Figure 3.

Molecular mechanisms underlying the synergistic effects of adiponectin and IL-1β. (A) The expression of IL-1 receptor 1 (IL-1R1) and adiponectin receptor 1 (AdipoR1) were determined by semi-quantitative PCR. (B) Time course activation of various signaling pathways in adiponectin-stimulated RA FLSs. FLSs cultured (2.5 × 10⁵ cells) in 60 mm dishes were serum-starved overnight and stimulated with either adiponectin or IL-1β for a given time. The cells were prepared for Western blot analysis. (C) IκB-α levels. (D) Nuclear levels of NF-κB in adiponectin and/or IL-1β-stimulated RA FLSs. IκB-α level was analyzed by Western blot after 60 min of stimulation. The nuclear level was evaluated after 90 min of stimulation, as described in the Materials and Methods section. For the Western analysis, three independent experiments were performed in one plate with FLSs from each patient. For the analysis of NF-κB level, three independent experiments were performed in quadruplicate with FLSs from each patient. The data shown are representative of three independent experiments, and similar results were obtained from all three. Values are expressed as mean ± S.E.M. ^*P < 0.05 versus treatment with IL-1β (100 pg/ml) or adiponectin (10 µg/ml) alone.