乳铁蛋白通过调节Keap1-Nrf2信号通路改善Aβ_(25-35)诱导的N2a细胞氧化应激和凋亡  

Lactoferrin ameliorates Aβ_(25-35) induced oxidative stress and apoptosis of N2a cells by regulating Keap1-Nrf2 signaling pathway

作　　者：汪晓语 吕嘉琦 赵儒花 单树方 成果 张伶俐 张林[1] WANG Xiao-yu;LV Jia-qi;ZHAO Ru-hua;SHAN Shu-fang;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;不详)

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,循证药学中心

出　　处：《现代预防医学》2023年第6期1098-1104,共7页Modern Preventive Medicine

基　　金：国家重点研发计划(2020YFC2006300);国家自然科学基金面上项目(82173512);四川省科技厅应用基础项目(2021YJ0156)。

摘　　要：目的探索外源性乳铁蛋白(lactoferrin,Lf)对淀粉样蛋白25-35(βamyloid 25-35,Aβ_(25-35))诱导的阿尔茨海默病(Alzheimer’s Disease,AD)细胞模型的相关作用及机制。方法将N2a细胞随机分为空白对照组,Lf组,Aβ组和Aβ+Lf组,应用CCK-8法检测细胞活力;Western Blot检测细胞Tau、p-Tau、Bax、Bcl-2、Caspase3、Cleaved-Caspase3、Nrf2、Keap1、HO-1等蛋白表达水平;Annexin FITC/PI双染法检测细胞凋亡比率;试剂盒检测细胞超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和过氧化氢酶(catalase,CAT)活性以及氧化产物活性氧(reactive oxygen species,ROS)荧光强度、乳酸脱氢酶(lactate dehydrogenase,LDH)释放量、丙二醛(malondialdehyde,MDA)含量。采用t检验和方差分析进行组间比较。结果(1)当Aβ_(25-35)干预浓度为20μmol/L,干预时间为24 h时,细胞活力降至80%,且Tau蛋白磷酸化水平较空白组显著增加,表明AD细胞模型构建成功;(2)与Aβ组相比,Aβ+Lf组Bcl-2表达量显著增加(t=-10.573,P<0.001),Bax(t=40.529,P<0.001)和Cleaved-Caspase3/Caspase3(t=15.376,P<0.001)表达量显著降低,差异有统计学意义;(3)与Aβ组相比,Aβ+Lf组ROS荧光强度显著减弱,MDA含量(t=10.175,P<0.001)和LDH释放量(t=7.453,P<0.001)显著降低;(4)与Aβ组相比,预先干预Lf后,Keap1表达量显著降低(t=15.766,P<0.001),Nrf2(t=-32.475,P<0.001)和HO-1表达量(t=-6.792,P<0.001)显著升高。结论Lf可通过调节Keap1-Nrf2/HO-1信号通路改善Aβ_(25-35)诱导的N2a细胞氧化应激和凋亡。Objective To investigate the effects and mechanisms of exogenous lactoferrin(Lf)on Alzheimer’s Disease(AD)cell model induced byβamyloid 25-35(Aβ_(25-35)).Methods N2a cells were randomly divided into control group,Lf group,Aβgroup and Aβ+Lf group.The cell viability was assessed by cell counting kit-8(CCK-8)method.Protein expression levels of Tau,p-Tau,Bax,Bcl-2,Caspase3,Cleaved-caspase3,Nrf2,Keap1,HO-1 were detected using Western Blot.The apoptosis rate was obtained via Annexin V-fluorescein isothiocyanate(FITC)/Propidium Iodide(PI)double staining method.The activities of antioxidant enzymes superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT)as well as the production levels of reactive oxygen species(ROS),lactate dehydrogenase(LDH)and malondialdehyde(MDA)were tested by detection kits.For comparisons between groups,t-test and ANOVA were used as appropriate.Results(1)When the concentration of Aβ_(25-35) was 20μM and the intervention period was 24 h,cell viability decreased to 80%while the phosphorylation level of Tau protein significantly increased compared with control group,indicating that the AD cell model was successfully constructed.(2)Expression levels of Bcl-2(t=-10.573,P<0.001)were increased while Bax(t=40.529,P<0.001)and Cleaved Caspase3/Caspase3 levels(t=15.376,P<0.001)were significantly decreased in Aβ+Lf group when compared with Aβgroup.(3)Levels of ROS,MDA content(t=10.175,P<0.001)and LDH release(t=7.453,P<0.001)in Aβ+Lf group were significantly decreased than those in Aβgroup.(4)Compared with Aβgroup,the expression of Keap1 in Lf group was decreased(t=15.766,P<0.001),while the expression of Nrf2(t=-32.475,P<0.001)and HO-1(t=-6.792,P<0.001)were significantly increased.Conclusion Lactoferrin may ameliorate oxidative stress and apoptosis of N2a cells induced by Aβ_(25-35) by regulating keap1-Nrf2/HO-1 signaling pathway.

关 键 词：阿尔茨海默病 氧化应激 乳铁蛋白 Β淀粉样蛋白 

分 类 号：R151[医药卫生—营养与食品卫生学]