摘要目的 二氮嗪预处理联合低温对大鼠海马神经元缺氧复氧时Bcl-2和Bax表达的影响.方法 清洁级SD大鼠,出生<24 h,体重5～6 g,离体培养海马神经元,接种于培养皿或96孔板.海马神经元随机分为8组,每组36孔或12皿:常温组(NT组)、二氮嗪预处理+常温组(DP+NT组)、浅低温组(MiH组)、二氮嗪预处理+浅低温组(DP+MiH组)、中低温组(MoH组)、二氮嗪预处理+中低温组(DP+MoH组)、深低温组(DeH组)和二氮嗪预处理+深低温组(DP+DeH组).DP+NT组、DP+MiH组、DP+MoH组和DP+DeH组培养液中加入二氮嗪,终浓度为100μmol/L,孵育1 h,1次/d,连续2 d,随后分别在37、34、30和22℃下缺氧4 h,37℃复氧48 hoNT组、MiH组、MoH组和DeH组分别在37、34、30和22℃下行缺氧4 h,37℃复氧48 h.于复氧48 h时测定海马神经元活力、凋亡率和Bcl-2和Bax 的表达水平,计算Bcl-2/Bax.结果 与NT组比较.DP+NT组、MiH组、MoH组和DeH组海马神经元活力升高,凋亡率降低,Bcl-2表达上调,Bax表达下调,Bcl/21Bax升高(P<0.05);与DP+NT组比较,DP+MiH组、DP+MoH组和DP+DeH组海马神经元活力升高,早期凋亡率降低,Bcl-2表达上调,Bax 表达下调,Bcl-21Bax升高(P<0.05),晚期凋亡率差异无统计学意义(P>0.05);与MiH组比较,DP+MiH组和DeH组海马神经元活力升高,早期凋亡率降低,Bcl-2表达上调,Bax表达下调,Bel-2/Bax升高(P<0.05),晚期凋亡率差异无统计学意义(P>0.05),MoH组上述指标差异无统计学意义(P>0.05);与DP+MiH组比较,DP+DeH组海马神经元活力升高,早期凋亡率降低,Bcl-2表达上调,Bax表达下调,Bcl-2/Bax升高(P<0.05).结论 二氮嗪预处理联合低温减轻大鼠神经元缺氧复氧损伤的机制可能与纠正Bcl-2与Bax失衡,抑制神经元早期凋亡有关.

abstractsObjective To investigate the effects of diazoxide preconditioning combined with hypothermia on the expression of Bcl-2 and Bax during anoxia-reoxygenation in rat hippocampal neurons. Methods The hippocampal neurons isolated from newborn SD rats ( < 24 h, weighing 5-6 g) were inoculated in the culture dish or 96 well plates. The hippocampal neurons were randomly assigned into 8 groups and each group contained 36 wells or 12 dishes of neurons: normal temperature .group (group NT), diazoxide preconditioning (DP) + NT group (group DP+ NT), mild hypothermia group (group MiH) , DP + MiH group (group DP + MiH) , moderate hypothermia group (group MoH), DP + MoH group (group DP + MoH), deep hypothermia group (group DeH) and DP+DeH group (group DP+ DeH). In group DP + NT, DP + MiH, DP+ MoH and DP + DeH, diazoxide was added to the culture media, the final concentration was 100 μmol/L,and the neurons were incubated for 1 h once a day for 2 d, and then subjected to 4 h of hypoxia at 37, 34, 30 and 22℃ , respectively, followed by 48 h of reoxygenation at 37℃ . The neuronal viability, apoptotic rates and expression of Bcl-2 and Bax were determined and the ratio of Bcl-2/Bax was calculated. Results The neuronal viability was significantly higher, apoptotic rate lower, Bcl-2 expression higher, Bax expression lower and Bcl-2/Bax ratio higher in group DP + NT, MiH, MoH and DeH than in group NT ( P < 0.05). The neuronal viability was significantly higher, early apoptotic rate lower, Bcl-2 expression higher, Bax expression lower and Bcl-2/Bax ratio higher in group DP + MiH, DP + MoH and DP+ DeH than in group DP + NT ( P < 0.05), but there was no significant difference in the late apoptotic rate between group DP + MiH, DP + MoH, DP + DeH and DP + NT ( P > 0.05). The neuronal viability was significantly higher, early apoptotic rate lower, Bcl-2 expression higher, Bax expression lower and Bcl-2/Bax ratio higher in group DP+ MiH and DeH than in group MiH (P < 0.05), but there was no significant difference in the late apoptotic rate between group DP + MiH, DeH and MiH, and no significant difference in the above indices between group MoH and MiH (P > 0.05) . The neuronal viability was significantly higher, early apoptotic rate lower, Bcl-2 expression higher, Bax expression lower and Bcl-2/Bax ratio higher in group DP+ DeH than in group DP+ MiH ( P < 0.05). Conclusion Diazoxide preconditioning combined with hypothermia can attenuate the anoxia-reoxygenation injury in rat hippocampal neurons possibly through correcting the imbalance of Bcl-2 and Bax and inhibiting the early apoptosis of hippocampal neurons.