Figure 8.

Four-HHE is a potent Nrf2 activator. Primary cultures were incubated with 4-HHE (n-3) or 4-HNE (n-6) at a concentration of 10 μm for the indicated duration. A, Representative Western blots of cytosolic HO-1 and nuclear Nrf2. B, C, Quantitative analyses of HO-1 (B) and Nrf2 (C) levels indicated that lipid electrophiles can directly induce Nrf2 nuclear translocation and HO-1 expression, and that 4-HHE (n-3) is more potent than 4-HNE (n-6) in inducing Nrf2 activation. Data are presented as mean ± SE, and analyzed with ANOVA and post hoc tests (*p ≤ 0.05 vs 4-HNE-treated group at the same time point). D, Representative microphotographs of Nrf2-EGFP transfected neuronal cultures, showing that 4-HHE was a stronger inducer of Nrf2 than 4-HNE. Primary neurons were infected with lenti-Nrf2-EGFP for 3 d followed by treatment with 4-HHE and 4-HNE (10 μm) for 1 h. Cells were then fixed and counterstained with DAPI (blue). E, F, Representative Hoechst staining (E) and dead cell counts (F), showing that 4-HHE pretreatment reduced neuronal death. Primary neurons were treated with vehicle, 4-HNE, and 4-HHE (10 μm) for 2 h. After overnight recovery, neurons were challenged with OGD for 60 min and then stained with Hoechst (p ≤ 0.05 vs control, vehicle-treated, and 4-HNE-treated OGD groups.