为假手术组、TBI常温组和亚低温治疗组（31～33℃），每组20只。采用Marmarou法建立大鼠TBI模型，给予常温或亚低温干预4h。各组大鼠于6，12，24及48h断头取脑，免疫荧光双标法检测LC3与神经元特异性核蛋白（NeuN）、LC3与哺乳动物雷帕霉素靶蛋白（P-mTOR）的共定位表达，Westernblot法检测海马区LC3、Beclin-1和磷酸化哺乳动物雷帕霉素靶蛋白（P—mTOR）的蛋白表达。结果（1）激光共聚焦扫描显微镜观测到LC3与NeuN（或P—roTOR）的共定位表达，红色荧光与绿色荧光大部分重合。（2）与假手术组比较，TBI常温组LC3、Beclin-1蛋白表达明显增加（P〈0．05），两者6h开始增加，24h达高峰，48h回落，但仍高于假手术组水平；而P—mTOR蛋白表达量明显减少（P〈0．05），于6h开始减少，12h达最低，24h及48h回升，但仍低于假手术组水平。（3）与TBI常温组比较，亚低温治疗组各时相点LC3、Beclin-1表达显著下调（P〈0．05），同时P-mTOR蛋白表达量上调（P〈0．05）。结论亚低温可通过P—mTOR信号通路抑制TBI后海马区神经元自噬，进而起到一定的脑保护作用。Objective To investigate the effect of mild hypothermia on expression of autophagy- related proteins LC3 and Beclin-1 in the hippocampus of rats with traumatic brain injury （TBI）. Methods Sixty healthy male adult SD rats were divided into sham group, TBI group, hypothermia- treated group （31-33℃） according to the random number, with 20 rats per group. Rat models of TBI were established using the Marmarou＇s method. After 4 h of normothermia or hypothermia, the rats were killed 6, 12, 24, and 48 h later. Co-localization of LC3 with neuron specific protein （NeuN） or mammalian target of rapamycinm （mTOR） was determined by double immunofluorescence. Levels of LC3, Beclin-1 and p-mTOR proteins were detected by Western blot analysis. Results LC3 co-localization with NeuN （or mTOR） was observed under confocal laser scanning microscope. In TBI group LC3 and Beclin-1 levels were seen to rise at 6 h, peaked at 24 h and fell slightly at 48 h, but the levels were still higher than these in sham group （ P 〈 0.05 ）. In TBI group p-mTOR level began to decrease at 6 h, reached the lowest point at 12 h, and then ascended at 24 and 48 h, but the level was still lower than that in sham group （P 〈 0.05）. Mild hypothermia contributed to remarkable decreases in LC3 and Beclin-1 expressions, while up-regulating p-mTOR after injury （ P 〈 0.05 ）. Conclusion Mild hypothermia appears to be neuroprotective as it inhibits autophagy in hippocampal neurons after TBI in rats through activation of roTOR signaling pathway.

关 键 词：脑损伤 亚低温 自噬 海马 

分 类 号：R651.15[医药卫生—外科学]