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

作　　者：汪晓语 单树方 吕嘉琦 赵儒花 周嗣全 成果 张伶俐[2] 张林 Wang Xiao-yu;Shan Shu-fang;LüJia-qi;Zhao Ru-hua;Zhou Si-quan;Cheng Guo;Zhang Ling-li;Zhang Lin(Key Laboratory of Birth Defects and Related Diseases of Women and Children(Sichuan University),Ministry of Education,West China Second University Hospital,Sichuan University,Chengdu,Sichuan 610041,China;Department of Pharmacy,West China Second University Hospital,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]四川大学华西第二医院西部妇幼医学研究院出生缺陷与相关妇儿疾病教育部重点实验室,四川成都610041 [2]四川大学华西第二医院药学部,四川成都610041

出　　处：《中国现代医学杂志》2023年第9期41-50,共10页China Journal of Modern Medicine

基　　金：国家自然科学基金面上项目(No:82173512);国家重点研发计划(No:2020YFC2006300);四川省科技厅应用基础项目(No:2021YJ0156)。

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�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.

关 键 词：帕金森病 乳铁蛋白 1-甲基-4-苯基吡啶 炎症损伤 

分 类 号：R742.5[医药卫生—神经病学与精神病学]