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

摘要:

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250 μmol/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-α mRNA的表达；Western blotting检测不同浓度MPP⁺（0、100、250、500、1 000 μmol/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-α mRNA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。

Abstract:

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 250 μmol/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.