黄芩苷增强N2a/APPswe细胞的抗氧化能力并促进核因子E2相关因子2(Nrf2)的核转位

Baicalin increases the antioxidant capacity via promoting the nuclear translocation of NF-E2-related factor 2(Nrf2) in N2a/APPswe cells

目的探讨黄芩苷对阿尔茨海默病(AD)转基因N2a/APPswe细胞的保护作用及可能机制。方法应用(0.1、0.5、1、5、10、20)μmol/L黄芩苷处理N2a/APPswe细胞,MTT法检测黄芩苷对细胞存活率的影响。用(1、5、10)μmol/L黄芩苷处理细胞48 h,通过黄嘌呤氧化酶法检测各组细胞超氧化物歧化酶(SOD)的活性,硫代巴比妥酸法检测各组细胞培养上清液的丙二醛(MDA)的含量。荧光实时定量PCR测定核因子E2相关因子2(Nrf2)的mRNA表达,Western blot法检测细胞总Nrf2、核内Nrf2和核因子κB(NF-κB)的蛋白水平。免疫荧光染色观察Nrf2在细胞内的分布。结果 MTT试验结果显示,黄芩苷能促进N2a/APPswe细胞增殖。与对照组相比,模型组SOD活性明显降低,MDA含量明显增多,而黄芩苷能提高SOD活性,抑制MDA的生成,尤其以高剂量组最为明显。黄芩苷处理组与模型组相比Nrf2 mRNA和总蛋白表达均无明显差异,但黄芩苷处理后能显著增加细胞核内Nrf2的含量,下调细胞核内NF-κB的水平。免疫荧光染色结果也证明,黄芩苷能促进Nrf2的核转位。结论黄芩苷增强N2a/APPswe细胞的抗氧化能力并促进Nrf2的核转位。

Objective To investigate the protective effect and related mechanism of baicalin in murine neuroblastoma N2 a cells stably expressing human Swedish mutant amyloid precursor protein（ APP）（ N2 a / APPswe cells）,a cellular model of Alzheimer＇s disease（ AD）. Methods MTT assay was performed to observe the effect of baicalin（ 0. 1,0. 5,1,5,10,20） μmol / L on the viability of N2 a / APPswe cells. After N2 a / APPswe cells were incubated with（ 1,5,10） μmol / L baicalin for48 hours,xanthine oxidase assay was used to test superoxide dismutase（ SOD） activity and thiobarbituric acid method to detect malondialdehyde（ MDA） content in each group. Real-time quantitative PCR was applied to determine nuclear factor erythroid 2-related factor 2（ Nrf2） mRNA,and Western blotting to examine protein levels of total Nrf2,nuclear Nrf2 and nuclear factor κB（ NF-κB） in N2 a / APPswe cells exposed to different doses of baicalin. Immunofluorescence staining was also used to observe the distribution of Nrf2. Results We found that baicalin pretreatment increased cell viability. Compared with the control group（ N2 a / wt cells）,SOD activity in N2 a / APPswe cells significantly decreased,and MDA content significantly increased; but SOD activity was improved and MDA production was inhibited after pretreatment with baicalin,especially with 10 μmol / L bacalin. Both mRNA and total protein expression of Nrf2 were not significantly changed in baicalin treatment group compared with N2 a / APPswe group,but the nuclear protein of Nrf2 distinctly increased after treatment with baicalin. In addition,baicalin decreased the level of nuclear NF-κB protein. Furthermore,immunofluorescence staining revealed that baicalin promoted the translocation of Nrf2 to the nucleus. Conclusion Baicalin has the protection against oxidative stress via activation of Nrf2 in N2 a / APPswe cells.