New approach of medicinal herbs and sulfasalazine mixture on ulcerative colitis induced by dextran sodium sulfate

Figure 1 Sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg) alleviated Dextran Sodium Sulfate-induced experimental ulcerative colitis. A: Change of body weight after induction of colitis by DSS; B: Length of colon and macroscopic viewpoint; Normal (non-DSS), Vehicle (DSS control), 30sulfa (sulfasalazine 30 mg/kg-treated), 60sulfa (sulfasalazine 60 mg/kg-treated), and SCPB (30sulfa plus Citrus unshiu peel and Bupleuri radix mixture at 30 mg/kg-treated); C: Hematoxylin and eosin staining of colon (magnification × 200), orange line meaned crypt destruction and loss. Data are presented as mean ± standard error of the mean for n = 7. ^aP < 0.05, ^eP < 0.001 vs initial body weight (b.w.) per group; ^fP < 0.001 vs normal (non-DSS) mice. SCPB: Sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg).

Figure 2 Sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg) reduced Serum reactive oxygen species and NADPH oxidase activity in colon. A: Serum reactive oxygen species (ROS); B: Western blot detection of NADPH oxidase proteins; NOX4, p47^phox, and Rac 1 protein expressions in mice from the groups of Normal (non-DSS), Vehicle (DSS control), 30sulfa (sulfasalazine 30 mg/kg-treated), 60sulfa (sulfasalazine 60 mg/kg-treated), and SCPB (30sulfa plus Citrus unshiu peel and Bupleuri radix mixture at 30 mg/kg-treated). Data are presented as mean ± standard error of the mean for n = 7. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001 vs normal (non-DSS and untreated) mice; ^cP < 0.05, ^fP < 0.001 vs DSS control mice. SCPB: Sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg); ROS: Reactive oxygen species.

Figure 3 Effect of sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg) on antioxidant proteins in Dextran Sodium Sulfate-induced colitis mice. Western blot detection of the antioxidant proteins; SOD, Catalase, and GPx-1/2 protein expression levels in mice from the groups of Normal (non-DSS), Vehicle (DSS control), 30sulfa (sulfasalazine 30 mg/kg-treated), 60sulfa (sulfasalazine 60 mg/kg-treated), and SCPB (30sulfa plus Citrus unshiu peel and Bupleuri radix mixture at 30 mg/kg-treated). Data are presented as mean ± standard error of the mean for n = 7. ^aP < 0.05, ^eP < 0.001 vs normal (non-DSS and untreated) mice; ^cP < 0.05, ^dP < 0.01 vs DSS control mice. SCPB: Sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg).

Figure 4 P-p38 and p-ERK protein expressions in Dextran Sodium Sulfate-induced colitis. Western blot detection of the mitogen-activated protein kinase-related proteins; p-p38 and p-ERK in mice from the groups of Normal (non-DSS), Vehicle (DSS control), 30sulfa (sulfasalazine 30 mg/kg-treated), 60sulfa (sulfasalazine 60 mg/kg-treated), and SCPB (30sulfa plus Citrus unshiu peel and Bupleuri radix mixture at 30 mg/kg-treated). Data are presented as mean ± standard error of the mean for n = 7. ^aP < 0.05, ^bP < 0.01 vs normal (non-DSS) mice; ^cP < 0.05 vs DSS control mice. SCPB: sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg).

Figure 5 NF-κB p65, COX-2, iNOS, TNF-α, IL-1β MCP-1, and ICAM-1 protein expressions in Dextran Sodium Sulfate-induced colitis. Western blot detection of the inflammatory proteins; NF-κBp65, COX-2, iNOS, TNF-α, IL-6, MCP-1, and ICAM-1 in mice from the groups of Normal (non-DSS), Vehicle (DSS control), 30sulfa (sulfasalazine 30 mg/kg-treated), 60sulfa (sulfasalazine 60 mg/kg-treated), and SCPB (30sulfa plus Citrus unshiu peel and Bupleuri radix mixture at 30 mg/kg-treated). Data are presented as mean ± standard error of the mean for n = 7. ^aP < 0.05, ^bP < 0.01, ^eP < 0.001 vs normal (non-DSS) mice; ^cP < 0.05, ^dP < 0.01, ^fP < 0.001 vs DSS control mice. SCPB: sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg).

Figure 6 Bax, Bcl-2, and caspase-3 protein expressions in Dextran Sodium Sulfate-induced colitis. Western blot detection of the apoptotic and anti-apoptotic proteins; Bax, Bcl-2, and caspase-3 in mice from the groups of Normal (non-DSS), Vehicle (DSS control), 30sulfa (sulfasalazine 30 mg/kg-treated), 60sulfa (sulfasalazine 60 mg/kg-treated), and SCPB (30sulfa plus Citrus unshiu peel and Bupleuri radix mixture at 30 mg/kg-treated). Data are presented as mean ± standard error of the mean for n = 7. ^aP < 0.05, ^bP < 0.01 vs normal (non-DSS) mice; ^cP < 0.05, ^dP < 0.01, ^fP < 0.001 vs DSS control mice. SCPB: sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg).

Figure 7 Possible mechanism of Citrus unshiu peel and Bupleuri radix mixture combined with sulfasalazine in Dextran Sodium Sulfate-induced ulcerative colitis. SCPB: 30sulfa plus Citrus unshiu peel and Bupleuri radix mixture 30 mg/kg-treated mice. DSS: Dextran Sodium Sulfate; SCPB: Sulfasalazine (30 mg/kg) + Citrus unshiu peel and Bupleuri radix mixture (30 mg/kg); ROS: Reactive oxygen species; UC: Ulcerative colitis.