Chen Dan, Kanthasamy Anumantha G, Reddy Manju B
Department of Food Sciences and Human Nutrition, Iowa State University, 220 Mackay Hall, Ames, IA 50010, USA.
Department of Biomedical Sciences, Iowa State University, Ames, IA 50010, USA.
Parkinsons Dis. 2015;2015:843906. doi: 10.1155/2015/843906. Epub 2015 Dec 6.
Background. Parkinson's disease (PD) is a progressive neurodegenerative disease that causes severe brain dopamine depletion. Disruption of iron metabolism may be involved in the PD progression. Objective. To test the protective effect of (-)-epigallocatechin-3-gallate (EGCG) against 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity by regulating iron metabolism in N27 cells. Methods. Protection by EGCG in N27 cells was assessed by SYTOX green assay, MTT, and caspase-3 activity. Iron regulatory gene and protein expression were measured by RT-PCR and Western blotting. Intracellular iron uptake was measured using (55)Fe. The EGCG protection was further tested in primary mesencephalic dopaminergic neurons by immunocytochemistry. Results. EGCG protected against 6-OHDA-induced cell toxicity. 6-OHDA treatment significantly (p < 0.05) increased divalent metal transporter-1 (DMT1) and hepcidin and decreased ferroportin 1 (Fpn1) level, whereas pretreatment with EGCG counteracted the effects. The increased (55)Fe (by 96%, p < 0.01) cell uptake confirmed the iron burden by 6-OHDA and was reduced by EGCG by 27% (p < 0.05), supporting the DMT1 results. Pretreatment with EGCG and 6-OHDA significantly increased (p < 0.0001) TH(+) cell count (3-fold) and neurite length (12-fold) compared to 6-OHDA alone in primary mesencephalic neurons. Conclusions. Pretreatment with EGCG protected against 6-OHDA-induced neurotoxicity by regulating genes and proteins involved in brain iron homeostasis, especially modulating hepcidin levels.
背景。帕金森病（PD）是一种进行性神经退行性疾病，会导致大脑多巴胺严重耗竭。铁代谢紊乱可能参与帕金森病的进展。目的。通过调节N27细胞中的铁代谢，测试（-）-表没食子儿茶素-3-没食子酸酯（EGCG）对6-羟基多巴胺（6-OHDA）诱导的神经毒性的保护作用。方法。通过SYTOX green检测、MTT法和半胱天冬酶-3活性评估EGCG对N27细胞的保护作用。通过逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测铁调节基因和蛋白质表达。使用（55）Fe测量细胞内铁摄取。通过免疫细胞化学在原代中脑多巴胺能神经元中进一步测试EGCG的保护作用。结果。EGCG可防止6-OHDA诱导的细胞毒性。6-OHDA处理显著（p<0.05）增加二价金属转运蛋白1（DMT1）和铁调素水平，并降低铁转运蛋白1（Fpn1）水平，而EGCG预处理可抵消这些作用。（55）Fe细胞摄取增加（96%，p<0.01）证实了6-OHDA导致的铁负荷，EGCG可使其降低27%（p<0.05），支持DMT1检测结果。与单独使用6-OHDA相比，EGCG和6-OHDA预处理在原代中脑神经元中显著增加（p<0.0001）TH（+）细胞计数（约3倍）和神经突长度（约12倍）。结论。EGCG预处理通过调节参与脑铁稳态的基因和蛋白质，特别是调节铁调素水平，防止6-OHDA诱导的神经毒性。
