摘要
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。
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.
基金
supported by the International Cooperation Project of Science and Technology Department of Heilongjiang Province, China (No.WB04301, No.WB08B05)
the Science and Technology Foundation of Education Department of Heilongjiang Province, China (No.11521076)
