Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCδ phosphorylation

Rottlerin通过抑制PKCδ磷酸化减轻6羟基多巴胺对多巴胺能细胞的毒性作用(英文)

Objective The present study aims to investigate the role of protein kinase C 5 subtype （PKCS） phosphorylation in the process of 6-hydroxydopamine （6-OHDA）-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson＇ s disease. Methods The pheochromocytoma （PC 12） cell line was employed in the present study. Cells were treated with 2 μmol/L PKC5 inhibitor Rottlerin, 10 nmol/L protein kinase C α subtype （PKCα） inhibitor bisindolylmaleimide 1, or 5 nmol/L G66976 that could specifically inhibit the calcium-dependent PKC isoforms, respectively. PKC8 activator phorbol-12-myristate-13-acetate （PMA, 100 nmol/L） was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotox- icity of 6-OHDA was determined by methyl thiazolyl tetrazolium （MTT） reduction assay and PKCδ phosphorylation levels in various groups were measured by western blotting. Results Bisindolylmaleimide I and Go6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCδ phosphorylation in PC 12 cells. However, Rottlerin could inhibit the phosphorylation of PKC5 and attenuate 6-OHDA-induced cell death, and the cell viability was raised to 69.6 ±2.63% of that in control group （P 〈 0.05）. In contrast, PMA induced a significant increase in PKC5 phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to 49.8±5.06% of that in control group （P 〈 0.001）. Conclusion Rottlerin can protect PC 12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKC δ phosphorylation. The results suggest that PKCδ may be a key regulator of neuron loss in Parkinson＇s disease.

目的观察蛋白激酶Cδ亚型(PKCδ)磷酸化在6羟基多巴胺(6-OHDA)引起的多巴胺能神经细胞死亡过程中的作用,探讨帕金森病中神经元缺失的分子机制。方法体外培养大鼠嗜铬细胞瘤细胞系PC12细胞,观察预先加入的PKC抑制剂(bisindolylmaleimide I,G6976及Rottlerin)和激动剂佛波酯对6-OHDA毒性作用的影响,噻唑蓝比色法检测细胞存活率,免疫印迹法观察磷酸化PKCδ的表达。结果PKCδ抑制剂Rottlerin(2μmol/L)可抑制PKCδ的磷酸化,减轻6-OHDA引起的细胞死亡,细胞存活率上升至69.6±2.63%(P<0.05)。PKCα抑制剂bisindolylmaleimide I和钙依赖性PKC抑制剂G6976对6-OHDA的毒性作用及PKCδ磷酸化均无显著影响,而PKCδ激活剂佛波酯(100nmol/L)能提高PKCδ磷酸化水平,加重6-OHDA的损害作用,使细胞存活率下降至单用6-OHDA组水平的49.8±5.06%(P<0.001)。结论Rottlerin能抑制PKCδ的磷酸化,进而减轻6-OHDA对多巴胺能神经细胞死亡的诱导作用,说明PKCδ505位点丝氨酸的磷酸化是6-OHDA发挥毒性作用的关键,提示PKCδ在帕金森病病人神经元缺失中起重要作用。