ATP敏感性钾通道开放剂二氮嗪对海马神经元氧糖剥夺损伤的保护作用及其分子机制  

Protection effect of ATP-sensitive K^＋ channel opener diazoxide on hippocampus neurons treated with oxygen glucose deprivation and its molecular mechanism

作　　者：雷小峰[1] 雷立健[2] 张志清[1] 程焱[3] 

机构地区：[1]天津市第四中心医院神经内科,300140 [2]山西医科大学公共卫生学院流行病学教研室,太原030001 [3]天津医科大学总医院神经内科,天津300052

出　　处：《中华神经医学杂志》2016年第5期445-451,共7页Chinese Journal of Neuromedicine

基　　金：天津市科委重点项目（13ZCZDSY01900）

摘　　要：目的探讨ATP敏感性钾通道（KATP）开放剂二氮嗪（DZX）对海马神经元氧糖剥夺（OGD）损伤的保护作用及其分子机制。方法构建海马神经元OGD模型和海马区脑片OGD模型，并采用MTT法和乳酸脱氢酶（LDH）释放实验检测海马神经元活性。将不经任何处理的海马神经元和海马区脑片设为正常对照组，将经OGD预处理的海马神经元和海马区脑片设为OGD组，将DZX或DZX＋5-羟基癸酸甘油酯（5-HD）处理的经OGD预处理的海马神经元和海马区脑片分别设为OGD＋DZX组和OGD＋DZX＋5-HD组。采用流式细胞术检测各组海马神经元凋亡情况，采用RT-PCR检测各组海马神经元凋亡相关因子Bax、Bcl-2、Caspase-3 mRNA表达水平；采用TUNEL染色检测各组海马区脑片中神经元凋亡情况，采用膜片钳检测各组海马区脑片中KATP电流情况。结果MTT检测发现：与对照海马神经元相比，经OGD预处理后0、4、8、12 h海马神经元的吸光度值明显下降，差异均统计学意义（P〈0.05）。LDH释放实验检测发现：与对照海马神经元相比，经OGD预处理后0、4、8、12 h海马神经元LDH的释放量明显增高，差异均有统计学意义（P〈0.05）。流式细胞术和TUNEL法检测共同显示：与正常对照组海马神经元相比，OGD组海马神经元凋亡率显著增多，差异有统计学意义（P〈0.05）；OGD＋DZX组海马神经元凋亡率比OGD组明显减少，差异有统计学意义（P〈0.05）；OGD＋DZX＋5-HD组海马神经元凋亡率比OGD＋DZX组显著增加，但仍比OGD组海马神经元凋亡率低，差异均统计学意义（P〈0.05）。RT-PCR检测显示：与正常对照组相比，OGD组Bax、Caspase-3 mRNA表达水平显著升高，差异均有统计学意义（P〈0.05）；DZX＋OGD组Bax、Caspase-3 mRNA表达水平比OGD组明显减少，差异均有统计学意义（P〈0.05）；OGD＋DZX＋5-HD组Bax、Caspase-3 mRNA表达水平明显高于DZX＋OGD组，差异均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, Bcl-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 an

关 键 词：ATP敏感性钾通道 二氮嗪 氧糖剥夺 海马神经元 细胞凋亡 

分 类 号：R749.13[医药卫生—神经病学与精神病学]