Li Jing, Fan Ying, Zhang Yi-Na, Sun Dian-Jun, Fu Song-Bin, Ma Lan, Jiang Li-Hong, Cui Can, Ding Hai-Feng, Yang Jun
Department of Geriatrics, the Second Affiliated Hospital of Harbin Medical University, Harbin, China.
Pharmazie. 2012 Aug;67(8):718-24.
6-Hydroxydopamine (6-OHDA) is a widely used dopaminergic neurotoxin that leads to cell apoptosis in vivo and in vitro, and is a widely accepted experimental model of neurodegeneration in Parkinson's disease. However, the molecular mechanisms responsible for 6-OHDA-induced cell apoptosis are unclear. We found that the treatment of PC12 cells with 6-OHDA resulted in a significant decrease in cell viability and elevated apoptosis as detected by MTT assay, Hoechst 33258 staining, and flow cytometry. In addition, 6-OHDA induced a time-dependent phosphorylation of ERK1/2 at Thr-202/Tyr-204 and of Raf-1 at Ser-338, but a decreased level of Raf-1 phosphorylation at Ser-259. Phosphorylation of ERK1/2 at Thr-202/Tyr-204 and Raf-1 at Ser-338 were inhibited by the Raf-1 inhibitor GW5074, while the ERK1/2 pathway inhibitor U0126 decreased phosphorylation of ERK1/2. Furthermore, 6-OHDA-induced PC12 cells apoptosis was suppressed by GW5074 and U0126. Our results suggest that GW5074 and U0126 act as neuroprotants against 6-OHDA toxicity in PC12 cells by modulating Raf-1/ERK1/2 signaling systems.
6-羟基多巴胺（6-OHDA）是一种广泛应用的多巴胺能神经毒素，可导致体内外细胞凋亡，是帕金森病神经退行性变广泛接受的实验模型。然而，6-OHDA诱导细胞凋亡的分子机制尚不清楚。我们发现，用6-OHDA处理PC12细胞导致细胞活力显著下降，通过MTT法、Hoechst 33258染色和流式细胞术检测凋亡增加。此外，6-OHDA诱导ERK1/2在Thr-202/Tyr-204位点和Raf-1在Ser-338位点的时间依赖性磷酸化，但Raf-1在Ser-259位点的磷酸化水平降低。ERK1/2在Thr-202/Tyr-204位点和Raf-1在Ser-338位点的磷酸化被Raf-1抑制剂GW5074抑制，而ERK1/2通路抑制剂U0126降低ERK1/2的磷酸化。此外，GW5074和U0126抑制了6-OHDA诱导的PC12细胞凋亡。我们的结果表明，GW5074和U0126通过调节Raf-1/ERK1/2信号系统，作为抗6-OHDA对PC12细胞毒性的神经保护剂。
