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

Figure 3

CORM-3-induced HO-1 expression is mediated via mitochondrial respiratory complex. (a) RBA-1 cells were pretreated with various concentrations of rotenone for 1 h and then incubated with 30 μM CORM-3 for 6 h. The levels of HO-1 and GAPDH (as an internal control) protein expressions were determined by western blot. (b) The cells were pretreated with 5 μM Rotenone for 1 h 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, subsequently pretreated with 5 μM Rotenone for 1 h, 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 pretreated with rotenone (5 μM) for 1 h, incubated with 30 μM CORM-3 for 10 min, and then labeled with H2DCF-DA. The fluorescence of DCF staining was detected using an ELISA assay. Data were expressed as of three independent experiments. ^#, as compared with the control or pretreatment with inhibitor indicated in the figure.

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