降低海马NO含量对凋亡相关因子的调节作用

Lowering Nitric Oxide in Hippocampus Regulating Apoptosis-Related Factors

目的探讨降低海马NO含量对凋亡相关因子的调节作用。方法体内和体外实验均分为4组:生理盐水对照组(NS组),海人酸致痫组(KA组),氨基胍预处理+海人酸组(AG+KA组)和氨基胍组(AG组)。采用免疫细胞化学法显示体外培养的海马神经元在不同处理因素作用24h和48h后bcl-2、bax的免疫反应性。采用Western blot法或半定量RT-PCR法检测各组大鼠造模后24h、48h时bcl-2、bax蛋白及Apaf-1mRNA的表达情况。结果在体外实验中,KA组bcl-2的免疫反应较NS组减弱而bax的免疫反应增强;AG+KA组bcl-2的免疫反应较KA组增强而bax的免疫反应减弱;AG组与NS组相比无明显差异。体内实验中,Western blot检测显示大鼠海马组织中bcl-2和bax蛋白的表达水平呈负相关,其趋势与体外实验结果一致;半定量RT-PCR检测显示Apaf-1mRNA的含量在造模后24h和48h时间点,KA组明显高于NS组(P<0.05),而AG预处理则明显拮抗KA诱导的Apaf-1mRNA高表达。结论降低NO含量可以上调bcl-2蛋白的表达,下调bax和Apaf-1mRNA的表达。

Objective To investigate the regulatory effect of lowering the nitric oxide in hippocampus on apoptosis-related factors.Methods The in vitro and in vivo experiments were randomly divided into 4 groups：normal saline control（NS）,kainic acid（KA）,aminoguanidine（AG）pretreatment plus KA（AG＋KA）,AG.The immunoreactivity of bcl-2 and bax in the in vitro cultured hippocampal neurons treated with different factors for 24 h and 48 h was detected by using immunohistochemistry.The levels of bcl-2 and bax proteins and Apaf-1 mRNA in rat hippocampus were detected by Western blot and semi-quantitative RT-PCR respectively at 24 and 48 h after modeling in rats.Results In the in vitro experiments,compared to the NS group,the protein expression of bcl-2 was decreased,and bax increased in KA group;compared to the KA group,the protein expression of bcl-2 was increased,and bax decreased in AG＋KA group.There was no significant difference in the immunoreactivity of bcl-2 and bax between NS group and AG group.In the in vivo experiments,there was negative correlation between the levels of bcl-2 and bax protein expression,which was consistent with that in the in vitro experiments.There was significant difference in Apaf-1 mRNA levels among 4 groups at 24 h and 48 h.The Apaf-1 mRNA levels in KA group were significantly higher than in NS group（P0.05）,and AG pretreatment obviously antagonized KA-induced Apaf-1 expression（P0.05）.Conclusion The decrease of NO concentration may up-regulate the expression of bcl-2 protein while down-regulate the expression of bax protein and Apaf-1 mRNA.