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目的观察蛋白激酶Cδ亚型(PKCδ)磷酸化激活在6羟基多巴胺(6-OHDA)引起的多巴胺能神经细胞死亡过程中所起作用,探讨帕金森病中神经元缺失的分子发病机制。方法体外培养多巴胺能神经细胞系SH-SY5Y细胞,观察预先加入的PKC抑制剂和激活剂对6-OHDA毒性作用的影响,噻唑蓝比色法观察细胞生存率,Western免疫印迹法观察磷酸化PKCδ的表达。结果PKCδ抑制剂rottlerin2μmol/L可抑制PKCδ磷酸化激活,减轻6-OHDA引起的细胞死亡约57%±6%(P<0.01)。总PKC抑制剂双吲哚亚酰胺(BisindolylmaleimideⅠ)和钙依赖性PKC抑制剂G6976不影响6-OHDA的毒性作用和PKCδ激活,而PKCδ激活剂100nmol/L佛波酯,增加磷酸化PKCδ水平,加重6-OHDA的损害,使细胞生存率下降至单用6-OHDA时生存率的66%±9%(P<0.01)。结论磷酸化激活的PKCδ是6-OHDA发挥毒性作用的关键一环,抑制PKCδ可减轻6-OHDA诱导的多巴胺能神经细胞死亡,说明PKCδ在帕金森病患者神经元缺失中可能起作用。
Objective To investigate the role of phosphorylation of protein kinase Cδ subtype (PKCδ) in dopaminergic neuronal death induced by 6-hydroxydopamine (6-OHDA) and to explore the molecular pathogenesis of neuronal loss in Parkinson’s disease. Methods The dopaminergic neuronal cell line SH-SY5Y cells were cultured in vitro. The effects of PKC inhibitor and activator added beforehand on the toxicity of 6-OHDA were observed. Cell viability was observed by thiazolyl blue colorimetric assay. The phosphorylation of PKCδ expression. Results PKCδ inhibitor rottlerin (2μmol / L) inhibited PKCδ phosphorylation and decreased cell death induced by 6-OHDA by about 57% ± 6% (P <0.01). Bisindolylmaleimide I, a total PKC inhibitor, and G6976, a calcium-dependent PKC inhibitor, did not affect the toxic effect of 6-OHDA and PKCδ activation, whereas PKCδ activator, 100nmol / L of phorbol ester, increased the level of phosphorylated PKCδ, Damage to 6-OHDA decreased cell survival to 66% ± 9% (p <0.01) of survival with 6-OHDA alone. CONCLUSION: Phosphorylation-activated PKCδ is a key factor in the toxic effect of 6-OHDA. Inhibition of PKCδ attenuates 6-OHDA-induced dopaminergic neuronal death, suggesting that PKCδ may play a role in neuronal loss in patients with Parkinson’s disease.