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

Figure 5

CORM-3-induced HO-1 expression is mediated via mTOR. (a) RBA-1 cells were incubated with various concentrations of rapamycin 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 1 μM rapamycin 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 1 μM rapamycin 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 transfected with mTOR siRNA and then challenged with 30 μM CORM-3 for 6 h. The protein levels of HO-1, mTOR, and GAPDH (as an internal control) were determined by western blot. (d) The cells were pretreated with rapamycin (1 μM), NAC (10 mM), APO (10 mM), DPI (10 μM), rotenone (5 μM), LY294002 (10 μM), or SH-5 (10 μM) for 1 h and then stimulated by 30 μM CORM-3 for the indicated time intervals. The levels of phosphorylated mTOR and total mTOR proteins were determined by western blot. Data are expressed as the of three independent experiments. ^#, as compared with the control, pretreatment with inhibitor, or siRNA indicated in the figure.

(a)

(b)

(c)

(d)