Fig. 4.

5Z-7-oxozeaenol selectively inhibits MSU-induced IL-1β, TNF-α, IL-8, and MCP-1 production and reduces expression of the proform of IL-1β and mature IL-1β in THP-1 macrophages. To study the effect of IRAK1, TRAF6, and TAK1 inhibitors on MSU-induced cytokine and chemokine production, THP-1 macrophages were pretreated with an inhibitor of IRAK1 [N-(2-morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, 0.5–10 µM], an inhibitor of TAK1 [5Z-7-oxozeaenol, 5Z-7-ox; 0.05–5 µM], a TRAF6 inhibitor (i) peptide [10–20 µM] or control (c) peptide, [10–20 µM] for 2 h, followed by MSU (100 μg/ml) stimulation for 24 h. Conditioned media were used to estimate the levels of a IL-1β, b TNF-α, c IL-8, and d MCP-1 production by ELISA. e–g THP-1 macrophages treated with MSU and/or 5Z-7-ox for e 30 min or f 4 h were lysed, and cell lysates were probed for the proform of IL-1β, mature IL-1β, or cleaved IL-1β (Asp¹¹⁶) by Western blotting. g Effect of MG132 on 5Z-7-ox-induced pro-IL-1β degradation. h Primary human macrophages were pretreated with 5Z-7-ox, followed by MSU (100 μg/ml) stimulation for 30 min. Cell lysates were probed for p-IRAK1 (Thr³⁸⁷) and pro-IL-1β expression. **p < 0.01 for NS (not significant) vs. MSU alone; ^#p < 0.05 or ^##p < 0.01 for MSU vs. inhibitors