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

缺血/再灌注后的氧化应激已被证明可诱导DNA损伤和随后的DNA修复活性。DNA修复酶无尿嘧啶/无嘧啶内切酶（或称氧化还原效应因子-1，APE/Ref-1）参与DNA氧化损伤后无尿嘧啶/无嘧啶位点的碱基切除修复。我们研究了该蛋白在缺血预处理后神经元对全脑缺血耐受的发展中的作用。成年雄性Sprague-Dawley大鼠进行5分钟的致死性全身缺血，同时进行或不进行3分钟的亚致死性缺血预处理，或者只进行3分钟的缺血。对神经元损伤进行组织学评估，并通过DNA片段原位标记和DNA凝胶电泳观察DNA损伤。免疫组织化学和Western blot检测APE的表达。海马CA1神经元在缺血5分钟后3天发生dna片段化细胞死亡。然而，这些神经元在缺血预处理后1至3天对缺血5分钟表现出很强的耐受性。免疫组化显示CA1神经元中几乎没有APE蛋白的组成性表达；然而，缺血预处理在1 ~ 3天后诱导神经元APE表达。Western blot同时证实该区域的ku70表达增加。APE的时空表达与海马CA1神经元对缺血的耐受性相对应，提示APE蛋白参与了缺血预处理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.