Effectiveness of a hydroxynaphthoquinone fraction from Arnebia euchroma in rats with experimental colitis

Abstract

Aim: To evaluate the potential effectiveness of hydroxynaphthoquinone mixture (HM) in rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Methods: Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) and mesalazine 100 mg/kg 24 h after TNBS instillation. Disease progression was monitored daily by observation of clinical signs and body weight change. At the end of the experiment, macroscopic and histopathologic lesions of rats were scored, and myeloperoxidase (MPO) activity was determined. We also determined inflammatory cytokine tumor necrosis factor (TNF)-α level by ELISA, Western blotting and immunochemistry to explore the potential mechanisms of HM.

Results: After intracolonic instillation of TNBS, animals developed colitis associated with soft stool, diarrhea and marked colonic destruction. Administration of HM significantly attenuated clinical and histopathologic severity of TNBS-induced colitis in a dose-dependent manner. It abrogated body weight loss, diarrhea and inflammation, decreased macroscopic damage score, and improved histological signs, with a significant reduction of inflammatory infiltration, ulcer size and the severity of goblet cell depletion (all P < 0.05 vs TNBS alone group). HM could reduce MPO activity. In addition, it also decreased serum TNF-α level and down-regulated TNF-α expression in colonic tissue. This reduction was statistically significant when the dose of HM was 10 mg/kg (P < 0.05 vs TNBS alone group), and the effect was comparable to that of mesalazine and showed no apparent adverse effect. The underlying mechanism may be associated with TNF-α inhibition.

Conclusion: These findings suggest that HM possesses favourable therapeutic action in TNBS-induced colitis, which provides direct pharmacological evidence for its clinical application.

Keywords: 2,4,6-trinitrobenzene sulfonic acid-induced colitis; Arnebia euchroma (Royle) Johnst; Hydroxynaphthoquinones; Inflammatory bowel disease; Tumor necrosis factor.

Figure 1

Treatment with hydroxynaphthoquinone mixture ameliorates body weight loss of 2,4,6-trinitrobenzene sulfonic acid-induced rats. Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) and mesalazine 100 mg/kg 24 h after TNBS instillation. Disease progression was monitored by observation of clinical signs and body weight change. Data are represented as mean ± SD of 8 animals of each group. ^bP < 0.01 vs TNBS alone. HM: Hydroxynaphthoquinone mixture; TNBS: 2,4,6-trinitrobenzene sulfonic acid.

Figure 2

Hydroxynaphthoquinone mixture reduces colonic macroscopic damage in rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. A: Intestinal macroscopic changes; B: Macroscopic pathological scores. Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) and mesalazine 100 mg/kg 24 h after TNBS instillation. 1: Control; 2: Rat treated with TNBS alone; 3: Rat treated with TNBS and mesalazine; 4-6: Rats treated with TNBS and HM (2.5, 5, 10 mg/kg). Data are represented as mean ± SD of 8 animals of each group. ^bP < 0.01 vs control; ^cP < 0.05 vs TNBS alone. HM: Hydroxynaphthoquinone mixture; TNBS: 2,4,6-trinitrobenzene sulfonic acid.

Figure 3

Hydroxynaphthoquinone mixture prevents 2,4,6-trinitrobenzene sulfonic acid-induced histopathological changes. A: Intestinal histopathological features; B: Pathological scores of representative colonic samples of each group. Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) and mesalazine 100 mg/kg 24 h after TNBS instillation. Histopathological analysis was performed in HE-stained sections of colons. 1: Control; 2: Rat treated with TNBS alone; 3: Rat treated with TNBS and mesalazine; 4-6: Rats treated with TNBS and HM (2.5, 5, 10 mg/kg). Data are represented as mean ± SD of 5 animals of each group. ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS alone. HM: Hydroxynaphthoquinone mixture (original magnification, × 100); TNBS: 2,4,6-trinitrobenzene sulfonic acid.

Figure 4

Hydroxynaphthoquinone mixture decreases myeloperoxidase activity (A) and serum tumor necrosis factor-α level (B) in rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) and mesalazine 100 mg/kg 24 h after TNBS instillation. TNF-α level was determined by ELISA. Data are represented as mean ± SD of 8 animals of each group. ^bP < 0.01 vs control; ^cP < 0.05, ^dP < 0.01 vs TNBS alone. HM: Hydroxynaphthoquinone mixture; TNBS: 2,4,6-trinitrobenzene sulfonic acid; MPO: Myeloperoxidase; TNF-α: Tumor necrosis factor-α.

Figure 5

Immunostaining for tumor necrosis factor-α. A: Immunostaining sections; B: Integrated optical density (A) values of representative colonic samples of each group. Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) 24 h after TNBS instillation. Colons were excised and stained with anti-TNF-α antibody. 1: Control; 2: Rat treated with TNBS alone; 3-5: Rats treated with TNBS plus HM (2.5, 5, 10 mg/kg). Data are represented as mean ± SD of 4 animals of each group. ^aP < 0.05 vs control; ^dP < 0.01 vs TNBS alone. HM: Hydroxynaphthoquinone mixture (original magnification, × 400); TNBS: 2,4,6-trinitrobenzene sulfonic acid; TNF-α: Tumor necrosis factor-α.

Figure 6

Hydroxynaphthoquinone mixture decreases tumor necrosis factor-α expression in colonic tissue of rats with 2,4,6-trinitrobenzene sulfonic acid-induced colitis. A: Tumor necrosis factor (TNF)-α protein bands; B: Integrated optical density (A) values of protein bands. Colitis was induced by intracolonic administration of TNBS (80 mg/kg, dissolved in 50% ethanol). Rats were treated daily for 7 d with HM (2.5, 5, 10 mg/kg) 24 h after TNBS instillation. Protein extracts were obtained from colons and TNF-α expression level was detected by Western blotting analysis. Lane 1: Controls; lane 2: Rats treated with TNBS alone; lane 3-5: Rats treated with TNBS plus HM (2.5, 5, 10 mg/kg). Data are represented as mean ± SD of 4 animals of each group. ^aP < 0.05 vs control; ^cP < 0.05 vs TNBS alone. HM: Hydroxynaphthoquinone mixture; TNBS: 2,4,6-trinitrobenzene sulfonic acid.