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目的观察树鼩脑缺血海马神经元Akt(p S473)和Akt(p T308)磷酸化改变对CA1区细胞凋亡的影响,探讨缺血后适应(PC)抑制细胞凋亡的可能机制。方法用光化学诱导法诱导树鼩脑缺血并建立缺血PC模型;用免疫组织化学TACS原位凋亡检测试剂盒检测皮层及海马CA1区神经元凋亡数量,用免疫组织化学法检测Akt(p S473)和Akt(p T308)表达的空间分布,用ELISA法检测海马CA1区神经元Akt(p S473)和Akt(p T308)磷酸化水平;用电子显微镜观察其神经元超微结构改变。结果脑缺血后神经元皱缩,核消失以缺血24h为著。脑缺血后4 h、24 h及72 h皮层及海马CA1区神经元TUNEL阳性细胞数明显增加(P<0.01),海马CA1区神经元Akt(p S473)和Akt(p T308)的表达及磷酸化水平明显升高,以脑缺血4 h的改变最明显,分别为(152.3±3.5)units/mg、(130.8±2.6)units/mg和(149.5±4.7)units/mg和(42.35±2.49)units/mg、(19.23±1.41)units/mg和(23.38±1.32)units/mg(P<0.01)。缺血PC组神经元损伤明显减轻,皮层及海马CA1区TUNEL阳性细胞明显减少(P<0.01),且与海马神经元Akt(p T308)活化水平呈平行改变(P<0.05)。结论树鼩脑缺血海马CA1区细胞凋亡与Akt(p S473)和Akt(p T308)过磷酸化的信号机制有关,缺血PC抑制海马神经元Akt(p S473)和Akt(p T308)双磷酸化可能具有抗凋亡作用。
Objective To observe the effect of phosphorylation of Akt (p S473) and Akt (p T308) on apoptosis of hippocampus CA1 area in hippocampus of tree shrew so as to explore the possible mechanism of apoptosis induced by postischemic conditioning (PC). Methods The cerebral ischemic model was induced by photochemical induction and the model of ischemic PC was established. The number of apoptotic neurons in cortex and hippocampal CA1 area was detected by TACS in situ immunohistochemistry. The expression of Akt p S430) and Akt (pT308). The phosphorylation levels of Akt (p S473) and Akt (p T308) in hippocampal CA1 neurons were detected by ELISA. The ultrastructural changes of neurons were observed with electron microscopy. Results After cerebral ischemia, neurons shrank and nuclei disappeared with ischemia 24h. The number of TUNEL positive cells in cortex and hippocampal CA1 neurons increased significantly at 4 h, 24 h and 72 h after cerebral ischemia (P <0.01), and the expressions of Akt (p S430) and Akt (p T308) (152.3 ± 3.5) units / mg, (130.8 ± 2.6) units / mg and (149.5 ± 4.7) units / mg and (42.35 ± 2.49) units / mg, (19.23 ± 1.41) units / mg and (23.38 ± 1.32) units / mg (P <0.01). Neuronal injury in ischemic PC group was significantly reduced, TUNEL positive cells in cortex and hippocampus CA1 area were significantly decreased (P <0.01), and were also changed in parallel with the activation of Akt (pT308) in hippocampal neurons (P <0.05). Conclusions The apoptosis of hippocampal CA1 area in tree shrew is related to the signal pathway of Akt (p S4308) and Akt (pT308) hyperphosphorylation. The ischemic PC inhibits the expression of Akt (p S473) and Akt Double phosphorylation may have anti-apoptotic effects.