乳铁蛋白对MPP^(+)诱导的帕金森病细胞模型炎症损伤的保护作用及机制研究

Ameliorative effect of lactoferrin on inflammatory injury in MPP^(+)induced cell model for Parkinson's disease and its mechanism

目的探讨外源性乳铁蛋白对1-甲基-4-苯基吡啶(MPP^(+))诱导的小鼠神经母细胞瘤N2a细胞帕金森病(PD)模型炎症损伤的保护作用及其机制。方法以250μmol/L的MPP^(+)诱导N2a细胞损伤构建PD细胞模型,将模型随机分为对照组、乳铁蛋白组、MPP^(+)组和乳铁蛋白+MPP^(+)组。采用CCK-8法检测细胞存活率;Annexin FITC/PI双染法检测细胞凋亡率;Hoechst33342染色检测细胞早期凋亡情况;检测各组的谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)水平;采用实时荧光定量聚合酶链反应(qRT-PCR)检测细胞IL-4、IL-6、IL-13、IL-1β、TNF-αmRNA的表达;Western blotting检测不同浓度MPP^(+)(0、100、250、500、1000μmol/L)N2a细胞的酪氨酸羟化酶(TH)和多巴胺转运体(DAT)蛋白的表达,检测各组的Bcl-2、Bax、Caspase-3、Cleaved Caspase-3、p38、p-p38、JNK、p-JNK、ERK、p-ERK蛋白的表达。结果MPP^(+)组细胞凋亡率较对照组上升(P<0.05),乳铁蛋白+MPP^(+)组细胞凋亡率较MPP^(+)组下降(P<0.05);MPP^(+)组细胞核亮染阳性数目较对照组增加(P<0.05),而乳铁蛋白+MPP^(+)组亮染阳性数目较MPP^(+)组减少(P<0.05);与对照组比较,MPP^(+)组Bax蛋白相对表达量升高(P<0.05),Bcl-2蛋白相对表达量减少(P<0.05),Cleaved Caspase-3/Caspase-3相对表达量升高(P<0.05);与MPP^(+)组比较,乳铁蛋白+MPP^(+)组Bax蛋白相对表达量降低(P<0.05),Bcl-2蛋白相对表达量升高(P<0.05),Cleaved Caspase-3/Caspase-3相对表达量降低(P<0.05)。与MPP^(+)组比较,乳铁蛋白+MPP^(+)组TH和DAT蛋白相对表达量升高(P<0.05),GSH-Px活性升高,MDA水平下降(P<0.05)。与MPP^(+)组比较,乳铁蛋白+MPP^(+)组促炎因子IL-6、IL-1β、TNF-αm RNA相对表达量降低,抑炎因子IL-4、IL-13 mRNA相对表达量升高(P<0.05)。与对照组比较,MPP^(+)组p-p38、p-JNK和p-ERK蛋白相对表达量升高,p-p38/p38、p-JNK/JNK、p-ERK/ERK比值增加(P<0.05);与MPP^(+)组比较,乳铁蛋白+MPP^(+)组p-p38、p-JNK和p-ERK蛋白相对表达量降低,p-p38/p38、p-JNK/JNK、p-ERK/ERK比值降低(P<0.05)。结论乳铁蛋白可能抑制MAPKs信号通路的活化,以及该通路活化所诱导的炎症反应,从而改善MPP^(+)所致N2a细胞的炎症损伤。

Objective To investigate the ameliorative impact and explore the underlying mechanism of exogenous lactoferrin(Lf)on inflammatory injury in the N2a cell model for Parkinson's disease(PD)induced by 1-methyl-4-phenylpyridinium(MPP^(+)).Methods N2a cell model for PD was established with 250μmol/L MPP^(+).Cells were treated with medium(control group),Lf(Lf group),MPP^(+)(model group)as well as Lf and MPP^(+)(Lf pretreatment group).The cell survival rate and apoptosis rate were assessed by cell counting kit-8(CCK-8)method and Annexin V-fluorescein isothiocyanate(FITC)/Propidium Iodide(PI)double staining,respectively.Hoechst33342 staining was used to examine early apoptosis of cells.Glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)level were assessed using detection kits.Meanwhile,the mRNA and protein expression of inflammatory cytokines were detected by real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting.Results Lf pretreatment reduced the apoptosis rate(P<0.05)and nuclear damage rate(P<0.05),up-regulated Bcl-2 protein expression(P<0.05),down-regulated Bax protein(P<0.05),and activated Caspase-3 expression(P<0.05)of N2a cells induced by MPP^(+).Compared with model group,the expressions of tyrosine hydroxylase(TH)and dopamine transporter(DAT)protein and GSH-Px activity in Lf pretreatment group were up-regulated(P<0.05),but MDA level was decreased(P<0.05).Compared to model group,cells in Lf pretreatment group had decreased mRNA expression levels of proinflammatory cytokines IL-6,IL-1β,and TNF-α,and increased mRNA expressions of anti-inflammatory cytokines IL-4 and IL-13(P<0.05).The phosphorylation levels of p38,c-JNK N-terminal kinase(JNK),and extracellular signal-regulated kinase(ERK)in Lf pretreatment group were lower than that in model group(P<0.05).Conclusions Lf may ameliorate the inflammatory damage to N2a cells induced by MPP^(+),through inhibiting the activation of mitogen-activated protein kinase(MAPKs)signaling pathway and the subsequent inflammatory response.