Crack Peter J, Taylor Juliet M, Ali Ugur, Mansell Ashley, Hertzog Paul J
Centre for Functional Genomics and Human Disease, Monash Institute of Medical Research, Monash University, Melbourne, Australia.
Stroke. 2006 Jun;37(6):1533-8. doi: 10.1161/01.STR.0000221708.17159.64. Epub 2006 Apr 20.
We have previously identified an increased susceptibility of Gpx1(-/-) mice to increased infarct size after middle cerebral artery occlusion (MCAO). This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state.
Gpx1(-/-) mice were exposed to transient MCAO and reperfusion by intraluminal suture blockade. Protein expression of the p65 subunit of transcription factor nuclear factor-kappaB (NF-kappaB) was examined by immunohistochemistry and Western Analysis. NF-kappaB DNA-protein activity was assessed by electrophoretic mobility shift assays (EMSA). Wild-type and Gpx1(-/-) mice were exposed to MCAO with or without the NF-kappaB inhibitor, pyrrolidinedithiocarbamate (PDTC).
Upregulation of the p65 subunit of NF-kappaB and subsequent p65 phosphorylation at serine 536 was detected in the Gpx1(-/-) brains after stroke. EMSAs revealed that increased ischemia-enhanced DNA binding of NF-kappaB was observed in Gpx1(-/-) mice compared with wild-type. Supershift assays indicated that the p50 and p65 subunits participated in the bound NF-kappaB complex. The NF-kappaB inhibitor PDTC, a potential antioxidant, was able to afford partial neuroprotection in the Gpx1(-/-) mice.
NF-kappaB is upregulated in the Gpx1(-/-) mouse, and this upregulation contributes to the increased cell death seen in the Gpx1(-/-) after MCAO. The activation of NF-kappaB may increase the expression of downstream target genes that are involved in the progression of neural injury after MCAO.
我们之前已经发现，谷胱甘肽过氧化物酶1基因敲除（Gpx1(-/-)）小鼠在大脑中动脉闭塞（MCAO）后梗死面积增加，易感性增强。本研究旨在阐明内源性氧化状态改变导致神经元细胞死亡增加所涉及的机制。
通过腔内缝线阻塞法使Gpx1(-/-)小鼠暴露于短暂性MCAO及再灌注过程中。采用免疫组织化学和蛋白质印迹分析检测转录因子核因子-κB（NF-κB）的p65亚基的蛋白表达。通过电泳迁移率变动分析（EMSA）评估NF-κB的DNA-蛋白质活性。野生型和Gpx1(-/-)小鼠在有或无NF-κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）的情况下接受MCAO。
中风后在Gpx1(-/-)小鼠脑内检测到NF-κB的p65亚基上调以及随后丝氨酸536位点的p65磷酸化。EMSA显示，与野生型相比，Gpx1(-/-)小鼠中缺血增强的NF-κB的DNA结合增加。超迁移分析表明p50和p65亚基参与了结合的NF-κB复合物。NF-κB抑制剂PDTC作为一种潜在的抗氧化剂，能够为Gpx1(-/-)小鼠提供部分神经保护作用。
在Gpx1(-/-)小鼠中NF-κB上调，这种上调导致了MCAO后Gpx1(-/-)小鼠中细胞死亡增加。NF-κB的激活可能会增加下游靶基因的表达，这些基因参与了MCAO后神经损伤的进展。
