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目的探讨内质网应激反应(endoplasmic reticulum stress response,ERS)及相关凋亡途径在多巴胺能神经元变性死亡中的作用。方法6-羟基多巴胺(6-OHDA)、N-甲基-4苯基吡啶离子(MPP~+)、鱼藤酮作用于神经生长因子(NGF)诱导的PC12细胞,MTT及流式细胞仪方法检测各种药物的神经毒性作用及细胞凋亡率,应用RT-PCR和Western印迹技术观察以上药物处理后ERS通路中XBP1、Grp78、CHOP表达水平的变化及相关凋亡因子caspase-12的活化和利血平耗竭胞内多巴胺水平后的影响。结果3种神经诱变剂处理PC12细胞后,细胞活力呈浓度依赖性下降,其中6-OHDA 100μmol/L、MPP~+75μmol/L、鱼藤酮20 nmol/L作用24 h使细胞活力分别下降52%、44%、40%。流式细胞仪显示的细胞凋亡率在8 h、16 h、24 h内逐渐增高(P＜0．01)。RT-PCR及免疫组化检测显示处理后XBP1、Grp78表达和CHOP的基因及蛋白表达水平明显增加,分别在8 h、16～24 h达到高峰(P＜10．01)。caspase-12 mRNA水平也在诱导后16 h显著升高(P＜0．01),利血平预处理使其基因表达减弱(P＜0．05)。结论内质网应激和相关凋亡途径是多巴胺能神经元选择性变性死亡的内在环节之一。
Objective To investigate the role of endoplasmic reticulum stress response (ERS) and related apoptotic pathways in the degeneration of dopaminergic neurons. Methods 6-OHDA, MPP + and rotenone were applied to PC12 cells induced by nerve growth factor (NGF) The neurotoxicity and apoptosis rate of these drugs were observed. The changes of XBP1, Grp78, CHOP expression in ERS pathway and the activation of caspase-12 and the activation of pro-apoptotic factor caspase-12 were observed by RT-PCR and Western blotting. The effect of flat depletion of intracellular dopamine levels. Results The viability of PC12 cells decreased with the concentration of 6-OHDA 100μmol / L, MPP + 75μmol / L and rotenone 20 nmol / L for 24 hours, respectively. The viability of PC12 cells decreased by 52% , 44%, 40%. Flow cytometry showed that the rate of apoptosis increased gradually within 8 h, 16 h and 24 h (P <0.01). RT-PCR and immunohistochemistry showed that the expression of XBP1, Grp78 and CHOP gene and protein were significantly increased after treatment, reaching the peak at 8 h and 16-24 h respectively (P <10.01). The level of caspase-12 mRNA was also significantly increased at 16 h after induction (P <0.01). Pretreatment with reserpine decreased the gene expression of caspase-12 (P <0.05). Conclusion Endoplasmic reticulum stress and related apoptosis pathways are one of the internal processes of selective denaturation of dopaminergic neurons.