泛素蛋白酶体抑制剂诱发多巴胺能神经元内质网应激反应及其凋亡的作用

Effects of ubiquitin proteasome inhibitor induced endoplasmic reticulum stress and apoptosis on dopaminergic neuron

目的 探讨泛素蛋白酶体(ubiquitin proteasome,UP)功能失调诱发的内质网应激反应(endoplasmic reticulum stress response,ERS)机制在多巴胺(dopamine,DA)能神经元变性死亡中的作用.方法 通过MTT及流式细胞仪检测UPS抑制剂Lactacystin对NGF诱导的PC12细胞的神经毒性作用,应用RT-PCR和Western blot技术观察Lactacystin处理后ERS通路中XBP1、Grp78、CHOP表达水平的变化及利血平耗竭胞内DA水平后的影响.结果 不同浓度的Lactacystin 5～20μmol/L处理PC12细胞后,细胞活力呈浓度依赖性下降,其中Lactacystin 10μmo1/L作用24h使细胞活力下降51%.在相同浓度条件下(10μmol/L)流式细胞仪显示的细胞凋亡率在4h、8h、1 6h、24h内逐渐增高(6.1%、14.1%、24.9%、30.9%)(P＜0.01).RT-PCR及免疫印迹检测显示UPS抑制剂处理后XBP1、Grp78和CHOP的基因及蛋白表达水平明显增加,分别在8h、16～24h达到高峰(P＜0.05).Caspase-12基因水平也在Lactacystin诱导后16h显著升高(P＜0.05).利血平预处理则使CHOP、caspase-12基因表达减弱(P＜0.05).结论 UPS功能抑制诱发的内质网应激和相关凋亡途径机制是DA能神经元选择性变性死亡的内在环节之一.

Objective To explore the mechanism of endoplasmic reticulum stress response(ERS) induced by ubiquitin proteasome(UP) dysfunction in dopaminergic neurons death. Methods The neurotoxicity of UPS inhibitor,lactacystin,on NGF treated-PC12 cells was measured by using MTT assay and flow cytometry. The expression of ERS-related gene XBP1,Grp78,CHOP,caspase-12 in lactacystin-treated group and reserpine preincubation group was determined with RT-polymerase chain reaction (RT-PCR)and Western blot. Results After exposed to different concentration of lactacystin(5～20μmol/L),PC12 cells showed dose-dependent decrease in cell vitality,51 decline at 10μmol/L concentration for 24 hours. FCM assay confirmed time-dependent cell apoptosis for 4h,8h,16h,24h under 10μmol/L lactacystin treated(6.1,14.1,24.9,30.9,respectively)(P< 0.01 ). The gene and protein expression of XBP1 and Grp78 in lactacystin treated group were significantly increased and reached peak at 8h (P< 0.05 ). The expression level of CHOP and transcription of caspase-12 were increased at 16～24h (P< 0.05 ),but alleviated by reserpine preincubayion(P< 0.05 ). Conclusion The dysfunction of UPS might induce dopaminergic neuron selective death resulted from excessive ERS and relative activated cell apoptosis pathway.