Figure 5.

Effect of WBP on the activation of the upstream signaling pathways. RAW264.7 cells (5 × 10⁶ cells ml^–1) were incubated with 100 μg ml^–1 WBP for the indicated periods of time. (a) The whole-cell lysates were extracted for immunoblotting to determine the levels of phospho- or total MAPKs (ERK, p38, and JNK) identified based on their antibodies. (b) Inhibitory effects of specific inhibitors p38 MAPK kinase (SB203580) on iNOS, COX-2, and TNF-α expression in RAW 264.7 cells. Cells were pre-treated with SB203580 followed by stimulation with 100 μg ml^–1 WBP for 6 h. Total RNA was isolated and real time-PCR was performed to determine the mRNA level of each gene with gene-specific primer. Within a column, any significant differences between p38 inhibitor-treated and -untreated groups were analyzed using the Student’s t-test (*p < 0.05). (c) RAW 264.7 cells (5 × 10⁶ cells ml^–1) were incubated with 100 μg ml^–1 WBP for the indicated periods of time. The whole-cell lysates were extracted for immunoblotting to determine the level of TLR4. (d) Inhibitory effects of anti-TLR4 on WBP-induced NO production in RAW 264.7 cells. Cells were treated with anti-TLR4 or control IgG fraction (10 μg ml^–1) for 30 min followed by stimulation with different concentration of WBP (25, 50, or 100 μg ml^–1) for 24 h. The level of NO was detected by Griess reagent. Any significant differences between treatments with anti-TLR4 and control antibody were analyzed using the Student’s t-test (*p < 0.05).