Carbon Monoxide Releasing Molecule-3 Enhances Heme Oxygenase-1 Induction via ROS-Dependent FoxO1 and Nrf2 in Brain Astrocytes

Figure 7

Nrf2 contributes to CORM-3-induced HO-1 expression. (a) RBA-1 cells were transfected with Nrf2 siRNA and then challenged with 30 μM CORM-3 for 6 h. The protein levels of HO-1, Nrf2, and GAPDH were determined by western blot. (b) The cells were transfected with Nrf2 siRNA and then incubated with 30 μM CORM-3 for 4 h. The levels of HO-1 and GAPDH mRNA were determined by real-time PCR (open bars). The cells were transiently transfected with HO-1 report gene together with a β-galactosidase plasmid, followed by transfected with Nrf2 siRNA, and then incubated with 30 μM CORM-3 for 1 h. Promoter activity was determined in the cell lysates (solid bars). (c) The cells were challenged with 30 μM CORM-3 for the indicated time intervals. The cell lysates were centrifuged to prepare nuclear fraction. The levels of Nrf2, phosphorylated-Nrf2, and lamin A were determined by western blot. (d) The cells were pretreated with NAC (10 mM), DPI (10 μM), APO (10 mM), or rotenone (5 μM) for 1 h and then incubated with 30 μM CORM-3 for 30 min. The nuclear fraction was prepared and analyzed by western blot. (e) Cells were pretreated with or without rotenone (5 μM), NAC (10 mM), APO (10 mM), or DPI (10 μM) for 1 h and then stimulated by CORM-3 for 30 min. These cells were stained using anti-p-Nrf2 antibodies and DAPI. The images of p-Nrf2 and nucleus were detected with a fluorescence microscope.  μm. (f) The cells were transiently transfected with ARE report gene together with a β-galactosidase plasmid and then incubated with 30 μM CORM-3 for the indicated time intervals. ARE promoter activity was determined by a luciferase reporter gene assay. (g) The cells were transfected with Nrf2 siRNA, followed by transiently transfected with ARE report gene together with a β-galactosidase plasmid, and then challenged with 30 μM CORM-3 for 1 h (upper panel). The cells transfected with ARE report gene together with a β-galactosidase plasmid were pretreated with NAC (10 mM), APO (10 mM), DPI (10 μM), or rotenone (5 μM) for 1 h and then stimulated by 30 μM CORM-3 for 1 h (lower panel). The levels of ARE promoter activity were determined in the cell lysates. (h, i) Cells were treated with 30 μM CORM-3 for the indicated time points (h) or pretreated with rotenone (5 μM), NAC (10 mM), APO (10 mM), or DPI (10 μM) for 1 h and then stimulated by CORM-3 for 2 h (i). The levels of Nrf2 binding to ARE region of the HO-1 promoter were detected by a ChIP assay. Data are expressed as the of three independent experiments. ^#, as compared with the control, pretreatment with inhibitor, or siRNA indicated in the figure. DAPI: 4,6-diamidino-2-phenylindole.

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