Biliverdin and bilirubin sulfonate inhibit monosodium urate induced sterile inflammation in the rat

Abstract

Background: Biliverdin, a by-product of haem catabolism, possesses potent endogenous antioxidant and anti-inflammatory properties. Bilirubin-C10-sulfonate (BRS), an active metabolite formed after enteral administration of BV in the rat, also possess antioxidant properties. Therefore, we investigated the anti-inflammatory and antioxidant activity of BV and BRS in an in vivo model of monosodium urate induced sterile inflammation.

Methods: Subcutaneous air pouches were created on the dorsal flanks of Wistar rats (10-12 weeks of age). Prior to stimulation of the 6-day old pouch with monosodium urate (25 mg), groups were pre-treated with intraperitoneal BRS (27 mg/kg) and BV (27 mg/kg). Total and differential leukocyte counts were determined in pouch fluid aspirate at 1, 6, 12, 24 and 48 h after monosodium urate stimulation. Biliverdin (BV), BRS and unconjugated bilirubin (UCB) concentrations in the serum and pouch fluid were quantified using liquid chromatography-mass spectrometry. Pouch fluid cytokine concentrations (IL-1β, IL-1α, TNF-α, IL-17A, IL-12, GM-CSF, IL-33, IFN-γ, IL-18, IL-10, MCP-1, CXCL-1 and IL-6) were assessed after 6 h. In addition, 24 h protein carbonyl and chloramine concentrations were assessed in pouch fluid using ELISA and spectrophotometry, respectively.

Results: BRS and BV significantly (p < 0.05) inhibited leukocyte (total, neutrophil and macrophage) infiltration into the pouch fluid from 6 to 48 h. For example, after 6 h neutrophil counts decreased following BRS (0.32 ± 0.11 × 10⁶ cells mL^-1) and BV (0.17 ± 0.03 × 10⁶ cells mL^-1) compared to MSU only (3.51 ± 1.07 × 10⁶ cells mL^-1). Both BV and BRS significantly (p < 0.05) reduced pouch GM-CSF (BV: 5.8 ± 1.2 pg mL^-1, BRS: 6.9 ± 1.5 pg mL^-1 vs MSU only: 13.0 ± 1.9 pg mL^-1) and MCP-1 concentrations at 6 h (BV: 1804 ± 269 pg mL^-1, BRS: 7927 ± 2668 pg mL^-1 vs MSU only: 17,290 ± 4503 pg ml^-1), whilst BV additionally inhibited IL-6 (4354 ± 977 pg mL^-1 vs MSU only: 25,070 ± 5178 pg mL^-1) and IL-18 (17.6 ± 2.0 pg mL^-1 vs MSU only: 81.5 ± 19.9 pg mL^-1) concentrations at 6 h (p < 0.05). Despite these differences, no change in pouch chloramine or protein carbonyl concentrations occurred at 24 h (p > 0.05). Serum BV concentrations rapidly diminished over 6 h, however, BRS was readily detected in the serum over 48 h, and in pouch fluid over 12 h.

Conclusions: This study is the first to elucidate anti-inflammatory activity of BRS and the efficacy of BV administration in a model of gouty inflammation. Reduced leukocyte infiltration and cytokine production in response to sterile inflammation further support the importance of these molecules in physiology and their therapeutic potential in sterile inflammation.

Keywords: Bile pigments; Bilirubin sulfonate; Biliverdin; Cytokines; Gout; Monosodium urate; Sterile inflammation.