p38MAPK磷酸化水平及Caspase-3在NMDA诱导的体外培养神经元中的激活及SB203580的保护作用

山东大学学报(医学版) ›› 2012, Vol. 50 ›› Issue (6): 51-56.

p38MAPK磷酸化水平及Caspase-3在NMDA诱导的体外培养神经元中的激活及SB203580的保护作用

刘学文1，朱锦莉2，田步先1，李熙东1，蔡爱民1，邢瑞仙1，张雪杰1，李秋实1

1.辽宁医学院附属第一医院神经内科, 辽宁锦州 121001；
2.锦州市中心医院神经内科, 辽宁锦州 121000

收稿日期:2011-09-13 出版日期:2012-06-10 发布日期:2012-06-10
作者简介:刘学文(1969- )，女，副主任医师，硕士生导师，主要从事癫痫的基础与临床研究
基金资助:
辽宁省科技厅博士启动基金资助项目(20091049)

Activations of P-p38MAPK and caspase-3 in NMDA-induced cultured
cortical neurons in vitro and the protective effect of SB203580

LIU Xue-wen1, ZHU Jin-li2, TIAN Bu-xian1, LI Xi-dong1, CAI Ai-min1,
XING Rui-xian1, ZHANG Xue-jie1, LI Qiu-shi1

1. Department of Neurology, The First Affiliated Hospital of Liaoning Medical College, Jinzhou 121001, Liaoning, China;
2. Department of Neurology, Jinzhou Central Hospital, Jinzhou 121000, Liaoning, China

Received:2011-09-13 Online:2012-06-10 Published:2012-06-10

摘要/Abstract

摘要：

目的探讨p38丝裂原活化蛋白激酶磷酸化(P-MAPK)水平及半胱氨酸蛋白酶-3(Caspase-3)在N-甲基-D-天冬氨酸(NMDA)诱导的体外培养神经元中的激活及p38MAPK抑制剂SB203580的保护作用。方法随机将原代培养7d的大鼠皮质神经元分为对照组， NMDA组，SB203580低、中、高剂量组。对照组仅用DMEM/F12完全培养基，NMDA组去除正常神经元培养液，加入50μmol/L NMDA，处理时间10min；SB203580低、中、高剂量组浓度分别为5、10、20μmol/L，继续培养24h，加入50μmol/L NMDA。采用MTT法评价细胞生存力，丫啶橙(AO)染色检测凋亡细胞数量及乳酸脱氢酶(LDH)含量；免疫细胞化学染色法(IHC)及免疫印记法(WB)检测皮质神经元内PMAPK及Caspase-3的表达水平。结果与对照组比较，NMDA组的吸光度值明显降低、神经元凋亡明显增加、P-MAPK及Caspase-3表达增加(P均<0.01)，SB203580低、中、高剂量组PMAPK及Caspase-3随着浓度的增加表达减少，以高剂量组为明显(P<0.05，P<0.01)；与NMDA组比较， SB203580低、中、高剂量组吸光度值均升高，以高剂量组为明显，细胞凋亡数量明显减少(P均<0.05)。结论 P-MAPK及Caspase-3在NMDA诱导的皮质神经元中表达增强，SB203580对NMDA诱导的神经元损伤具有保护作用，其作用机制可能与p38MAPK的活化，进而直接或间接激活Caspase-3的表达有关。

关键词: p38丝裂原活化蛋白激酶磷酸化；半胱氨酸天冬氨酸蛋白酶3；受体，N-甲基-D-天冬氨酸；细胞凋亡

Abstract:

Objective To investigate the activations of phosphorylation of p38MAPK(P-p38MAPK) and caspase-3 in cultured cortical neurons after NMDA injury and the protective effect of SB203580. Methods Primary cortical neurons cultured for 7d were randomly divided into 5 groups: the control group, the NMDA group and low， medium and high doses of SB203580 groups. The cortical neurons were pre-cultured with regular media including different doses of SB203580 (5,10 and 20μmol/L) for 24h before being exposed to NMDA (50μmol/L). MTT assay was used to evaluate cell survival. Apoptotic cortical neurons were examined using acridine fluorescent staining. Content of lactic dehydrogenase(LDH) was detected. P-p38MAPK and caspase-3 protein expressions were detected by immunocytochemical(IHC) staining and Western blot (WB). Results Compared with the control group, the OD value of the NMDA group was significantly increased(P<0.01), while it was decreased in the low, medium and high dose groupsof SB203580(P<0.05), and apoptotic cortical neurons were significantly increased in the NMDA group(P<0.01). Compared with the NMDA group, the number of apoptotic cortical neurons was decreased in SB203580 groups(P<0.05). Content of LDH was significantly increased in the NMDA group compared with the control group, (P<0.01)， while it was significantly decreased in SB203580 groups compared with the NMDA group(P<0.05). Expressions of P-p38MAPK and caspase-3 in cultured neuron were remarkably up-regulated in the NMDA-induced group than in the control group, while they were significantly down-regulated in SB203580 groups than in the NMDA group (P<0.05 or P<0.01). Conclusion P-p38MAPK and caspase-3 are activated in cultured cortical neurons after NMDA-induced injury, and SB203580 has a protective effect through activation of P-p38MAPK and down-regulation of caspase-3.

Key words: Phosphorylation of p38mitogen-activated protein kinase; Caspase 3; Receptors, N-methyl-D-aspartate; Apoptosis

中图分类号:

R322.81

刘学文1，朱锦莉2，田步先1，李熙东1，蔡爱民1，邢瑞仙1，张雪杰1，李秋实1. p38MAPK磷酸化水平及Caspase-3在NMDA诱导的体外培养神经元中的激活及SB203580的保护作用[J]. 山东大学学报(医学版), 2012, 50(6): 51-56.

LIU Xue-wen1, ZHU Jin-li2, TIAN Bu-xian1, LI Xi-dong1, CAI Ai-min1, XING Rui-xian1, ZHANG Xue-jie1, LI Qiu-shi1. Activations of P-p38MAPK and caspase-3 in NMDA-induced cultured
cortical neurons in vitro and the protective effect of SB203580[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2012, 50(6): 51-56.

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