Oxygen Glucose Deprivation Post-conditioning Protects Cortical Neurons against Oxygen-glucose Deprivation Injury: Role of HSP70 and Inhibition of Apoptosis

In the present study,we examined the effect of oxygen glucose deprivation(OGD)post-conditioning(PostC)on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro.Four-h OGD was induced by OGD by using a specialized and humidified chamber.To initiate OGD,culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke’s medium.After OGD treatment for 4 h,cells were then allowed to recover for 6 h or 20 h.Then lactate dehydrogenase(LDH)release assay,Western blotting and flow cytometry were used to detect cell death,protein levels and apoptotic cells,respectively.For the PostC treatment,three cycles of 15-min OGD,followed by 15 min normal cultivation,were applied immediately after injurious 4-h OGD.Cells were then allowed to recover for 6 h or 20 h,and cell death was assessed by LDH release assay.Apoptotic cells were flow cytometrically evaluated after 4-h OGD,followed by re-oxygenation for 20 h(O4/R20).In addition,Western blotting was used to examine the expression of heat-shock protein 70(HSP70),Bcl-2 and Bax.The ratio of Bcl-2 expression was(0.44±0.08)%and(0.76±0.10)%,and that of Bax expression was(0.51±0.05)%and(0.39±0.04)%,and that of HSP70 was(0.42±0.031)%and(0.72±0.045)%respectively in OGD group and PostC group.After O4/R6,the rate of neuron death in PostC group and OGD groups was(28.96±3.03)%and(37.02±4.47)%,respectively.Therefore,the PostC treatment could up-regulate the expression of HSP70 and Bcl-2,but down-regulate Bax expression.As compared with OGD group,OGD-induced neuron death and apoptosis were significantly decreased in PostC group(P<0.05).These findings suggest that PostC inhibited OGD-induced neuron death.This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.