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目的研究预缺血对蛋白伴侣hsp70表达和蛋白聚集物形成的影响,探讨其可能的脑保护机制。方法采用大鼠双侧颈总动脉暂时夹闭法建立全脑缺血模型。大鼠分为3min缺血组,10min缺血组以及预缺血组。苏木素-伊红染色,光镜下随机计数分析预缺血后海马CA1区死亡神经元数量变化。免疫组织化学及激光扫描共聚焦显微镜法观察蛋白伴侣hsp70在CA1区神经元内的分布。差速离心分离细胞浆、细胞核及蛋白聚集物。蛋白印迹法检测不同缺血状态下海马CA1神经元内蛋白聚集物含量的变化,以及胞浆、胞核及蛋白聚集物内蛋白伴侣hsp70含量的变化。结果组织学检查显示预缺血能够显著减少海马CA1区神经元死亡数量。预缺血诱导海马CA1区神经元内蛋白伴侣hsp70在再灌注后24h表达。预缺血处理后,海马CA1区神经元内蛋白聚集物显著减少。预缺血诱导的蛋白伴侣hsp70与再缺血形成的异常蛋白结合在一起并防止其聚集。结论预缺血可能通过诱导蛋白伴侣hsp70的表达和抑制再缺血后蛋白聚集物的形成,减少再缺血引起的神经元死亡。
Objective To investigate the effect of ischemic preconditioning on the expression of hsp70 and the formation of protein aggregates, and to explore its possible mechanism of brain protection. Methods The model of global cerebral ischemia was established by transient occlusion of bilateral common carotid arteries in rats. Rats were divided into 3min ischemia group, 10min ischemia group and ischemic group. Hematoxylin - eosin staining, random counting under light microscope to analyze the changes of the number of neurons in CA1 area of hippocampus after preconditioning. Immunohistochemistry and laser scanning confocal microscopy were used to observe the distribution of hsp70, a protein chaperone, in CA1 neurons. Differential centrifugation of cytoplasm, nucleus and protein aggregates. Western blotting was used to detect the changes of protein aggregates in hippocampal CA1 neurons under different ischemic conditions and the change of hsp70 protein content in cytoplasm, nucleus and protein aggregates. Results Histological examination showed that ischemic preconditioning can significantly reduce the number of neuronal death in hippocampal CA1 area. Preischemic induction of hsp70 expression in hippocampal CA1 neurons during 24 h after reperfusion. After pre-ischemic treatment, there was a significant reduction of protein aggregates in neurons in CA1 hippocampus. The preischemic-induced chaperone hsp70 binds to and prevents the aggregation of aberrant proteins formed by re-ischemia. Conclusions Preconditioning may reduce neuronal death induced by re-ischemia by inducing the expression of hsp70, a protein chaperone, and inhibiting the formation of protein aggregates after reperfusion.