Ifenprodil对肌萎缩侧索硬化离体模型中运动神经元的保护作用

摘要

目的 探讨Ifenprodil对苏-羟天冬氨酸(THA)诱导的肌萎缩侧索硬化(ALS)离体模型中运动神经元的保护作用.方法 在离体培养的脑组织片中加入THA造成慢性谷氨酸损伤,模拟肌萎缩侧索硬化的特征性病理改变.将脑片分为6组:(1)对照组,不添加THA; (2)THA损伤组,细胞换液时加入100 μmol/L THA,持续1周;(3)不同剂量Ifenprodil组(分别在THA损伤基础上添加0.1、1、10、100 μmol/L Ifenprodil);每组6个培养皿.Ifenprodil 于THA损伤前2h加入到各组脑片中.根据PI染色的荧光亮度测定Ifenprodil保护作用的最佳浓度.在损伤的不同时间点用丙二醛(MDA)试剂盒测定培养体系中MDA的含量,Western blotting测定运动皮层中半胱氨酸天冬氨酸酶-3(caspase-3)蛋白表达量.结果 PI染色显示10 μmol/L Ifenprodil对运动神经元有明显保护作用.THA损伤后,培养体系中MDA含量明显增加,运动皮层中caspase-3活化亚基(相对分子量为17 000)表达量增高,与其他组别比较差异有统计学意义(P＜0.05).10μmol/L Ifenprodil能有效阻止MDA和活化caspase-3的增加.结论 Ifenprodil能够通过减轻运动神经元的氧化应激反应,抑制caspase-3活化,进而有效保护ALS离体模型中运动神经元的损伤.%Objective To investigate the protective effect of Ifenprodil on motor neurons in in vitro threo-hydroxyaspartate (THA)-induced amyotrophic lateral sclerosis (ALS) models.Methods THA was added into the isolated culture of brain slices to induce chronic glutamate damage and mimic the typical changes of ALS pathology.Cultured brain slices were separated randomly to 6 different groups,including control group (without THA),THA inducement group (adding 100 μmol/L THA for one week) and THA and Ifenprodil treatment groups (adding 0.1,1,10 and 100 μmol/L of Ifenprodil 2 h before 100 μmol/L THA inducement); 6 culture dishes were employed in each group.According to PI fluorescence intensity,the most effective concentration of Ifenprodil was selected.The concentration of malonaldehyde (MDA) in supematant of cultured tissues was evaluated by MDA testing kit and the protein expression of cysteine-aspartic proteases-3 (caspase-3) in motor cortex was tested by Western blotting at different injury times.Results PI staining showed that 10 μmol/L Ifenprodil could produce prominent protective effect on motor neurons in the ALS models.Levels of MDA in supernatant and activated 17KD caspase-3 in the THA treatment group obviously increased as compared with those in the other groups (P＜0.05); 10 μmol/L Ifenprodil could block the increment of MDA and activated caspase-3 levels effectively.Conclusion Ifenprodil can effectively protect motor neurons in in vitro THA-induced ALS models by reducing oxidative stress and inhibiting caspase-3 activation.