Figure 4.

Curcumin induces HO-1 expression and ARE activation in HAECs by activating Nrf2 and the p38-MAPK signalling pathway. (a) HAECs were incubated with 100 μmol/L curcumin for the indicated times. The Nrf2 protein expression in the nuclear extracts was analysed by Western blotting. The results are quantified as the Nrf2 intensity relative to Lamin A. (b) Ten hours after transfection with an ARE luciferase or control vector, HAECs were incubated with the indicated concentrations of curcumin for 6 hours. Cell lysates were assayed for luciferase activity as the fold induction by normalising the transfection efficiency and dividing the values of each experiment relative to the control. The ARE-luciferase activity (c) and HO-1 protein expression (d) were analysed by luciferase assay and Western blotting and, respectively, in lysates of HAECs transfected with scrambled siRNA or Nrf2 siRNA for 48 hours, then incubated with or without 100 μmol/L curcumin for a further 6 hours. The results are quantified as the HO-1 intensity relative to β-actin or the luciferase activity fold induction relative to the control. (e) HO-1 protein expression was analysed by Western blotting in lysates of HAECs preincubated with 10 μmol/L SB203580, PD098059, SP600125, or LY294002 for 1 hour and then incubated with or without 100 μmol/L curcumin for a further 6 hours. (f) Phosphorylated p38-MAPK and p38-MAPK were analysed by Western blotting in lysates of HAECs incubated with 100 μmol/L curcumin for the indicated times. The results are quantified as the phosphorylated p38-MAPK relative to the total p38. (g) The Nrf2 protein expression in the nuclear extracts was analysed by Western blotting in lysates of HAECs incubated with 100 μmol/L curcumin with or without p38-MAPK inhibitor SB203580. The results are quantified as the Nrf2 intensity relative to Lamin A. All data are expressed as the mean ± SEM of three independent experiments.^∗ indicates P < 0.05 compared to cells incubated without control.