间歇低氧导致海马miR-26b上调表达与神经元凋亡机制研究

Mechanism of miR-26b up-regulated expression and neuron apoptosis in the hippocampus induced by intermittent hypoxia

目的探讨间歇低氧损伤后SD大鼠认知功能损伤与海马神经元凋亡及miR．26b上调表达的相关性。方法健康雄性SD大鼠20只按随机数字表法分为常氧对照组、间歇低氧1周组、2周组、4周组，每组5只。后3组大鼠分别给予不同时间的间歇低氧刺激，常氧对照组大鼠持续给予常氧。Morris水迷宫实验对间歇低氧4周组和常氧对照组大鼠进行空间学习和记忆力检测，Western blotting检测4组大鼠海马组织凋亡相关蛋白Caspase3、Bax及抗凋亡蛋白Bcl-2的表达。RT-PCR检测4组大鼠海马组织miR．26b的表达。结果定位航行实验显示间歇低氧4周组大鼠前5d寻找平台所需时间（平均逃避潜伏期1均较常氧对照组大鼠延长，差异有统计学意义（P〈0．05）。空间探索实验显示，间歇低氧4周组大鼠在60S内进入目标象限时间为（22．0±6．7）S，明显短于常氧对照组大鼠[（39．8±8．8）s]，差异有统计学意义（P〈0．05）；Western blotting检测显示，与常氧对照组比较．间歇低氧1、2、4周组大鼠海马组织凋亡蛋白Bax及Caspase3表达上调，抗凋亡蛋白Bcl-2表达下调．间歇低氧1周组大鼠海马凋亡蛋白Bax及Caspase3的表达较间歇低氧2、4周组高．而抗凋亡蛋白Bcl-2表达较间歇低氧2、4周组低，差异有统计学意义（P〈0．05）；RT—PCR检测显示。与常氧对照组比较，间歇低氧1、2、4周组大鼠海马组织miR-26b的表达均上调，且间歇低氧1周组大鼠海马凋亡miR-26b的表达较间歇低氧2、4周组高，差异有统计学意义（P〈0．05）。结论间歇低氧损伤后海马组织miR-26b上调表达可能涉及间歇低氧导致的海马神经元Bcl-2／Bax相关的线粒体抗凋亡／凋亡机制，其可能作为干预治疗间歇低氧损伤有效的基因治疗手段。

Objective To study the cognitive impairment in SD rats after intermittent hypoxia （IH）, and explore the relation of miR-26b up-regulated expression and neuron apoptosis in the hippocampus of SD rats after IH. Methods Eight-week-old male SD rats （n=20, each weighing approximately 300±10 g） were randomly divided into normal oxygen control group, IH 1-week group, IH 2-weeks group and IH 4-weeks group （n=5）. Rats in the later three groups were given 1H for different times, and rats in the normal oxygen control group were given normal oxygen. The spatial learning and memory abilities were detected by Morris Water Maze （MWM） in the normal oxygen control group and 1H 4-weeks group. The levels of apoptosis proteins Caspase3 and Bax and anti-apoptosis protein Bcl-2 in the hippocampus of 4 groups were detected by Western blotting. The miR-26b expression level in the 4 groups was detected by real time-PCR. Results （1） The results of MWM revealed that the mean escape latency in the IH 4-weeks group was significantly prolonged as compared with that in the normal oxygen control group （/9〈0.05）; the time entering into the target quadrant in the IH 4-weeks group （[22.0± 6.7] s） was significantly shorter than that in the normal oxygen control group （[39.8±8.8] s, P〈0.05）. （2） Western blotting indicated that up-regulated expressions of apoptosis proteins Bax and Casepase3 and down-regulated expression ofanti-apoptosis protein Bcl-2 in the IH 1-week group, IH 2-weeks group and IH 4-weeks group were noted as compared with those in the control group, with significant differences （P〈0.05）; significantly higher apoptosis protein Bax and Casepase3 expressions in the IH 1-week group were noted as compared with those in the IH 2-weeks group and IH 4-weeks group （P〈0.05）, while significantly decreased Bcl-2 expression in the IH 1-week group was noted as compared with that in the IH 2-weeks group and IH 4-weeks group （P〈0.05）. （3） The results of real time-PCR revealed that the miR-26b expression level in the hippocampus was up-regulated in the IH 1-week group, IH 2-weeks group and IH 4-weeks group as compared with that in the control group, with significant differences （P〈0.05）; miR-26b expression level in the IH 1-week group was significantly higher as compared with that in the IH 2-weeks group and IH 4-weeks group （P〈0.05）. Conclusion The miR-26h up-regulated expression in the hippocampus might refer to Bax /Bcl-2-related mitochondrial apoptotic signaling pathway after IH brain injury; miR-26b could be a potential mean of gene therapy after IH brain injury.