槲皮素对N2a/APP细胞的神经保护作用机制

Mechanism study of neuroprotective effect of quercetin in N2a/APP cells

目的探究槲皮素在稳定表达人瑞典淀粉样蛋白前体蛋白APP695的小鼠神经母细胞瘤N2a细胞中神经保护作用的机制。方法采用稳转APP695瑞士突变的N2a细胞(N2a/APP)为研究对象,通过CCK-8法检测槲皮素对N2a/APP细胞活力的影响;选择不同浓度槲皮素处理N2a/APP细胞,并用蛋白质印迹法(Western blot)法检测槲皮素处理后N2a/APP细胞内β-淀粉样蛋白前体蛋白(APP)、25kDa突触关联蛋白(SNAP25)、突触素(Synaptophysin,SYP)和突触后致密区蛋白95(PSD95)、细胞外调节蛋白激酶(ERK1/2)通路蛋白、丝氨酸/苏氨酸蛋白激酶B(AKT)通路蛋白、胱氨酸/谷氨酸逆向转运系统的轻链亚基(xCT)蛋白及NADPH氧化酶4(NOX4)蛋白的表达水平;使用流式细胞术检测槲皮素处理后细胞活性氧自由基(ROS)和线粒体膜电位变化,免疫荧光法检测槲皮素处理后N2a/APP细胞内脂质过氧化和DNA损伤程度。结果槲皮素处理N2a/APP细胞后,APP表达显著降低,SNAP25和Synaptophysin表达明显升高,磷酸化ERK1/2水平显著降低,磷酸化AKT水平升高,NOX4表达降低,xCT表达明显升高,线粒体膜电位恢复,氧化应激、脂质过氧化和DNA氧化损伤程度降低,差异均有统计学意义(P<0.05)。结论槲皮素对N2a/APP细胞的神经保护作用机制可能与ERK1/2和AKT通路减轻氧化应激、恢复受损突触和损伤线粒体的功能有关。

Objective To explore the mechanism of the neuroprotective effect of quercetin on mouse neuroblastoma N2a cells with stably expressing human Swedish amyloid precursor protein APP695.Methods N2a cells with stably expressing Swiss mutated amyloid precursor protein(APP)cells(N2a/APP)were taken as research object.CCK-8 assay was used to examine the effect of quercetin on N2a/APP cell viability.N2a/APP cells were treated with different concentrations of quercetin.Westemm blot was performed to detect the expression protein levels of APP,synaptosomal-associated proteins:Synaptosomal-associated protein 25(SNAP25),Synaptophysin(SYP)and postsynaptic density protein 95(PSD95),the proteins in Extracellular Signal-Regulated Kinase 1 and 2(ERK1/2)pathway and Serine/threonine protein kinase B(AKT)pathway,cystine/glutamate antiporter system light chain subunit(xCT)and NADPH Oxidase 4(NOX4).Flow cytometry was used to detect cellular reactive oxygen species(ROS)levels and mitochondrial membranepotential(MMP).Immunofluorescent staining was used to examine lipid peroxidation and DNA-damage in N2a/APP cells after quercetin treatment.Results Quercetin treatment notably reduced APP protein expression,significantly increased SNAP25 and Synaptophysin protein expressions,reduced phosphorylated ERK1/2 level,elevated AKT phosphorylation level,decreased NOX4 expression,increased xCT expression,restored MMP,reduced oxidative stress,lipid peroxidation and DNA damage(P<0.05).Conclusion The neuroprotective effect of quercetin on N2a/APP cells may be related to reducing oxidative stress and restoring the function of damaged synapses and damaged mitochondria via ERK1/2 and AKT pathways.