Objective： To explore the mechanism of endoplasmic reticulum stress（ERS） response and related apoptosis in dopaminergic neurons death. Methods： Nerve growth factor （NGF）-treatedPC12 cells were treated with 6-OHDA, MPP^＋ and rotenone. MTT assay and flow cytometry were used to measure the cell viability and the rate of celluar apoptosis induced by those neurotoxins. The expression of ERS-related gene XBP1, Grp78, CHOP, caspase-12 in drug-treated group and reserpine preincubafion group was determined with RT-polymerase chain reaction（RT-PCR） and immunohistochemistry. Results： After the exposure to different toxins, the viability of PC12 cells were decreased by 52%, 44%, 40% at 100μM6-OHDA, 75 μM MPP^＋, 20 nM rotenone for 24 h respectively. FCM assay confirmed time-dependent cell apoptosis （P 〈 0.01 ）. The gene and protein expression of XBP1, Grp78 in drug-treated group were significantly increased and reached their peaks 8 h after the treatment（P 〈 0.05）. The expression levels of CHOP and caspase-12 gene were increased 16-24 h after the treatment（P 〈 0.01 ）, but the expression level of caspase-12 was inhibited by reserpine preincubayion（P 〈 0.05）. Conclusion： The excessive ERS and relative activated cell apoptosis pathway may be associated with selective death of dopaminergic neurons.
Lan Wang~, Shenggang Sun~, Xuebing Cao~, Zhentao Zhang~ and Li Xu~ Shenggang Sun Xuebing Cao Zhentao Zhang Li Xu
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 PC 12 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 PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 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 PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.
This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine （6-OHDA） in PC12 cells. By using neural differentiated PC12 cells treated with 6-OHDA, the apoptosis model of dopaminergic neurons was established. Cell viability was measured by MTT. Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry. Western blot was used to detect the activation of extracellular regulator kinasel/2 （ERK1/2） pathway and the phosphorylation of retinoblastoma protein （RB）. Our results showed that after PC12 cells were treated wtih 6-OHDA, the viability of PC12 cells was declined in a concentration-dependent manner. Flow cytornetry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner. The percentage of ceils in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased. Simultaneously, ERK1/2 pathway was activated and phosphorylated RB increased. It was concluded that 6-OHDA could induce cell cycle reentry of dopaminergic neurons through the activation of ERK1/2 pathway and RB phosphorylation. The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.
