Xue Rong-Liang, He Jia-Xuan, Wang Ning, Yao Feng-Zhen, Lv Jian-Rui, Wu Gang
Department of Anesthesiology, Second Affiliated Hospital, Xioan Jiaotong University, Xioan 710004, China.
Neurosci Bull. 2009 Jun;25(3):115-21. doi: 10.1007/s12264-009-8818-9.
To investigate the protein levels of phospho-ERK and phospho-APE/Ref-1 in hippocampal neurons after global cerebral ischemia reperfusion in rats, and observe the relationship between transmembrane signal transduction and repair of DNA damage. The role of ERK signal transduction pathway following global cerebral ischemia reperfusion in rats is further discussed.
Ninety healthy male SD rats were divided into 3 groups randomly: Sham group (S group), Ischemia reperfusion group (IR group) and Pd98059 pretreatment/ischemia reperfusion group (PD group). Global cerebral ischemia reperfusion model was established by four-vessel occlusion (4-VO) method, and reperfusion was performed 5 minutes following ischemia. Protein levels of phospho-ERK and phospho-APE/Ref-1 were detected using immunohistochemical method at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after reperfusion, and neuron apoptosis was observed by HE and TUNEL staining.
In CA1 region of IR group, TUNEL positive cells began to appear at 6 h after IR, and reached the apex during 24 h to 48 h. However, TUNEL positive was most strongly exhibited in PD group. In IR group, phospho-ERK was obviously detected in CA3 region at 2 h after IR, and its level was gradually decreased from 6 h until totally absent at 48 h. Besides, phospho-ERK expression in PD group was weaker than that in IR group. For phospho-APE/Ref-1, its expression began to appear in CA1 region in IR group at 2 h after IR, with no obvious changes during 2 h to 12 h. Phospho-APE/Ref-1 expression began to decrease at 24 h and this decrease continued thereafter. Expression level of phospho-APE/Ref-1 in PD group was lower than that in IR group. Results showed the concurrence of decreased phospho-ERK expression level and increased neuron apoptosis after cerebral ischemia reperfusion, the former of which was consistent with the decrease of phospho-APE/Ref-1 expression. Also, the greater the inhibition of ERK phosphorylation was, the greater decrease of APE/Ref-1 expression occurred.
Activation of ERK signal transduction pathway increased the expression of phospho-APE/Ref-1, and thus faciliated the repair of DNA damage. So, activation of ERK signal transduction pathway may protect neurons from apoptosis after cerebral ischemia reperfusion.
探讨大鼠全脑缺血再灌注后海马神经元中磷酸化细胞外信号调节激酶（phospho-ERK）和磷酸化脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子1（phospho-APE/Ref-1）的蛋白水平，观察跨膜信号转导与DNA损伤修复之间的关系。进一步探讨大鼠全脑缺血再灌注后ERK信号转导通路的作用。
将90只健康雄性SD大鼠随机分为3组：假手术组（S组）、缺血再灌注组（IR组）和PD98059预处理/缺血再灌注组（PD组）。采用四血管闭塞（4-VO）法建立全脑缺血再灌注模型，缺血5分钟后进行再灌注。再灌注后2小时、6小时、12小时、24小时、48小时和72小时，采用免疫组织化学方法检测磷酸化ERK和磷酸化APE/Ref-1的蛋白水平，并用苏木精-伊红（HE）和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色观察神经元凋亡情况。
IR组海马CA1区TUNEL阳性细胞在再灌注后6小时开始出现，24小时至48小时达到高峰。然而，PD组TUNEL阳性表现最为明显。IR组再灌注后2小时在CA3区可明显检测到磷酸化ERK，其水平从6小时开始逐渐下降，至48小时完全消失。此外，PD组磷酸化ERK的表达低于IR组。对于磷酸化APE/Ref-1，IR组再灌注后2小时在CA1区开始出现表达，2小时至12小时无明显变化。磷酸化APE/Ref-1的表达在24小时开始下降，并持续下降。PD组磷酸化APE/Ref-1的表达水平低于IR组。结果表明，脑缺血再灌注后磷酸化ERK表达水平降低与神经元凋亡增加同时发生，前者与磷酸化APE/Ref-1表达的降低一致。而且，ERK磷酸化的抑制越强，APE/Ref-1表达的下降就越大。
ERK信号转导通路的激活增加了磷酸化APE/Ref-1的表达，从而促进了DNA损伤的修复。因此，ERK信号转导通路的激活可能在脑缺血再灌注后保护神经元免于凋亡。
