Cytotoxicity of BAY 60-7550 and LPS in RAW 264.7 cells. The cells were treated with BAY 60-7550 (1, 3, 10, and 30 μM) for 15 h (A). The cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 4, 15, or 24 h to observe the cytotoxicity of BAY 60-7550 and LPS (B). The number of viable cells was assessed by MTT assay. Results were expressed as a percentage (%) in comparison with the control. Each value represents the mean ± S.D from four independent experiments.|@|~(^,^)~|@|The inhibitory effect of BAY 60-7550 on the LPS-induced intracellular oxidative stress in RAW 264.7 cells. The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg/mL) for 15 h. The expression levels of iNOS were determined by western blot analysis (A). The culture supernatant was analyzed for NO. The NO content was determined by Griess reagent (B). The cells were pretreated with various concentrations of BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h and treated with LPS (0.1 μg /mL) for 15 h. ROS levels were assessed with the H2DCF-DA assay. ROS levels were expressed as a percentage (%) relative to the level in the vehicle control (C). Each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 & ***p < 0.001).|@|~(^,^)~|@|The inhibitory effect of BAY 60-7550 on LPS-induced overexpression of COX-2 and PGE2 in RAW 264.7 cells.
The protein expression levels of COX-2 were confirmed by western blot assay (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1h. The band density of COX-2 was quantified relative to the β-actin. PGE2 production was evaluated by ELISA assay in LPS-treated RAW 264.7 cells (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 1 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the PGE2 absorbance standard curve. Each value represents the mean ± S.D from experiments. # & ### indicate significant differences from the vehicle control, which was treated with DMSO alone (#p < 0.05 and ###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).|@|~(^,^)~|@|The inhibitory effect of BAY 60-7550 on LPS-induced overproduction of pro-inflammatory cytokines in RAW 264.7 cells. The levels of TNF-α, IL-6, and IL-1β were confirmed by ELISA. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the TNF-α absorbance standard curve (A). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-6 absorbance standard curve (B). RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 15 h. The absorbance at 450 nm was measured by ELISA protocol, and the concentration was measured according to the IL-1β absorbance standard curve (C). Each value represents the mean ± S.D from three separate experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). * and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05 and ***p < 0.001).|@|~(^,^)~|@|The inhibitory effect of BAY 60-7550 on LPS-induced activation of MAPK-ERK1/2 and NF-κB signaling pathways in RAW 264.7 cells. The levels of phosphorylated-ERK1/2, total-ERK1/2 (A), phosphorylated-NF-κB, and total-NF-κB (B) were confirmed by western blotting assay. RAW 264.7 cells were pretreated with BAY 60-7550 (1, 3, 10, and 30 μM) for 1 h, then treated with 0.1 μg/mL of LPS for 4 h. The band density of phosphorylated-ERK1/2 and the phosphorylated-NF-κB protein level was quantified relative to the total-ERK1/2 and total-NF-κB, respectively. NF-κB levels were expressed as a percentage (%) relative to the level in the vehicle control, and each value represents the mean ± S.D from experiments. ### indicates a significant difference from the vehicle control, which was treated with DMSO alone (###p < 0.001). *, **, and *** indicate significant differences from the cells treated with 0.1 μg/mL of LPS alone (*p < 0.05, **p < 0.01, and ***p < 0.001).
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