AFB1抑制NrF2信号通路诱导牛乳腺上皮细胞抗氧化应激

AFB1 induced oxidative stress in bovine mammary epithelial cells by inhibiting NrF2 signaling pathway

利用不同浓度的黄曲霉毒素B1(aflatoxinB1,AFB1)作用于牛乳腺上皮细胞(MAC-T)培养24 h后,通过相关检测试剂盒检测细胞内氧化损伤标志物活性氧(reacive oxygen species,ROS)、总超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、丙二醛(malondialdehyde,MDA)、总抗氧化能力(total antioxidation capability,T-AOC)的活性;实时荧光定量聚合酶链式反应(real-time quantitative polymerasechain reaction,RT-qPCR)方法检测核因子E2相关因子(nuclear factor erythroid-2-related factor 2,NrF2)、血红素氧合酶-1(heme oxygenase-1,HO-1)和NAD(P)H醌氧化还原酶(NAD(P)H quinone oxidoreductase 1,NQO-1)的mRNA相对表达水平;利用蛋白免疫印迹(Western blot)试验检测NrF-2、HO-1和NQO-1的蛋白相对表达水平。结果显示,与0μmol/L相比,10,20μmol/L AFB1处理组显著升高MAC-T细胞内ROS和MDA的含量,降低SOD、T-AOC和GSH-Px的活性。RT-qPCR结果显示,与0μmol/L组相比,不同浓度AFB1组显著下调MAC-T细胞中NrF2、HO-1、NQO-1的mRNA表达水平。Western blot结果显示,10μmol/L AFB1组MAC-T细胞中NrF2、HO-1、NQO-1的蛋白表达水平显著低于0μmol/L组。结果表明,AFB1通过抑制NrF2信号通路诱导牛乳腺上皮(MAC-T)细胞发生氧化应激性损伤。

This study aims to investigate the effects of aflatoxin B1(AFB1) on the antioxidant function of bovine mammary epithelial cells(MAC-T).The MAC-T cells in logarithmic growth phase were cultured with AFB1 at different concentrations for 24 h.Reactive oxygen species(ROS),superoxide dismutase activity(SOD),glutathione peroxidase activity(GSH-Px),malondialdehyde(MDA),total antioxidation capability(T-AOC) were tested using the relative detection kit.The relative expression level of Nuclear factor erythroid-2-related factor 2(NrF-2),Heme oxygenase-1(Oxygenase-1),Heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase 1(NQO-1) mRNA were detected by real-time quantitative polymerase chain reaction(RT-qPCR) method.The relative expression levels of NrF-2,HO-1 and NQO-1 protein were detected by Western blot assay.The results showed that compared to the 0 μmol/L group,the contents of ROS and MDA in MAC-T cells were significantly increased in 10,20 μmol/L AFB1 treatment groups,while the activities of SOD,T-AOC and GSH-px were decreased.The results of RT-qPCR assay indicated that compared with the 0μmol/L group,the expression levels of NrF-2,HO-1and NQO-1mRNA in MAC-T cells were significantly down-regulated in AFB1 with different concentrations.Western blot results showed that 10μmol/L AFB1significantly decreased the protein expression levels of NrF2,HO-1and NQO-1in MAC-T cells compared with the 0μmol/L group.These results indicate that AFB1could induce oxidative stress injury of bovine mammary epithelial cells(MAC-T)by inhibiting NrF2signaling pathway.