吗啡预处理减轻小鼠海马神经元氧-糖缺失/恢复损伤的影响因素

目的 探讨吗啡预处理减轻小鼠海马神经元氧-糖缺失/恢复损伤的影响因素(剂量和作用时间窗).方法 急性分离小鼠海马组织,制备脑片,行4部分实验,实验Ⅰ:应用吗啡0.1、0.3、0.5、1.0、3.0、10.0 μmol/L孵育脑片30 min,再常规培养30 min,缺氧无糖20 min,复氧复糖2 h.实验Ⅱ:应用吗啡3.0 μmol/L孵育脑片5、15、30、45、60 min,再常规培养30 min,缺氧无糖20 min,复氧复糖2 h.实验Ⅲ:应用吗啡3.0 μmol/L孵育脑片30 min,再常规培养即刻、5、15、30、60、120 min时行缺氧无糖20 min,复氧复糖2 h.实验Ⅳ:应用蛋白激酶C(PKC)非特异性抑制剂白屈菜赤碱10 μmol/L、选择性新奇型PKCε(nPKCε)抑制剂εVI.2 2 μmol/L、特异性钙-钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)抑制剂AIP 2 μmol/L、N.甲基-D-天冬氨酸(NMDA)受体特异性阻断剂MK-801 10 μmol/L孵育脑片30 min,再与吗啡3.0 μmol,L共同孵育30 min,常规培养30 min,缺氧无糖20 min,复氧复糖2 h.计算神经元存活率.结果 与氧-糖缺失/恢复组比较,吗啡0.5、1.0、3.0、10.0 μmol/L预处理组、吗啡预处理15、30、45、60 min组、吗啡预处理后常规培养即刻、5、15、30、60 min组神经元存活率升高(P<0.05).PKC、nPKGt、CaMKⅡ抑制剂和NMDA受体阻断剂可部分抑制吗啡预处理升高神经元存活率的作用.结论 有效减轻小鼠海马神经元氧-糖缺失,恢复损伤的吗啡预处理方式为:浓度0.5～1.0 μmol/L,持续时间15～60 min,间隔时间小于60 min;其保护作用可能部分依赖于PKC、nPKCε、CaMKⅡ的活化和NMDA受体的激活.

Factors affecting the protective effect of morphine preconditioning on murine hippocampal neurons against anoxia-reoxygenation injury

Objective To investigate the factors affecting the protective effects of morphine preconditioning on murine hippocampal neurons against anoxia/reoxygenation (A/R) injury and the underlying mechanisms.Methods Hippocampal slices (400 μm thick) were prepared using hippocampi isolated from decapitated mice. A/R injury was simulated in vitro using artificial cerebral spinal fluid (ACSF) deprived of O_2 and glucose for 20 min followed by reoxygenation and glucose supply for 2 h. The experiment was performed in 4 parts: I .The slices were incubated with 5 different concentrations of morphine (0.1, 0.3, 0.5, 1.0, 3.0, 10.0 /μmol/L) for 30 min at 30 min before A/R; Ⅱ. The slices were incubated with morphine 3.0 /μmol/L for 5 different periods of time (5, 15, 30, 45, 60 min) at 30 min before A/R; Ⅲ. The slices were incubated with morphine 3.0 μmol/L for 30 min followed by A/R at 6 different intervals (0, 5, 15,30,60, 120 min); Ⅳ. The slices were incubated with (a) chelerythrine (a non-selective PKC antagonist) 10 /μmol/L or (b) εVl-2 (a selective nPKCe isoform antagonist) 2 μmol/L or (c) AIP 2 μmol/L (a selective CaMK Ⅱ antagonist) or (d) MK-801 10 μmol/L (a non-competitive NMDA receptor blocker) for 30 min and then for another 30 min together with morphine 3.0 μmol/L before A/R at 30 min interval. The survival rates of the hippocampal neurons were assessed by TTC staining. Results Neuronal survival rates were significantly higher in morphine preconditioning groups which preconditioned with morphine (0.5-10.0 μmol/L) for 15-60 min at an interval of 0-60 min before A/R than in A/R group. Increase in neuronal survival rate induced by morphine preconditioning was partially blocked by chelerythrine or εV1-2 or AIP or MK-801. Conclusion Preconditioning with appropriate concentrations of morphine (0.5-10.0 μmol/L) for appropriate period of time (15-60 min) at appropriate interval (within 60 min) before A/R can protect hippocampal neurons against A/R injury through activation of nPKCε, NMDA receptor and CaMKⅡ.