Fig. 3.

Effects of MSU on TLR signaling and NS on TLR4 expression. (A) BMDMs were treated with MSU (0.5 mg/mL) in the presence of TLR inhibitors (CU for TLR1/2, αTLR2 for TLR2, and TAK and PMB for TLR4) or NF-κB inhibitor (Bay) for 3 h. The protein levels of pro-IL-1β were measured as a readout of MSU–TLR interaction. The below bar graph indicates band intensity. Mice (n = 6 per group) fed Non (200 μL of water) or NS (1.1 mg/mouse/day) for 7 days and then ip injected with MSU (10 mg/mouse) to induce peritonitis. PECs were isolated from mice, and (B) TLR4 mRNA and (C) protein expression was analyzed by qPCR and immunoblotting. BMDMs were treated with NS for 3 h, and (D) mRNA and (E) protein expression levels of TLR4 were measured using RT-PCR/qPCR and immunoblotting. (F) BMDMs were treated with NS in the presence of TLR2, TLR4, NF-κB, or MyD88 (MyDi) inhibitor, and TLR4 mRNA expression was assayed by RT-PCR/qPCR. Bar graph presents the mean ± SD. All data shown are representative of at least two independent experiments. MW, molecular weight (100 bp ladder).

BMDM, bone marrow–derived macrophages; IL, interleukin; ip, intraperitoneally; MSU, monosodium urate; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NS, nonsaponin fraction; PEC, peritoneal exudate cell; qPCR, quantitative real-time polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation; TLR, toll-like receptor.