P057 Intestinal Anti-Inflammatory Activity of Indigo Alkaloid in TNBS-Induced Colitis
Authors: de Almeida Ana Cristina , de-Faria Felipe , Dunder Ricardo , Manzo Luis , Socca Eduardo Augusto , Souza-Brito Alba Regina , Luiz-Ferreira Anderson 
Published: December 2019

BACKGROUND:
Various drugs have been developed for treating inflammatory bowel disease (IBD), but there are still limitations in the treatment due to the insufficient responses and significant adverse effects. The aim of this study was to evaluate the effect of Indigo in experimental ulcerative colitis. Indigo is a bis-indolic alkaloid that has antiulcerogenic, antioxidant and anti-inflammatory activities.

METHODS:
Male Wistar HanUnib rats (200-300 g) were divided into five groups: SAL (non-colitic control), SAL + TNBS (colitic control), IND3+TNBS (colitic test), IND10 + TNBS (colitic test) and IND30 + TNBS (colitic test). After a ketamine (50 mg/kg) and xylazine (10 mg/kg) anesthesia, the SAL + TNBS and IND + TNBS groups received a single dose of TNBS (10 mg) dissolved in 0.25 mL of 50% ethanol (v/v) by rectal administration. The SAL group rectally received 0.25 mL of physiological saline. The physiological saline (10 mL/Kg) and Indigo (3, 10 and 30 mg/Kg) were administered 72, 48, 24, and 2 hours before and 24 hours after colitis induction by gavage. Animals were euthanized by cervical dislocation (under halothane anesthesia) 48 h after inflammation induction, and colons were removed for measuring the macroscopic inflammation, the weight/length ratio of colon, and the COX-1, COX-2 and nuclear factor-kappa B expressions, as well as for quantifying IL-10 by ELISA. The occurrence of adhesions (score 0-2) and/or diarrhea (score 0-1) was also evaluated. Data are expressed as median (minimum-maximum) or mean +/- SEM. Statistical analysis: Kruskal-Wallis test, followed by Duns or one-way ANOVA, followed by Tukey. The experiments were approved by the Ethics Commission in Animal Use - CEUA/Unicamp (CEUA number 2399-1).

RESULTS:
The TNBS group was characterized by increased colonic wall thickness, edema and diffuse inflammatory cell infiltration. In contrast, the Indigo (3 mg/kg) treatment significantly reduced the macroscopic inflammation score (P-value less than 0.05) and reduced the histological damage promoted by TNBS instillation. Indigo (30 mg/kg) prevented the increase in the weight/length ratio of the rat colon (as an indicator of inflammation; P-value less than 0.001), but did not prevent the presence of adhesions to adjacent organs and diarrhea. COX-1 expressions remained unchanged in all groups, indicating that COX-1 protein was constitutively expressed in the colonic tissue and was not significantly changed after TNBS induction or in the presence of Indigo treatment. On the other hand, COX-2 protein was significantly increased by the hapten, indicating that the inducible isoenzyme expression could be induced at the early stage of colonic lesion caused by TNBS. Nevertheless, oral administration of Indigo (3 mg/kg) decreased the up-regulation of COX-2 expression. Nuclear factor-kappa B expression was higher in the SAL + TNBS group, which was not prevented after treatment with Indigo. Colon damage caused by TNBS administration was also characterized by a decrease in the level of anti-inflammatory cytokine IL-10 compared to the SAL group, and treatment with Indigo (3 mg/kg) did not inhibit this reduction.

CONCLUSION(S):
Indigo decreased the macroscopic inflammation score and decreased weight/length ratio of the colon. Indigo action on colitis should mainly involve inhibiting or reducing the release of inflammatory mediators such as COX-2. These results suggest a potential role of Indigo in colitis therapy.