脑源性神经营养因子经由环磷酸腺苷反应元件结合蛋白磷酸化对缺氧大鼠胚脑皮质神经元细胞的保护作用

The Neuroprotective Role of Brain-derived Neurotrophic Factor for Embryonic Rat Cortical Neurons Against Hypoxia Via CREB Phosphorylation

转录因子环磷酸腺苷反应元件结合蛋白（Cyclic AMP response element-binding protein，CREB）是胚脑皮质神经元细胞内脑源性神经营养因子（Brain-derived neurotrophic factor，BDNF）诱导基因表达的重要调节因子。我们前期的研究表明BDNF对缺氧性神经元损伤具有保护作用，为了阐明BDNF对缺氧性神经元损伤的保护作用是否经过了核蛋白CREB的磷酸化，本研究采用蛋白质免疫印迹法检测单纯缺氧组和BDNF干预组胚脑皮质神经元细胞内CREB及Ser^133磷酸化CREB在不同时间点表达水平的变化。结果显示缺氧及BDNF均能刺激胚脑皮质神经元细胞内Ser^133磷酸化CREB表达增加，在不同缺氧时间点BDNF干预组Ser^133磷酸化CREB表达水平较单纯缺氧组明显增强（P〈0．01），BDNF干预组1h后Ser^133磷酸化CREB表达至高峰，以后持续表达维持6h以上，维持时间较单纯缺氧组明显延长；在缺氧0～3h，BDNF干预组细胞内总CREB表达水平与单纯缺氧组基本一致；随着缺氧时间的延长，单纯缺氧组细胞内CREB表达明显减少，以第5～6h最明显。结果表明，BDNF对缺氧性神经元损伤的保护作用经过了核蛋白CREB的磷酸化。

Transcription factor cyclic AMP response element-binding protein（CREB） in embryonic cortical neurons is an important modulator of Brain-derived neurotrophic factor（BDNF） induced gene expression. Meanwhile, our early researches have indicated that BDNF possesses neuroprotective role for hypoxic neurons against hypoxia. In order to disclose whether the neuroprotective role of BDNF for embryonic rat cortical neurons against hypoxia is fulfilled via nucleoprotein CREB phosphorylation, we used western blotting method to detect the expression of CREB and phosphorylated CREB in experimental groups （with BDNF） and hypoxic control group （without BDNF） with the time changes of exposure to hypoxia. Results indicated that hypoxia and BDNF both could induce phosphorylation of CREB in embryonic cortical neurons. Phosphorylation of CREB in experimental group （with BDNF） was much higher than that in hypoxic control group at the same time points [ P 〈 0.01 ）. The expression of phosphorylated CREB reached the highest level at the first hour af- ter being exposed to hypoxia in experimental groups, then phosphorylated CREB decreased slowly and remained at the level for much longer time in experimental groups than in control group. The total amount of CREB in embryonic cortical neurons at the first 0-3 hours after being exposed to hypoxia in experimental groups were the same as that in hypoxic control group. CREB decreased more quieldy in hypoxic control group at 5-6 hours after hypoxia. This in vitro research demonstrates that BDNF plays its neuroprotective role for embryonic rat cortical neurons against hypoxia via CREB phosphorylation.