姜黄素激活转录因子Nrf2对人肝细胞氧化应激的影响

Effect of curcumine on intracellar ROS in cultured human hepatocyte by upregulating the activity of Nrf2

目的观察姜黄素对人肝细胞系L02中细胞转录因子NF-E2相关因子2(Nrf2)核转位及细胞氧化应激水平的影响。方法用15μmol/L和30μmol/L姜黄素干预L02细胞,用RPMI1640培养作为对照,干预6h和12h,间接免疫荧光法观察核转位水平;将人肝细胞L02分成对照组、模型组、干预组三组,对照组正常培养未给予葡萄糖氧化酶(GO)和姜黄素干预,模型组仅加入100U/LGO干预2h,制备细胞氧化应激模型,干预组加入最佳浓度的姜黄素干预12h,然后给予100U/LGO干预2h。免疫荧光法观察L02细胞Nrf2核转位情况,分光光度法检测细胞MDA、GSH水平,速率法检测细胞培养液AST和ALT的水平,流式细胞术检测细胞内活性氧簇(reactive oxygen species,ROS)水平的变化。结果姜黄素能够诱导Nrf2的核转位,其中30μmol/L的作用优于15μmol/L,各浓度时12h作用优于6h。模型组MDA、AST、ALT较对照组显著升高(P<0.01),干预组MDA、AST、ALT较模型组显著降低(P<0.01),模型组GSH较对照组显著降低(P<0.01),干预组GSH较模型组显著升高(P<0.01)。模型组ROS阳性细胞荧光强度较对照组显著升高(P<0.01),干预组阳性细胞水平较模型组显著降低(P<0.01)。结论姜黄素通过促进肝细胞L02的Nrf2核转位,降低细胞内活性氧的水平,进而减轻细胞氧化应激损伤。

Objective To investigate the effect of curcumine on intracellar ROS in cultured human hepatocyte L02 by upregulating the activity of Nrf2.Methods Hepatocyte L02 grown on cover slips were treated with 15 and 30 μmol/L curcumine,or vehicle （RPMI1640） as control.After 6 or 12 h treatment,the level of nuclear translocation was analyzed by immunocytochemistry.Hepatocyte L02 were divided into control group,model group and curcumine-treated group.Hepatocytes in control group were only incubated with RPMI 1640,hepatocytes in model group were treated with 100 mU/mL glucose oxidase （GO） for 2 h,hepatocytes in curcumine-treated group were treated with 30 μmol/L curcumine for 12 h and subsequently treated with 100 U/L GO for 2 h.Then the hepatocytes were analyzed by immunocytochemistry to observe Nrf2 nuclear translocation.The levels of MDA and GSH were measured by spectrophotometric method.The levels of AST and ALT were measured by kinetic rate method using commercially available reagent kits.The ROS level was analyzed on a flow cytometer by using Dihydroethidium （DHE）.Results Curcumine resulted in nuclear translocation.The effect of the treatment with 30 μmol/L curcumine was better than that in the treatment with 15 μmol/L curcumine.At the same concentration,the effect of 12 h treatment was better than that of 6 h treatment.Compared with control group,the levels of MDA,AST and ALT increased significantly in model group （P〈0.01）.However,the levels of MDA,AST and ALT in curcumine-treated group decreased significantly compared with model group （P〈0.01）.Compared with control group,the level of GSH decreased significantly in model group （P〈0.01）.However,the levels of GSH in curcumine-treated group increased significantly compared with model group （P〈0.01）.Compared with control group,the level of intracellar ROS increased significantly in model group （P〈0.01）.However,the ROS levels in curcumine-treated group decreased significantly compared with model group （P〈0.01）.Conclusion Curcumine can reduce the intracellar ROS level in human hepatocytes through enhanced Nrf2 nuclear translocation.