Zhang Qi-Shan, Liao Yuan-Gao, Ji Zhong, Gu Yong, Jiang Hai-Shan, Xie Zuo-Shan, Pan Su-Yue, Hu Ya-Fang
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China; Department of Neurology, Chenzhou First People's Hospital, Chenzhou, Hunan 423000, P.R. China.
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China.
Exp Ther Med. 2016 Oct;12(4):2594-2598. doi: 10.3892/etm.2016.3658. Epub 2016 Sep 5.
The present study aimed to investigate the protective effect of a modified p5 peptide, TFP5, on 1-methyl-4-phenyl pyridine ion (MPP)-induced neurotoxicity in cortical neurons and explore the therapeutic effect of TFP5 on Parkinson's disease (PD). MPP was applied to a primary culture of mouse cortical neurons to establish the cell model of PD. Neurons were divided into four groups: Control, model (MPP), scrambled peptide (Scb) (Scb + MPP) and TFP5 (TFP5 + MPP) groups. Pretreatment with Scb or TFP5 was applied to the latter two groups, respectively, for 3 h, while phosphate-buffered saline was applied to the control and model groups. MPP was then applied to all groups, with the exception of the control group, and neurons were cultured for an additional 24 h. Neuron viability was evaluated using a Cell Counting kit-8 (CCK8) assay. To explore the mechanism underlying the protective effects of TFP5, the expression levels of p35, p25 and phosphorylated myocyte enhancer factor 2 (p-MEF2D) were determined by western blotting. Fluorescence microscopy showed that TFP5 was able to pass through cell membranes and distribute around the nucleus. CCK8 assay showed that neuronal apoptosis was dependent on MPP concentration and exposure time. Cell viability decreased significantly in the model group compared with the control group (55±7 vs. 100±0%; P<0.01), and increased significantly in the TFP5 group compared with the model group (98±2 vs. 55±5%; P<0.01) and Scb group (98±2 vs. 54±4%; P<0.01). Scb exhibited no protective effect. Western blotting results showed that MPP induced p25 and p-MEF2D expression, TFP5 and Scb did not affect MPP-induced p25 expression, but TFP5 reduced MPP-induced p-MEF2D expression. In summary, TFP5 protects against MPP-induced neurotoxicity in mouse cortical neurons, possibly through inhibiting the MPP-induced formation and elevated kinase activity of a cyclin-dependent kinase 5/p25 complex.
本研究旨在探讨修饰的p5肽TFP5对1-甲基-4-苯基吡啶离子（MPP）诱导的皮质神经元神经毒性的保护作用，并探索TFP5对帕金森病（PD）的治疗效果。将MPP应用于小鼠皮质神经元原代培养物以建立PD细胞模型。神经元分为四组：对照组、模型组（MPP）、乱序肽组（Scb）（Scb + MPP）和TFP5组（TFP5 + MPP）。后两组分别用Scb或TFP5预处理3小时，而对照组和模型组给予磷酸盐缓冲盐水。然后除对照组外，对所有组施加MPP，并将神经元再培养24小时。使用细胞计数试剂盒-8（CCK8）测定法评估神经元活力。为了探究TFP5保护作用的潜在机制，通过蛋白质印迹法测定p35、p25和磷酸化的肌细胞增强因子2（p-MEF2D）的表达水平。荧光显微镜检查显示TFP5能够穿过细胞膜并分布在细胞核周围。CCK8测定法显示神经元凋亡取决于MPP浓度和暴露时间。与对照组相比，模型组细胞活力显著降低（55±7 vs. 100±0%；P<0.01），与模型组相比，TFP5组细胞活力显著增加（98±2 vs. 55±5%；P<0.01），与Scb组相比也显著增加（98±2 vs. 54±4%；P<0.01）。Scb没有显示出保护作用。蛋白质印迹结果显示，MPP诱导p25和p-MEF2D表达，TFP5和Scb不影响MPP诱导的p25表达，但TFP5降低了MPP诱导的p-MEF2D表达。总之，TFP5可保护小鼠皮质神经元免受MPP诱导的神经毒性，可能是通过抑制MPP诱导的细胞周期蛋白依赖性激酶5/p25复合物的形成和激酶活性升高来实现的。
