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MAPKs介导NMDA诱导的大鼠皮层神经元凋亡

杨小荣, 孙萍, 秦花萍, 司沛沛, 孙雪菲, 张策

山西医科大学生理学系；第一医院营养科，太原 030001

摘要

NMDA诱导兴奋毒造成的神经损伤，包括细胞的凋亡和坏死。本研究旨在探讨神经元凋亡在NMDA兴奋毒所致大鼠皮层神经元死亡中的所占比例，并分析了NMDA致神经元凋亡的信号通路机制。通过使用Caspase抑制剂和测定乳酸脱氢酶活性，研究NMDA (100 μmol/L, 2 h)兴奋毒所致的神经元凋亡；并使用MAPKs选择性抑制剂，分别采用Caspase-3活性检测，TUNEL和Annexin V染色方法，进一步观察MAPKs通路中细胞外信号调节激酶(ERK)、c-Jun N-末端激酶(JNK)和p38 MAPK三条不同途径在NMDA所致神经元凋亡中的作用。结果显示：(1) Caspase依赖的凋亡占NMDA所致细胞死亡总数的22.49%；(2) p38 MAPK抑制剂SB203580 (10 μmol/L)使NMDA诱导的caspase-3活性降低30.43% (P < 0.05)；而ERK抑制剂PD98059 (20 μmol/L)和JNK抑制剂SP600125 (20 μmol/L)不影响caspase-3的活性；(3) SB203580 (10 μmol/L)使NMDA所致的TUNEL阳性细胞数减少33.10% (P < 0.05)；而PD98059 (20 μmol/L)或SP600125 (20 μmol/L)都没有作用；(4) Annexin V 染色结果显示，SB203580 (10 μmol/L)使NMDA所致的早期凋亡细胞减少55.56% (P < 0.05)；SP600125 (20 μmol/L)使NMDA所致的晚期凋亡/死亡细胞减少67.59% (P < 0.05)；PD98059 (20 μmol/L)对细胞凋亡/死亡没有明显作用。以上结果表明，NMDA介导的大鼠皮层神经元死亡除坏死外，还包含有一小部分神经元凋亡；p38 MAPK途径，而非JNK和ERK途径，介导了NMDA诱导的神经元凋亡，抑制与此相关的凋亡信号通路可发挥神经保护作用；JNK途径可能介导了NMDA所致的神经元坏死而非凋亡。

关键词： NMDA; 兴奋性神经毒; 凋亡; MAPKs

分类号：R338

Involvement of MAPK pathways in NMDA-induced apoptosis of rat cortical neurons

YANG Xiao-Rong, SUN Ping, QIN Hua-Ping, SI Pei-Pei, SUN Xue-Fei, ZHANG Ce

Department of Physiology； Department of Nutrition of the First Hospital, Shanxi Medical University, Taiyuan 030001, China

Abstract

NMDA-induced excitotoxicity cause severe neuronal damage including apoptosis and necrosis. The present study was aimed to evaluate the proportion of NMDA-induced apoptosis of rat cortical neurons and discover signal transduction mechanism. Caspase inhibitor and lactate dehydrogenase (LDH) assay were used to study the NMDA-induced apoptosis. To explore the involved signal pathways, the primary culture of rat cortical neurons were pretreated by the inhibitors of three MAPK pathways, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. With 2 h of NMDA treatment, cellular apoptosis was measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and Annexin V staining. The results showed that: (1) Caspase-dependent apoptosis accounted for 22.49% in NMDA-induced neuronal death; (2) Pretreatment with p38 MAPK inhibitor SB203580 (10 μmol/L) significantly decreased NMDA-mediated caspase-3 activity by 30.43% (P < 0.05). However, ERK inhibitor PD98059 (20 μmol/L) or JNK inhibitor SP600125 (20 μmol/L) did not influence caspase-3 activity; (3) Pretreatment with SB203580 significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05). PD98059 (20 μmol/L) or SP600125 (20 μmol/L) did not show obvious effect; (4) Pretreatment with SB203580 (10 μmol/L) significantly reduced the number of NMDA-induced early apoptotic neurons by 55.56% (P < 0.05). Also, SP600125 (20 μmol/L) significantly decreased the amount of late apoptotic/dead cells by 67.59% (P < 0.05). There was no effect of PD98059 (20 μmol/L). These results indicate that: (1) NMDA induces neuronal apoptosis besides necrosis; (2) p38 MAPK, but not JNK and ERK, is involved in NMDA-induced neuronal apoptosis, and inhibition of the apoptotic signaling pathway contributes to neuroprotection; (3) JNK activation might contribute to NMDA-induced neuronal necrosis rather than apoptosis.

Key words: NMDA; excitotoxicity; apoptosis; MAPKs

收稿日期：2012-03-10　　录用日期：2012-05-28

通讯作者：张策　　E-mail: cezh2002@yahoo.com

引用本文：

杨小荣, 孙萍, 秦花萍, 司沛沛, 孙雪菲, 张策. MAPKs介导NMDA诱导的大鼠皮层神经元凋亡[J]. 生理学报 2012; 64 (6): 609-616.

YANG Xiao-Rong, SUN Ping, QIN Hua-Ping, SI Pei-Pei, SUN Xue-Fei, ZHANG Ce. Involvement of MAPK pathways in NMDA-induced apoptosis of rat cortical neurons. Acta Physiol Sin 2012; 64 (6): 609-616 (in Chinese with English abstract).