Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats
Figure 7
Immunocytochemistry localization and functional assay (antioxidant response element (ARE) binding) of Nrf2 in CGNs exposed to curcumin, curcumin/hemin, and hemin. Curcumin (C) was capable of inducing nuclear translocation and binding of nuclear Nrf2 to ARE before and after hemin (H)-exposure. (a) Representative images with bright-field (40x, left panel), anti-Nrf2 signal (middle panel) and Hoechst stain (right panel). The same field is shown in each condition. CGNs were incubated (1–24 h) with 15 μM curcumin (C 1 h, C 4 h, C 6 h, C 16 h, C 24 h). In addition, cells were pretreated for 24 h with curcumin and then exposed to 30 μM hemin for 1 h (C 24 h + H). Additional conditions were the following: CGNs were incubated by 24 h with 15 μM curcumin and 24 h of recovery was allowed (C 24 h + 24 h), CGNs were incubated by 24 h with 15 μM curcumin, then curcumin was removed, and 30 μM hemin was added by 1 h and removed and 24 h of recovery was allowed (C 24 h + H + 24 h) and CGNs were incubated by 30 μM hemin by 1 h and then removed and 24 h of recovery was allowed (H + 24 h). (b) Intensity of fluorescence was measured in five different fields per well per condition. (c) Curcumin was incubated for 24 h or hemin for 1 h and the transcriptional activity of Nrf2 was measured at 450 nm using immobilized oligonucleotides containing the ARE consensus binding site. COS-7 nuclear extract was used as a positive control of this assay. Data are expressed as mean ± SEM, . versus control (untreated) and versus hemin.
