[Relationship between hypoxia-inducible factor-1alpha expression and extracellular signal-regulated kinase in hypoxic-ischemic cortical neurons].
OBJECTIVE
To explore the change tendency of hypoxia-inducible factor-1alpha (HIF-1alpha) and extracellular signal-regulated kinase 1/2 (ERK1/2) in fetal rat cerebral cortex neurons cultured in vitro after hypoxia-ischemia reperfusion and to investigate their mutual relationship.


METHODS
Cortical neurons obtained from cerebral cortex of 15 pregnant SD rats at 16-18 days of gestation underwent primary culture. The primary neurons 5 days after culture were adopted to establish model of oxygen and glucose deprivation (OGD). The experiment was divided into 4 groups: the experimental group 1, culture medium was changed to neuron complete medium containing glucose after the preparation of OGD model to form reperfusion, and the neurons were observed 0, 2, 4, 8, 12 and 24 hours after reperfusion; the control group 1, the neurons were treated with normal medium; the experimental group 2, the neurons were pretreated with U0126 followed by the preparation of OGD model, and the neurons were observed 4 and 8 hours after reperfusion; the control group 2, the neurons were pretreated with DMSO, and other treatments were the same as the experimental group 2. Expressions of HIF-1alpha, VEGF protein, ERK1/2 and p-ERK1/2 were detected by Western blot. Expression and distribution of p-ERK1/2 and HIF-1alpha protein were detected by SABC immunocytochemistry method.


RESULTS
Complicated synaptic connections between cortical neurons processes were observed 5 days after culture. The expression of HIF-1alpha and VEGF were increased gradually, peaked at 8 hours, and decreased gradually after 12 hours in the experimental group 1, and there were significant differences between the experimental group 1 and the control group 1 (P < 0.05). There was no significant difference between the experimental group 1 and the control group 1 in terms of ERK1/2 protein expression (P > 0.05). The p-ERK1/2 protein expression in the experimental group 1 started to increase at 2 hours, peaked at 4 hours, and started to decrease at 8 hours, showing significant differences compared with the control group 1 (P < 0.01). In the experimental group 2, the p-ERK1/2 protein decreased, and HIF-1 and VEGF protein expression subsequently decreased, showing significant differences compared with the control group 2 (P < 0.05). There was no significant difference between the experimental group 2 and the control group 2 in terms of ERK1/2 protein expression at each time point (P > 0.05). Immunocytochemistry staining showed that p-ERK1/2 and HIF-1alpha expression decreased, and the yellow-brown staining of the neurons was reduced.


CONCLUSIONS
Expressions of HIF-1alpha and its target-gene VEGF protein in the cortex neurons after OGD reperfusion are time-dependent. Their expressions decrease when ERK1/2 signaling pathway is inhibited, indicating the pathway plays an important role in the regulation of HIF-1alpha and VEGF induced by OGD of cortical neurons.
Zhang
Li
L
Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu Sichuan 610041, PR China.
Qu
Yi
Y
Zhang
Xiaolan
X
Yang
Chunlei
C
Mao
Meng
M
Mu
Dezhi
D
D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005260:Female; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases