纳洛酮对离体人脑神经元缺氧损伤谷氨酸释放的影响

Effects of Naloxone on Glutamate Release in Combined Oxygen-glucose Deprivation of Primary Cultured Human Embryo Neuron

目的建立原代培养人脑神经元缺氧模型,探讨纳洛酮对神经元缺氧损伤中谷氨酸释放的影响。方法应用无血清原代培养人胚胎大脑神经细胞,经神经微丝染色证实可获得富含神经元的混合培养细胞。将神经元细胞随机分为对照组、缺氧组和给药组(纳洛酮终浓度分别为0.25、5、10μg/m l)。对照组为正常培养,缺氧组和给药组细胞进行缺氧结合无糖处理(氧糖剥夺)1h并继续复氧培养24h。于复氧24h后观察细胞形态学变化,测定细胞外液乳酸脱氢酶(LDH)浓度,应用四甲基偶氮唑蓝法(M TT)观察细胞存活率,应用高效液相色谱法测定细胞外液谷氨酸浓度。结果缺氧组细胞外液LD H、谷氨酸浓度显著高于对照组(P<0.01),M TT法测得光密度(OD)值则显著低于对照组(P<0.01);应用纳洛酮后,随药物浓度增加,各组细胞外液谷氨酸浓度与缺氧组比较呈现逐渐降低趋势(P<0.05,P<0.01),M TT法测得O D值呈现逐渐升高趋势(P<0.01),至纳洛酮10μg/m l时恢复至对照组水平(P>0.05)。结论纳洛酮可以减少缺氧神经元的谷氨酸释放,减轻兴奋性神经毒性,对神经元缺氧损伤具有保护作用。

Objective To investigate the effects of naloxone on glutamate release in combined oxygen-glucose deprivation of primary cultured human embryo neurons. Methods The primary cultured embr-yonic human cortical neurons were demonstrated by immunocytochemical stain of neural filament(NF). The neurons were randomly allocated into control group, hypoxic group, and experimental group. The experimental group was further divided into three subgroups pretreated with different concentrations of naloxone(0.25, 5, 10 μg/ml). The neurons of hypoxic group and experimental group were deprived both oxygen and glucose for 1 hours followed by 24 hours of reoxygenation. Meanwhile, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay, high performance liquid chromato-graphy(HPLC), and biological analysis to study the survival rate of neurons and the changes of extra-cellular glutamate and lactate dehydrogenase(LDH)levels after 24 hours of reoxygenation. Results One hour of oxygen-glucose deprivation followed by 24 hours of reoxygenation was associated with a large increase in extracellular LDH and glutamate and a significant decrease of cell vitality(P < 0.01). Naloxone exerted a concentration-dependent protection against neuronal injury provoked by combined oxygen-glucose deprivation. After reoxygenation, the extracellular concentrations of glutamate gradually decreased(P <0.05, P < 0.01, respectively)and cell vitality increased(P < 0.01)with increase of the concentration of naloxone compared with control group. All of them returned to control level when naloxone was up to 10 μg/ml(P > 0.05). Conclusion Naloxone protects neurons from hypoxic injury by inhibiting the release of glutamate and therefore alleviating the exciting toxicity.