【摘要】 目的 探索外源性乳铁蛋白（lactoferrin, Lf）对淀粉样蛋白25-35(β amyloid 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。更多还原

【Abstract】 Objective To investigate the effects and mechanisms of exogenous lactoferrin（Lf） on Alzheimer’s Disease（AD） cell model induced by β amyloid 25-35(Aβ_25-35). Methods N2a cells were randomly divided into control group, Lf group, Aβ group 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 20 μM 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 Aβ group.（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 Aβ group.（4）Compared with Aβ group, 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.更多还原