| Adequate oxygen delivery is essential for neural tissue to generate its normal functions. Neuronal injury can be observed under hypoxic conditions, and hypoxia is correlated with the pathogenesis of many neural diseases. Recently, most studies in pharmacological and neural scientific areas were highly interested in neural protector.Hypoxia inducible factor 1(HIF1) is a nuclear protein with the function of transcriptional regulation. HIF1 plays an important role in increasing tissue resistance to hypoxic stress via transcriptional up-regulation of its downstream genes. HIF1 consists of two subunits ?HIFla and HIFl. HIFla plays an important role in the biological activity of HIF1, and the expression of HIFla was strictly regulated by O2 concentration. Our previous study found that HIF1 was to some extent correlated with chemically hypoxic/reoxygenational injury in PC12 cells, and HIFla antisense oligonucleotide could inhibit expression of HIFla protein. In this research, we studied the relationship between HIF1 protein expression and oxygen free radicals in chemically hypoxic PC12 cells. We further studied the effect of hypoxia/reoxygenation and HIFla antisense oligonucleotide on HIFla protein expression and cell injury in cultured cortical neurons to infer the relationship between HIF1 and hypoxic neuronal injury.Subcultured PC 12 were seeded in 24 well dishes. Chemical hypoxia was made by supplement of cobalt chloride to DMEM with the final concentration of 125 molL. PC12 cells were incubated in the medium for different time periods(0, 1, 2, 4, 8, 12, 16 h).Reoxygenation was performed by substitution of medium containing cobalt c hloride w ith fresh D MEM, and c ells w ere further i ncubated for different time periods(0, 4, 8, 12 h). Cells were lysised and SOD activity and MDA content were assayed. After hypoxia, SOD activity was decreased continuously and the declining process was terminated after 8 h of hypoxia. MDA content was increased gradually and peaked at 8 h of hypoxia. In the latter 8 hours, MDA content was maintained at a constant level. At every time point after reoxygenation, SOD activity were lower than those at the correspondent time point of hypoxia group. MDA content in the reoxygenation group were much higher than those in the hypoxia group. In addition, we treated PC12 cells with HIF1 a antisense oligonucleotide to further study the effect of HIF1 inhibition on the generation of oxygen free radicals in chemically hypoxic PC12 cells. PC12 cells were divided into four experimental groups(antisense, sense, control and blank). Cells were coincubated with HIF1 a antisense and sense oligonucleotide(5 molL) respectively. Chemical hypoxia was made for 8 hours. SOD activity and MDA content in each experimental group were assayed. The results implicated that SOD activity in antisense group was much lower than that in the control, whereas MDA content was obviously higher in comparison with the control.We cultured cortical neurons of neonatal SD rats to verify the neural protective effect of HIF1. Hypoxia was made upon exposure of cells to N2 for different time periods(0, 4, 8, 12, 16 h). Reoxygenation was performed by incubating cells in normal atmosphere (95% air and 5% CO2) after 4 h of hypoxia for different time periods(0, 4, 8, 12 h). Immunocytochemical fluorescence was used to assay HIF1 a protein expression in neurons after hypoxic and reoxygenational treatment. M oreover, L DH 1 eakage, S OD a ctivity a nd M DA c ontentin each group were assayed. We found that HIFl a was accumulated rapidly in hypoxic neurons and peaked at 4 h of hypoxia. Meanwhile, increasing of LDH leakage and MDA content and reduction of SOD activity could be observed. In comparison with hypoxic group, expression of HIFl a decreased rapidly after reoxygenation. LDH leakage and MDA content elevated obviously, and SOD activity was much lower.we treated neurons with HIFl a antisense oligonucleotide, and experimental cells were divided into four groups(antisense, sense, control and blank). Neurons were coincubated with HI... |
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