Role of DNA repair proteins in development of the tolerance after ischemic preconditioning

研究表明，缺血/再灌注后的氧化应激可诱导DNA损伤和随后的DNA修复活动。DNA修复酶，脱嘌呤/脱嘧啶核酸内切酶(或氧化还原效应因子-1，APE/Ref-1)，参与DNA氧化损伤后脱嘌呤/脱嘧啶位点的碱基切除修复。我们研究了该蛋白在缺血预适应后神经元对全脑缺血耐受性的形成中的作用。成年雄性SD大鼠接受5min的致死性全脑缺血，或3min的亚致死性缺血预适应，或仅3min的缺血。组织学观察神经元损伤情况，DNA片段原位标记和DNA凝胶电泳法观察DNA损伤情况。免疫组织化学和Western印迹分析检测APE的表达。脑缺血5min后3d，大鼠海马CA1区神经元DNA片段化死亡。然而，在缺血预适应后1~3d，这些神经元对5min的缺血表现出很强的耐受性。免疫组织化学显示CA1区神经元中几乎没有APE蛋白的组成性表达；然而，缺血预适应在1~3天后诱导神经元APE蛋白表达。Western印迹证实该区域的Ku-70在同一时间增加。APE在时间和空间上的表达与海马CA1区神经元对随后缺血的耐受性相对应，提示APE蛋白参与了缺血预适应后神经元耐受性的形成。

Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. The DNA repair enzyme, apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1), is involved in base excision repair of apurinic/apyrimidinic sites after oxidative DNA damage. We investigated the involvement of this protein in the development of neuronal tolerance to global cerebral ischemia after ischemic preconditioning. Adult male Sprague-Dawley rats were subjected to either 5 minutes of lethal global ischemia with or without 3 minutes of sublethal ischemic preconditioning or 3 minutes of ischemia only. Neuronal injury was histologically assessed, and DNA damage was visualized by in situ labeling of DNA fragmentation and DNA gel electrophoresis. APE expression was also examined by immunohistochemistry and Western blot analysis. Hippocampal CA1 neurons underwent DNA-fragmented cell death 3 days after 5 minutes of ischemia. However, these neurons showed a strong tolerance to 5 minutes of ischemia 1 to 3 days after ischemic preconditioning. Immunohistochemistry showed virtually no constitutive expression of APE proteins in CA1 neurons ; however, ischemic preconditioning induced neuronal APE expression 1 to 3 days later. Western blot confirmed an increase in Ku 70 in this region at the same time. The temporal and spatial expression of APE corresponded to tolerance of the hippocampal CA1 neurons to subsequent ischemia, suggesting the involvement of APE protein in the development of neuronal tolerance after ischemic preconditioning.