Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin.
OBJECTIVE
Hypoxic preconditioning is an endogenous protection against subsequent lethal hypoxia, but the mechanism involved is not understood. Hypoxia is followed by reactive oxygen species (ROS) production and induces hypoxia-inducible factor (HIF) and its downstream factor erythropoietin (Epo), which is associated with neuroprotection. We hypothesized that these endogenous processes may contribute to hypoxic preconditioning.


METHODS
We used a mouse neuronal culture model, with 2 hours of hypoxia as preconditioning followed by 15 hours of hypoxic insult, and examined the expression of HIF-1alpha, Epo, and their downstream proteins by Western blotting. Copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) mice were used to detect the effect of ROS. Cell survival and apoptosis were detected by mitogen-activated protein 2 quantification, apoptotic-related DNA fragmentation, and caspase-3 fragmentation. Antisense Epo was used to block endogenously produced Epo.


RESULTS
Hypoxic preconditioning was protective in wild-type (Wt) neurons but not in neurons obtained from SOD1 Tg mice. In Wt neurons, HIF-1alpha and Epo expression showed a greater increase after hypoxia compared with Tg neurons and reached a higher level with preconditioned hypoxia, followed by pJak2, pStat5, and nuclear factor kappaB (NF-kappaB) expression. Antisense Epo decreased these downstream proteins and the neuroprotection of hypoxic preconditioning.


CONCLUSIONS
Hypoxic preconditioning induces ROS, which may downregulate the threshold for production of HIF-1alpha and Epo expression during subsequent lethal hypoxia, thus exerting neuroprotection through the Jak2-Stat5 and NF-kappaB pathways.
Liu
Jing
J
Department of Neurosurgery, Stanford University School of Medicine, Stanford, California 94305-5487, USA.
Narasimhan
Purnima
P
Yu
Fengshan
F
Chan
Pak H
PH
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004921:Erythropoietin / Q000494:pharmacology*; D006579:Heterozygote; D000860:Hypoxia / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism; D019194:Ischemic Preconditioning; D053614:Janus Kinase 2; D051379:Mice; D008822:Mice, Transgenic; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018384:Oxidative Stress; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D051793:Hypoxia-Inducible Factor 1; D016328:NF-kappa B; D018696:Neuroprotective Agents; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; D004921:Erythropoietin; D013482:Superoxide Dismutase; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases