NMDA经p38MAPK信号通路诱导体外培养的皮层神经元损伤

NMDA induces cortical neuron injury in vitro via p38MAPK signaling pathway

目的观察MK801及SB203580对N-甲基-D-天门冬氨酸(NMDA)诱导的皮层神经元损伤的影响,探讨p38丝裂原活化蛋白激酶(p38MAPK)信号通路在神经元损伤中的作用和机制。方法培养7 d的新生SD大鼠皮层神经元,分为5组:对照组、NMDA损伤组、MK801干预组、SB203580干预组、联合干预组(SB203580+MK801)。噻唑蓝(MTT)比色实验评价细胞生存力,乳酸脱氢酶(LDH)释放率测定细胞损伤程度,吖啶橙/溴化乙锭(AO/EB)双重荧光染色观察细胞凋亡形态和数目,免疫细胞化学染色法和Western blot法检测大鼠皮层神经元各组p-p38MAPK、Bcl-2和BAX表达情况。结果同对照组比较,NMDA损伤组神经细胞存活力显著降低,培养上清液中LDH含量明显增加,凋亡细胞增多,p-p38MAPK、BAX表达增加,Bcl-2表达降低(P<0.05,P<0.01);同NMDA损伤组比较,各个干预组细胞生存力提高,LDH释放率降低,凋亡减少,p-p38MAPK及BAX的表达减少,Bcl-2表达增多(P<0.05,P<0.01)。结论 MK801、SB203580对NMDA诱导的皮质神经元损伤具有保护作用,p38MAPK通路可能参与介导MK801的保护作用,其共同机制部分是通过抑制p38MAPK信号通路介导的凋亡相关蛋白实现的。

Objective To observe the effects of MK801 and SB203580 on N-methyl-D-aspartic acid （NMDA）-induced cerebral cortical neuron injury and to determine the role and possible action mechanism of p38 mitogen-activated protein kinases （p38MAPK） signaling pathway in neuron injury. Methods Newborn SD rat cortical neurons were cultured for 7 d, and then randomly assigned to 5 groups： a control group, an NMDA injury group, an MK801 interventional group, an SB203580 interventional group, and a combined inter- ventional group （ SB203580 ＋ MK801 ）. MT＂ assay and lactate dehydrogenase （ LDH ） release assay were employed to assess primary neuron viability and cell membrane damage, respectively. Acridine orange/ethidium bromide （AO/EB） double fluorescent staining was used to observe morphology and cell apoptosis. Expression of p-p38MAPK, Bcl-2 and BAX was observed separately by IHC and Western blotting. Results Compared to the control group, markedly decreased cell viability, significantly increased LDH leakage and apoptotic cell number, increased expression of p-p38MAPK and BAX, and decreased expression of Bcl-2 were observed in the NMDA injury group （P 〈 0.05, P 〈 0. 01 ）. Compared to the NMDA injury group, MK801 and/or SB203580 improved cell viability, reduced LDH release rate and apoptosis, decreased the expression of p-p38MAPK and BAX, and increased the expression of Bel-2 （P 〈 0.05, P 〈 0. 01 ） in the interventional groups. Conclusion MK801 and SB203580 have protective effects on neurons with NMDA-induced injury. MK801 may protect the neurons from NMDA-indueed injury through p38MAPK signaling pathway. The common mechanism involves partial inhibition of p38MAPK signaling pathway-mediated apoptosis-retated proteins.