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 preincubation 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 100 μM6-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
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 kinase1/2 (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 cytometry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner. The percentage of cells 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 phos-phorylated RB increased. It was concluded that 6-OHDA could induce cell cycle reentry of dopa-minergic neurons through the activation of ERK1/2 pathway and RB phosphorylation. The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.
