Unconjugated bilirubin alleviates experimental ulcerative colitis by regulating intestinal barrier function and immune inflammation

BACKGROUND Unconjugated bilirubin (UCB) is generally considered toxic but has gained recent prominence for its anti-inflammatory properties. However, the effects of it on the interaction between intestinal flora and organisms and how it influences immune responses remain unresolved. AIM To investigate the role of UCB in intestinal barrier function and immune inflammation in mice with dextran-sulfate-sodium-induced colitis. METHODS Acute colitis was induced by 3% (w/v) dextran sulfate sodium salt in drinking water for 6 d followed by untreated water for 2 d. Concurrently, mice with colitis were administered 0.2 mL UCB (400 mu mol/L) by intra-gastric gavage for 7 d. Disease activity index (DAI) was monitored daily. Mice were sacrificed at the end of the experiment. The length of the colon and weight of the spleen were recorded. Serum level of D-lactate, intestinal digestive proteases activity, and changes to the gut flora were analyzed. In addition, colonic specimens were analyzed by histology and for expression of inflammatory markers and proteins. RESULTS Mice treated with UCB had significantly relieved severity of colitis, including lower DAI, longer colon length, and lower spleen weight (colon length: 4.92 +/- 0.09 cm vs 3.9 +/- 0.15 cm; spleen weight: 0.33 +/- 0.04 vs 0.74 +/- 0.04, P < 0.001). UCB administration inactivated digestive proteases (chymotrypsin: 18.70 +/- 0.69 U/g vs 44.81 +/- 8.60 U/g; trypsin: 1.52 +/- 0.23 U/g vs 9.05 +/- 1.77 U/g, P < 0.01), increased expression of tight junction (0.99 +/- 0.05 vs 0.57 +/- 0.03, P < 0.001), decreased serum level of D-lactate (31.76 +/- 3.37 mu mol/L vs 54.25 +/- 1.45 mu mol/L, P < 0.001), and lowered histopathological score (4 +/- 0.57 vs 7 +/- 0.57, P < 0.001) and activity of myeloperoxidase (46.79 +/- 2.57 U/g vs 110.32 +/- 19.19 U/g, P < 0.001). UCB also regulated the intestinal microbiota, inhibited expression of tumor necrosis factor (TNF) alpha and interleukin 1 beta (TNF-alpha: 52.61 +/- 7.81 pg/mg vs 105.04 +/- 11.92 pg/mg, interleukin 1 beta: 13.43 +/- 1.68 vs 32.41 +/- 4.62 pg/mg, P < 0.001), decreased expression of Toll-like receptor 4 (0.61 +/- 0.09 vs 1.07 +/- 0.03, P < 0.001) and myeloid differentiation primary response gene 88 (0.73 +/- 0.08 vs 1.01 +/- 0.07, P < 0.05), and increased expression of TNF-receptor-associated factor 6 (0.79 +/- 0.02 vs 0.43 +/- 0.09 P < 0.05) and inhibitor of kappa B alpha (0.93 +/- 0.07 vs 0.72 +/- 0.07, P < 0.05) in the colon. CONCLUSION UCB can protect intestinal barrier function, regulate normal intestinal homeostasis, and suppress inflammation via the Toll-like receptor 4/nuclear factor-kappa B signaling pathway.