Erythropoietin downregulates bax and DP5 proapoptotic gene expression in neonatal hypoxic-ischemic brain injury.
BACKGROUND
Perinatal asphyxia is an important cause of neonatal mortality and subsequent serious sequelae such as motor and cognitive deficits and seizures. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. Recent studies also confirm the antiapoptotic effect of Epo in a variety of in vitro and in vivo neuronal injury models including hypoxic-ischemic brain injury. However, molecular mechanisms of Epo protection and antiapoptotic effect in this model are unclear. Epo may exert its antiapoptotic effect via the differential regulation of the expression of genes involved in the apoptotic process.


OBJECTIVE
Thus, in the present study, we studied the effects of systemically administered Epo on antiapoptotic (bcl-2, bcl-XL), proapoptotic (bax and DP5) gene expression following hypoxic-ischemic brain injury in neonatal rats.


METHODS
Seven- day-old Wistar rat pups were divided into three groups: control group (n=15), saline-treated group (n=17), and Epo-treated group (n=18). Rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Epo-treated group received an intraperitoneal injection of recombinant human Epo at a dose of 1,000 units/kg, saline-treated group received an intraperitoneal injection of saline at the same volume of Epo. Forty-eight hours after hypoxia, 3 animals in each group were killed for histopathological evaluation. To detect DNA fragmentation in cell nuclei, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling reaction was applied. Bcl-2 and bax protein expression were also analyzed with immunohistochemistry. For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, rats were sacrificed 4, 12, and 24 h after hypoxia. Bcl-2, bcl-XL, bax, and DP5 mRNA expression were analyzed by RT-PCR.


RESULTS
Epo significantly prevented hypoxia-ischemia-induced bax and DP5 mRNA upregulation in brain tissue. Epo did not show any effect on bcl-XL transcription altered by injury. However, Epo reversed injury-induced downregulation in bcl-2 transcription. Modulating effects of Epo on bcl-2 and bax protein expression were also revealed by immunohistochemistry.


CONCLUSIONS
These results suggest that Epo exerts a neuroprotective effect against hypoxic-ischemic brain injury, at least partially, via the differential regulation of the expression of genes involved in apoptotic process.
Kumral
Abdullah
A
Department of Pediatrics, School of Medicine, Dokuz Eylul University, Inciralti, Izmir, Turkey.
Genc
Sermin
S
Ozer
Erdener
E
Yilmaz
Osman
O
Gokmen
Necati
N
Koroglu
Tolga F
TF
Duman
Nuray
N
Genc
Kursad
K
Ozkan
Hasan
H
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D004921:Erythropoietin / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D007150:Immunohistochemistry; D009479:Neuropeptides / Q000235:genetics*; D018696:Neuroprotective Agents; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein / Q000235:genetics*
D051017:Apoptosis Regulatory Proteins; C484585:Hrk protein, rat; D009479:Neuropeptides; D018696:Neuroprotective Agents; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D004921:Erythropoietin