Protective effect of necrostain-1 on oxygen-glucose deprivation induced caspase-independent cell death in primary cortical neurons

Objective To explore whether hypoxia-inducible factor-1α( HIF-1α )is involved in oxygenglucose deprivation (OGD)induced caspase-independent cell death in primary cortical neurons and whether their expression is infected by necrostatin-1 ( Nec-1 ).Methods ( 1 ) Primary cerebrocortical neurons were cultured for 14 days.Pretred z-VAD.Fmk (z-VAD)and Nec-1 with 0.1,1,5,10,25 and 50 μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours,then cell viability was determined by measure LDH level.(2)Pretred z-VAD before the neurons were exposed to OGD for 2 hours,then reoxygenated for 0,2,6,12,24and 48 hours.Then western blot analysis protein level of HIF-1 α ;rt-PCR check its RNA level.(3)Pretred z-VAD and Nec-1 with 25μ mol/L before the neurons were exposed to OGD for 2 hours and reoxygenated for 12 hours.Then western blot analysis protein level of HIF-1 α; rt-PCR check its RNA level.Result ( 1 ) When cells were pretread Nec-1 with 5 μ mol/L(6.97 ± 0.06),the level of LDH was lower than cells untreated( 14.23 ± 0.08 ) (P＜ 0.05);At 25 μmol/L( 2.21 ± 0.05),the level of LDH was essentially the same as that of the control( 1.03 ±0.03 ) (P＞0.05).(2)The protein level of HIF-1 αwas different from normal (0.24 ±0.01 ) when exposed to OGD for 2 hours and reoxygenated for 2 hours (0.57 ± 0.09) and was highest after cells were exposed to OGD for 2 hours and reoxygenated for 12 hours(0.91 ± 0.08 ) (P＜ 0.05 ).The RNA level of HIF-1 α when cells were exposed to OGD was not deferent from normal (P ＞ 0.05 ).( 3 ) When cells were pretread with Nec-1 (0.32 ± 0.04 ),the protein level of HIF-1α were lower than untreated(0.83 ±0.03) (P＜0.05),but the RNA level of HIF-1α had no deference(P ＞ 0.05).Conclusion HIF-1α was involved in cell' s caspase-independent cell death;Nec-1 can protect neurons through inhibiting the expression of HIF-1α. Key words: Hypoxia-inducible factor-1 α;  Necrostain-1 ;  Oxygen-glucose deprivation;  Caspase-independent death