Potential contribution of NF-kappaB in neuronal cell death in the glutathione peroxidase-1 knockout mouse in response to ischemia-reperfusion injury.
OBJECTIVE
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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Crack
Peter J
PJ
Centre for Functional Genomics and Human Disease, Monash Institute of Medical Research, Monash University, Melbourne, Australia. pcrack@unimelb.au
Taylor
Juliet M
JM
Ali
Ugur
U
Mansell
Ashley
A
Hertzog
Paul J
PJ
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002545:Brain Ischemia / Q000503:physiopathology*; D016923:Cell Death; D002544:Cerebral Infarction / Q000473:pathology; D004247:DNA / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000172:deficiency*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D010766:Phosphorylation; D011759:Pyrrolidines / Q000494:pharmacology; D015427:Reperfusion Injury / Q000503:physiopathology*; D020521:Stroke / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology; D014018:Tissue Distribution; D051996:Transcription Factor RelA / Q000378:metabolism; D015854:Up-Regulation; D000094765:Glutathione Peroxidase GPX1
D000975:Antioxidants; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D018696:Neuroprotective Agents; D011759:Pyrrolidines; D013859:Thiocarbamates; D051996:Transcription Factor RelA; C020972:pyrrolidine dithiocarbamic acid; D004247:DNA; D005979:Glutathione Peroxidase; D000094765:Glutathione Peroxidase GPX1; C000722068:Gpx1 protein, mouse