Protection effect of ATP-sensitive K+ channel opener diazoxide on hippocampus neurons treated with oxygen glucose deprivation and its molecular mechanism / 中华神经医学杂志

ABSTRACT

Objective To explore the effect of ATP-sensitive potassium channel opener,diazoxide (DZX),on protecting hippocampal neurons of oxygen-glucose deprivation (OGD) and its molecular mechanism.Methods OGD hippocampal neurons and hippocampal brain slices were used as models,MTT and lactic dehydrogenase (LDH) assays were used to determine activities of these neurons.Hippocampal neurons and hippocampal brain slices without any treatment were used as normal control group,OGD hippocampal neurons or hippocampal brain slices were used as OGD group,DZX and DZX+5-HD were used to treat the OGD hippocampal neurons or hippocampal brain slices as OGD+DZX and OGD+DZX+5-HD groups,respectively.The apoptosis rate of each group was determined by flow cytometry;expressions of apoptotic related genes,including Bax,Bc1-2 and Caspase-3,were detected by real time-PCR;TUNEL assay was used to examine the apoptotic cells in the hippocampal brain slices and patch clamp was used to record the average current density of each group.Results MTT results showed that the optical density in OGD group,OGD+DZX group and OGD+DZX+5-HD group at 0,4,8 and 12 h after OGD pretreatment was significantly lower than that in the normal control group (P＜0.05).LDH release results revealed that LDH release in OGD group,OGD+DZX group and OGD+DZX+5-HD group at 0,4,8 and 12 h after OGD pretreatment was significantly higher than that in the normal control group (P＜0.05).Flow cytometry and TUNEL assay results revealed that the apoptosis rate of hippocampal neurons in OGD group was significantly higher than that in normal control group (P＜0.05),and that in OGD+DZX group was significantly reduced as compared with that in the OGD group (P＜0.05);that in the DZX+5-HD+OGD group was significantly higher than that in the OGD+DZX group (P＜0.05),but significantly lower than that in the OGD group (P＜0.05).Real time-PCR results suggested that the expressions ofapoptotic genes Bax and Caspase-3 in OGD group were significantly higher than those in the normal control group,those in OGD+DZX group were significantly lower than those in the OGD group,those in OGD+DZX+5-HD group were significantly higher than those in the OGD+DZX group,but still significantly lower than those in the OGD group (P＜0.05).While the expression of anti-apoptotic gene Bcl-2 was contrary to apoptotic gene Bax and Caspase-3.The patch clamp tests demonstrated that the average current density of OGD group (93.2±5.4) was dramatically increased than that in normal control group (53.3±5.3),while the average current density of OGD+DZX group (53.2±3.0) was reduced to normal level,and the average current density of OGD+DZX+5-HD group (83.4±4.8) was close to OGD group,all of the results had significant statistical differences (P＜0.05).Conclusion DZX is able to protect hippocampal neurons against apoptosis via inhibiting the expressions ofapoptotic genes Bax and Caspase-3.