Shen Wanhua, Zhang Chunyi, Zhang Guangyi
Research Center of Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jinagsu 221002, China.
Brain Res. 2002 Apr 12;933(1):23-30. doi: 10.1016/s0006-8993(02)02291-6.
Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.
最近的研究表明，核因子NF-κB可能参与兴奋性毒素诱导的细胞凋亡。为了分析NF-κB的变化，在大鼠接受30分钟四血管闭塞并在选定的再灌注时间点处死之后，检测NF-κB的水平。分别用抗p65、p50抗体通过蛋白质印迹法检测主要由p65和p50亚基组成的NF-κB的诱导情况。通过电泳迁移率变动分析检测NF-κB的DNA结合活性。我们的研究表明，缺血诱导的NF-κB核转位具有时间依赖性。与对照组相比，核内NF-κB的诱导或结合活性在6至12小时内增加了约10倍，然后在接下来的24、72小时逐渐下降。为了进一步分析体内离子型谷氨酸受体和L型电压门控钙通道（L-VGCC）的调节作用，在缺血30分钟前20分钟给予N-甲基-D-天冬氨酸（NMDA）受体拮抗剂氯胺酮、α-氨基-3-羟基-5-甲基-4-异恶唑丙酸/海人藻酸受体拮抗剂6,7-二硝基喹喔啉-2,3（1H，4H）-二酮和L-VGCC拮抗剂硝苯地平。缺血/再灌注6小时后，这三种拮抗剂以剂量依赖性方式完全阻断了NF-κB核转位。通过蛋白质印迹法检测到NF-κB调节单位IκB-α的磷酸化增加。全脑缺血后再灌注3小时，在细胞质中发现IκB-α减少，这也被这三种拮抗剂阻断。这些结果说明，谷氨酸门控离子型NMDA或非NMDA受体以及电压门控钙通道是脑缺血损伤期间介导NF-κB激活的重要途径。活跃的NF-κB可能继而参与兴奋性毒素诱导的细胞死亡。我们的研究还表明，IκB-α是一个重要的调节单位，在全脑缺血后大鼠海马体中，它在磷酸化、降解和再合成后控制NF-κB的激活。
