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

Figure 1 UCB significantly relieved severity of dextran sulfate sodium induced colitis. A: Body weight changes, data plotted as percentage of basal body weight; B: Disease activity index; C: Changes in colon length; D: Relative spleen weight. Data are expressed as means ± SEM (n = 5). ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 vs control group, ^dP < 0.05, ^eP < 0.01, and ^fP < 0.001 vs dextran sodium sulfate group. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; SEM: Standard error of the mean.

Figure 2 UCB inactivated digestive protease activity in DSS treated mice. A: Trypsin activity in feces; B: Chymotrypsin activity in feces. Data are expressed as means ± SEM (n = 5). ^cP < 0.001 vs control group and ^eP < 0.01 vs dextran sodium sulfate group. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; SEM: Standard error of the mean.

Figure 3 UCB ameliorated colon tissue injury in DSS induced colitis mice. A: Light microscopic assessment of hematoxylin and eosin stained sections; B: Histological scores (images acquired at 10 × using Olympus BX60; scale bar =100 μM; images acquired at 20 × using Olympus BX60, scale bar = 50 μM, n = 3); C: MPO activity of colon tissues (n = 5). Data are expressed as means ± SEM. ^cP < 0.001 vs control group, and ^fP < 0.001 vs DSS. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; MPO: Myeloperoxidase; SEM: Standard error of the mean.

Figure 4 UCB increased expression of tight junction proteins in the colon and decreased intestinal permeability. A: Protein expression of tight junction occludin (n = 3); B: D-Lactate level in serum (n = 5). Data are expressed as means ± SEM. ^cP < 0.001 vs control group, and ^fP < 0.001 vs DSS group. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; SEM: Standard error of the mean.

Figure 5 UCB maintained the steady state of intestinal flora of DSS induced colitis mice. Data are expressed as means ± SEM (n = 5). ^aP < 0.05 and ^bP < 0.01 vs control group; ^eP < 0.01, and ^fP < 0.001 vs dextran sodium sulfate group. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; SEM: Standard error of the mean.

Figure 6 UCB reduced production of proinflammatory cytokines in DSS induced colitis mice. A and B: Protein expression of TNF-α and IL-1β; C and D: mRNA transcript expression of TNF-α and IL-1β. Data are expressed as means ± SEM (n = 5). ^cP < 0.001 vs control group and ^fP < 0.001 vs DSS. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; TNF-α: Tumor necrosis factor α; IL-1β: Interleukin 1β; SEM: Standard error of the mean.

Figure 7 UCB inhibited the TLR4/MyD88/TRAF6/nuclear factor-κB signaling in DSS induced colitis mice. A: Total protein from colon samples was extracted, and TLR4, MyD88, TRAF6, and IκBα protein expression was measured by western blotting; B-E: Quantification of TLR4, MyD88, TRAF6, and IκBα protein expression was performed by densitometric analysis of the blots. Data are expressed as means ± SEM (n = 3). ^aP < 0.05 and ^cP < 0.001 vs control group; ^dP < 0.05 and ^fP < 0.001 vs dextran sodium sulfate group. UCB: Unconjugated bilirubin; DSS: Dextran sodium sulfate; TLR4: Toll-like receptor 4; MyD88: Myeloid differentiation primary response gene 88; TRAF6: Tumor necrosis factor receptor-associated factor 6; IκBα: Anti-inhibitor of nuclear factor-κB alpha.