Huang Feng-Peng, Wang Zhi-Qiu, Wu Du-Chu, Schielke Gerald P, Sun Yi, Yang Guo-Yuan
Department of Surgery (Neurosurgery), University of Michigan, Ann Arbor, USA.
J Neurosci Res. 2003 Sep 1;73(5):698-707. doi: 10.1002/jnr.10654.
Our previous study demonstrated that the inhibition of interleukin-1beta (IL-1beta) reduces ischemic brain injury; however, the molecular mechanism of the action of IL-1 in cerebral ischemia is unclear. We are investigating currently the role of NFkappaB during focal cerebral ischemia, using mutant mice deficient in the interleukin-1 converting enzyme gene (ICE KO) in a middle cerebral artery occlusion (MCAO) model. Adult male ICE KO and wild-type mice (n = 120) underwent up to 24 hr of permanent MCAO. Cytoplasmic phospho-NFkappaB/p65 expression in ischemic brain was examined using Western blot analysis and immunohistochemistry. NFkappaB DNA-binding activity was detected using electrophoretic mobility shift assay (EMSA). Furthermore, ICAM-1 expression was examined in both the ICE KO and wild-type mice (WT). Western blot analysis and immunostaining showed that the level of cytosolic phosphorylated NFkappaB/p65 increased after 2 and 4 hr of MCAO in WT mice; however, NFkappaB/p65 was significantly reduced after MCAO in the ICE KO mice (P < 0.05). EMSA showed that NFkappaB DNA-binding activity increased after MCAO in WT mice; but this effect was reduced in the ICE KO mice. The number of ICAM-1-positive vessels in the ischemic hemisphere was greatly attenuated in the ICE KO mice (P < 0.05), which paralleled the results of immunohistochemistry. Our results demonstrate that NFkappaB phosphorylation is reduced in ICE KO mice, suggesting that ICE or IL-1 are involved in early NFkappaB phosphorylation. Because cerebral ischemia induced infarction is significantly reduced in ICE KO mice, we conclude that early NFkappaB phosphorylation plays a disruptive role in the ischemic process.
我们之前的研究表明，抑制白细胞介素-1β（IL-1β）可减轻缺血性脑损伤；然而，IL-1在脑缺血中作用的分子机制尚不清楚。我们目前正在使用白细胞介素-1转化酶基因缺陷的突变小鼠（ICE基因敲除小鼠），在大脑中动脉闭塞（MCAO）模型中研究核因子κB（NFκB）在局灶性脑缺血中的作用。成年雄性ICE基因敲除小鼠和野生型小鼠（n = 120）接受长达24小时的永久性MCAO。采用蛋白质免疫印迹分析和免疫组织化学方法检测缺血脑组织中细胞质磷酸化NFκB/p65的表达。采用电泳迁移率变动分析（EMSA）检测NFκB的DNA结合活性。此外，检测了ICE基因敲除小鼠和野生型小鼠（WT）中细胞间黏附分子-1（ICAM-1）的表达。蛋白质免疫印迹分析和免疫染色显示，野生型小鼠在MCAO 2小时和4小时后，细胞质磷酸化NFκB/p65水平升高；然而，ICE基因敲除小鼠在MCAO后NFκB/p65水平显著降低（P < 0.05）。EMSA显示，野生型小鼠在MCAO后NFκB的DNA结合活性增加；但在ICE基因敲除小鼠中这种作用减弱。ICE基因敲除小鼠缺血半球中ICAM-1阳性血管的数量显著减少（P < 0.05），这与免疫组织化学结果一致。我们的结果表明，ICE基因敲除小鼠中NFκB磷酸化水平降低，提示ICE或IL-1参与早期NFκB磷酸化。由于ICE基因敲除小鼠中脑缺血诱导的梗死明显减少，我们得出结论，早期NFκB磷酸化在缺血过程中起破坏作用。
