Abstract
Objective
To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP+) and to explore the potential mechanisms.
Methods
The viability and apoptosis of PC12 cells were determinded by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4′,6′-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2).
Results
The cell viability decreased and the number of apoptotic cells increased dramatically in MPP+ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP+-treated PC12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC12 cells induced by HPP.
Conclusion
HPP protects PC12 cells against MPP+ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.
Keywords: hydrogen peroxide preconditioning, 14-3-3 protein, ERK1/2, p38 mitogen-activated protein kinase, PC12 cell
摘要
目的
探讨H2O2预处理对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium, MPP+)诱导PC12细胞毒性损伤的保护作用及其可能的机制。
方法
分别用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑嗅盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]和4,6-二氨基-2-苯基吲哚(4′,6′-diamidino-2-phenylindole, DAPI)染色技术检测PC12细胞的活力及细胞凋亡核形态改变; Western blot检测PC12细胞中14-3-3蛋白、 磷酸化p38 MAPK的水平。 酶联免疫吸附实验(Enzyme-linked immunosorbent assay, ELISA)试剂盒检测ERK1/2磷酸化水平。
结果
MPP+处理PC12细胞24 h使细胞活力显著下降(51.6%), DAPI染色显示(51.3±6.6)%的PC12细胞呈现核固缩等细胞凋亡形态改变; H2O2预处理能显著增加PC12细胞活力(83.4%), 减少凋亡细胞数[(24.9±4.3)%], 说明H2O2预处理能对抗MPP+毒性, 对PC12细胞具有保护作用。 Western blot结果显示, H2O2预处理在减少PC12细胞凋亡的同时伴随有14-3-3蛋白及磷酸化p38 MAPK表达上调; ELISA实验显示ERK1/2磷酸化水平显著增加。 相关分析显示14-3-3蛋白表达的上调与ERK1/2磷酸化水平呈正相关(r = 0.923, P < 0.01), 而且PD98059抑制ERK1/2磷酸化后, H2O2预处理诱导14-3-3蛋白表达上调的作用消失。
结论
H2O2预处理能对抗MPP+对PC12细胞的毒性作用, 这种保护作用与细胞内14-3-3蛋白表达上调有关; 而14-3-3蛋白表达上调与ERK1/2和p38 MAPK信号通路的激活有关。
关键词: H2O2预处理, 14-3-3蛋白, ERK1/2, p38有丝分裂原激活蛋白激酶, PC12细胞
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