丹皮酚对缺糖缺氧再灌注损伤大鼠海马神经元EAA、NMDA受体表达的影响及机制探讨

Effect of paeonol on EAA,NMDA receptor in cultured rat hippocampal neurons subjected to oxygen-glucose deprivation / reperfusion-induced injury

目的观察丹皮酚对缺糖缺氧（OGD）再灌注损伤大鼠海马神经元兴奋性氨基酸（EAA）、N．甲基．D．天门冬氨酸（NMDA）受体表达的影响，探讨该药的神经保护作用机制。方法原代培养新生大鼠海马神经元8～12d后随机分为4组，其中对照组正常换液培养；模型组、丹皮酚组、地佐环平（MK-801）组均制备OGD再灌注损伤模型，在此基础上丹皮酚组于每个再灌注时间点前分别加入丹皮酚，MK-801组则加入MK-801溶液；倒置相差显微镜下观察神经元一般形态学变化，用MTr法测定神经元存活率，分别采用放射配基结合实验及RT-PCR法测定NMDA受体结合力、NR1亚基mRNA表达，以氨基酸分析仪检测EAA含量。结果模型组神经元肿胀或变形，突起缩短并逐渐消失，胞质内颗粒变性明显、甚至完全崩解；丹皮酚组及MK-801组神经元形态改变均较模型组减轻，胞体完整，突起较清楚，基本保持完整。与对照组比较，模型组神经元存活率明显降低，NMDA受体结合力、NR1亚基mRNA表达及E从含量明显升高，而丹皮酚组及MK_801组上述指标均较模型组明显改善（P均〈0．05）。结论丹皮酚对离体培养的大鼠海马神经元OGD再灌注损伤具有保护作用，其作用机制可能与抑制EAA及NMDA受体表达有关。

Objective To observe the effect of paeonol on excitatory amino acid （EAA）, N-methyl-D-aspartate （NMDA） receptor expression in cultured rat hippocampal neurons subjected to oxygen-glucose deprivation/reperfusion （OGD/R）-indueed injury and to investigate its neuroprotective mechanism. Methods We divided rat hippocampal neurons which were primarily cultured for 8-12 d into four groups： the control group, OGD/R group, paeonol treatment group and MK-801 group, and then we established the models of OGD/R-induced injury, paeonol and MK-801 solution were separately injected into rats of the paeonol treatment group and MK-801 group at each reperfusion time points. The morphological changes of neurons were observed by the inverted phase-contrast microscopy, the neuron viability was measured by 1WIT assay, the binding force of NMDA receptor was valuated by liquid scintillation counting, the expression of NMDA receptor NR1 subunit mRNA was detected by RT-PCR, and the content of EAA was detected by amino acid analyzer. Results Neurons in the OGD/R group were swollen or deformed, with short processes and then gradually disappeared; the granules of cytoplasm were significantly degenerative, and even completely disintegrated. The morphological changes of neurons in paeonol group and MK-801 group were reduced as compared with the OGD/R group, and with integrated cell body, clear neurite; compared with the control group, the neuron survival rate was sig nificantly decreased, binding capacity, NMDA receptor NR1 subunit mRNA expression and EAA content were significantly in creased in the OGD/R group. Indexes were significantly improved in the paeonol group and MK-801 group as compared with that in OGD/R group （all P 〈0. 05 ）. Conclusion The paeonol has the protective effect on rat neurons subjected to OGD/R-in duced injury, whose mechanism may be associated with the inhibition of EAA and NMDA receptor expression.