Figure 5.

1,25-Dihydroxyvitamin D [1,25(OH)₂D₃] promotes hemeoxygenase-1 (HO-1) transcription via increasing NF-E2-related factor 2 (Nrf2) activation in macrophages. (A) The inductions of Nrf2 by the 1,25(OH)₂D₃ time course (0, 4, 8, 16, and 32 h) treatment were detected by Western blotting. (B) The induction of Nrf2 in LPS time course (0, 4, 8, 16, and 32 h) treatment in absent or present with 1,25(OH)₂D₃. Band intensities were quantified from three independent experiments. (C) Nrf2 translocation in absent or present with 1,25(OH)₂D₃ following LPS stimulation was detected by immunofluorescence. The cells were stained with anti-Nrf2 antibody (red), and the nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Scale bar = 10 μm. More than 60 cells in each independent experiment were quantified. Each bar is the mean of three independent experiments. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. (D) Illustration of the ARE1 and ARE2 cis-DNA elements and the chromatin immunoprecipitation (ChIP) primers within the promoter of the HO-1 gene for ChIP assays. LPS promotes Nrf2 binding to the ARE site, and 1,25(OH)₂D₃ various dose treatment (0.2, 2, and 20 nM) enhances Nrf2 binding in bone marrow-derived macrophages. The DNA fragment precipitated by anti-Nrf2 antibody was assessed by regular-PCR. IgG as negative control. Data are representative of at least three independent experiments.