Objective To investigate the role of phosphatidylinositol 3-kinase (PI3K) signaling pathway in nuclear factor E2-related factor 2 (Nrf2) translocation induced by curcumine in human L02 hepatocytes.Methods L02 hepatocytes were treated with 30 μmol/L curcumine and wortmannin (PI3K inhibitor).After 12 h treatment, the levels of Nrf2 in cytosolic and nuclear fractions were assayed by Western blot analysis.L02 hepatocytes were divided into control group, model group, curcumine group and inhibitor group.Hepatocytes in control group were only incubated with RPMI 1640, hepatocytes in model group were treated with 100 U/L glucose oxidase (GO) for 2 h, hepatocytes in curcumine group were pretreated with 30 μmoL/L curcumine for 12 h and subsequently treated with 100 U/L GO for 2 h, hepatocytes in inhibitor group were pretreated with 0.1 μmoL/L wortmannin, then treated as the same as hepatocytes in curcumine group.The intercellular ROS level was analyzed with a flow cytometer by dihydroethidium.The levels of MDA,GSH and LDH were measured by spectrophotometric method.The level of AST was measured by kinetic rate method.Results Curcumine treatment induced markedly Nrf2 nuclear translocation in hepatocytes, and pretreatment with wortmannin partly blocked curcumine-derived Nrf2 nuclear translocation.The levels of ROS, MDA, AST and LDH were increased significantly in model group compared with control group (P ＜0.01 ), all of which were significantly decreased in curcumine group compared with model group (P ＜ 0.01 ).However, the levels of ROS, MDA, AST and LDH in inhibitor group were significantly higher than those in curcumine group, lower than those in model group ( P ＜ 0.01 ).Compared with control group, the level of GSH was decreased significantly in model group (P ＜0.01 ), the GSH level in model group was significantly lower than that in curcumine group ( P ＜ 0.01 ), and the level of GSH in curcumine group was significantly higher than that in inhibitor group ( P ＜ 0.01 ).Conclusion PI3 K is a very important pathway involved in curcumine-derived Nrf2 nuclear translocation in L02 hepatocytes.Wortmannin reduces the protective effects of curcumine on hepatocytes partly through by inhibiting Nrf2 nuclear translocation.%目的 研究磷脂酰肌醇3-激酶(PI3K)信号通路在姜黄素诱导人肝细胞Nrf2核转位中的作用.方法 用30 μmol/L姜黄素和Wortmannin(PI3K抑制剂)干预L02肝细胞12 h,Western blot观察Nrf2核转位水平;将L02肝细胞分为对照组、模型组、干预组和抑制剂组,对照组用RPMI1640正常培养,模型组用100 U/L 葡萄糖氧化酶(GO)干预2 h,干预组用30 μmol/L姜黄素干预12 h后给予100 U/L GO干预2 h,抑制剂组先用0.1 μmol/L Wortmannin预处理1h,余处理同干预组.流式细胞术检测细胞内活性氧簇(ROS).分光光度法检测细胞MDA、GSH及培养液LDH水平,速率法检测细胞培养液AST的水平.结果 姜黄素明显诱导Nrf2核转位,其诱导作用可被Wortmannin部分阻断.模型组ROS、MDA、AST、LDH水平较对照组显著升高(P<0.01),干预组ROS、MDA、AST、LDH水平较模型组显著降低(P<0.01),抑制剂组ROS、MDA、AST、LDH水平显著高于干预组,低于模型组(P<0.01).模型组GSH水平较对照组显著降低(P<0.01),干预组GSH水平较模型组显著升高(P<0.01),抑制剂组GSH水平显著低于干预组(P<0.01),高于模型组(P<0.01).结论 PI3K信号通路是姜黄素诱导Nrf2核转位的重要信号通路,通过抑制该通路可减弱姜黄素对肝细胞氧化应激损伤的保护作用.
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