Figure 5.

HO-1 is necessary for antioxidant and anti-inflammatory effects of 4-PG. RAW 264.7 cells were transfected with scramble RNA (scRNA) and siRNA against HO-1 (siHmox-1) for 36 h and then pretreated with either 4-PG (10 μM) or PTER (10 μM) for 6 h followed by stimulation of LPS (100 ng/ml) for another 4 h. (a) The production of ROS was determined by confocal microscopy. Scale bar = 10 μm. (b) The mRNA and protein levels of HO-1 were determined by RT-PCR and Western blot, respectively. The conditioned supernatants were harvested, and the secreted levels of TNF-α (c) and IL-6 (d) in Hmox-1^+/+ and Hmox-1^−/− BMDM were assessed by ELISA. (e) mRNA levels of HO-1, TNF-α, and IL-6 were determined by RT-PCR. (f–h) BMDMs, extracted from Hmox-1^+/+ and Hmox-1^−/− mice, were pretreated with 4-PG (10 μM) for 6 h and MitoTEMPO (100 μM) for 30 min, respectively, and then cells were stimulated with LPS (100 ng/ml) for an additional 4 h. (i–k) A549 cells were pretreated with 4-PG (10 μM) for 4 h or with MitoTEMPO (100 μM) for 30 min, respectively, and then stimulated with LPS (10 μg/ml) for an additional 6 h. (f and i) Mitochondrial ROS were stained with MitoSOX and measured by flow cytometry. (g and j) The fold change of fluorescence intensity is presented as mean ± SD (n = 5, determined in five independent experiments). (h and k) The mRNA level of TNF-α was determined by RT-PCR. (l and m) BMDMs isolated from Hmox-1^+/+ and Hmox-1^−/− mice were pretreated with 4-PG (10 μM) for 6 h followed by the challenge of LPS (100 ng/ml) for 4 h, and for Hmox-1^−/− BMDMs, cells were copretreated with 4-PG (10 μM) and HO-1 catabolites such as CO-releasing molecule- (CORM-) 2 (20 μM), bilirubin (5 μM), and ferrous sulfate (10 μM), respectively, in the presence of LPS stimulation. Data were expressed as mean ± SD (n = 5 determined in five independent experiments); ^∗∗∗p < 0.001. NS: not significant. Comparisons between scRNA and siHO-1-transfected groups were made by two-way ANOVA with Bonferroni post-tests.