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

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

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7 d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10 min;SB203580低、中、高剂量组浓度分别为5、10、20μmol/L,继续培养24 h,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。

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 20 μmol/L） for 24 h 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 immunocyto- chemical（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 con- trol 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 in- jury, and SB203580 has a protective effect through activation of P-p38MAPK and down-regulation of caspase-3.