Anti-inflammatory effect of ondansetron through 5-HT3 receptors on TNBS-induced colitis in rat

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestinal tract whose etiology has not yet been fully elucidated. Available medicines for treatment of IBD are not universally effective and result in marked deleterious effects. This challenge has thus heightened the need for research in order to adopt new therapeutic approaches for the treatment of IBD. 5-HT3 receptor antagonists have shown analgesic and anti-inflammatory properties in vitro and in vivo. Our aim was to investigate the effect of ondansetron, 5-HT3 receptor antagonist, in an immune-based animal model of IBD, trinitrobenzene sulfonic acid (TNBS)-induced rat colitis and probable involvement of 5-HT3 receptors. Two hours after induction of colitis (instillation of 50 mg/kg of TNBS dissolved in 0.25 ml of ethanol 50 % v/v) to male Wistar rats, ondansetron (2 mg/kg), dexamethasone (1 mg/kg), meta-chlorophenylbiguanide (mCPBG, 5 mg/kg), a 5-HT3 receptor agonist, or ondansetron + mCPBG were administrated intraperitoneally (ip) and continued daily for six days. The animals were sacrificed and distal colons were assessed macroscopically, histologically and biochemically [myeloperoxidase (MPO), tumor necrosis factor-alpha, interleukin-6 and interleukin-1 beta]. Ondansetron and dexamethasone resulted in a decrease in macroscopic and microscopic colonic damage significantly. In addition a dramatic reduction in MPO activity and colonic levels of inflammatory cytokines were seen. The protective effects of ondansetron were antagonized by concurrent administration of mCPBG. Our data suggests that the beneficial effects of ondansetron in TNBS-induced colitis could be mediated by 5-HT3 receptors.

Keywords: 5-HT3 receptor; TNBS-induced colitis; inflammatory bowel diseases; ondansetron.

Table 1. Scoring criteria for assessment of macroscopic colonic injuries

Table 2. Macroscopic and histologic parameters of colitis induced by TNBS (50mg/kg) in rats. All treatments were made daily for six days after induction of colitis.

Figure 1. Measurement of ulcer area by ImageJ program. Horizontal and vertical charting rulers as horizontal and vertical scale were recognized and interpreted by the ImageJ. With regard to ulcer color differentiation, some template photographs with a series of red colors were applied in order to enable the program for recognition of ulcers and calculate the area automatically.

Figure 2. Changes in diarrheal status before (day 0) and during 6 days of treatment after induction of colitis (TNBS, 50 mg/kg) in rats. Values are means ± SEM (n=6). ^**P<0.01 and ^*P<0.05 compared with normal group. Dex= Dexamethasone; TNBS-control= TNBS-control group; Ond= ondansetron group; mCPBG= meta chlorophenylbiguanide group; Ond+mCPBG= ondansetron+metachlorophenylbiguanide group. TNBS= 2,4,6-trinitrobenzenesulfonic acid. The rats were checked daily for stool consistency (1. Formed stools, 2. Loosed stools and 3. Diarrhea).

Figure 3. Microscopic presentation of TNBS-induced colitis in rats (hematoxylin and eosin staining; original magnification 10×). (A) Normal group: Colon tissue with normal architecture; (B) TNBS-control group: epithelial distortion, architectural destruction of the crypts and inflammatory cell infiltrates; (C & D) Dexamethasone and ondansetron groups respectively: mild to moderate mucosal and submucosal inflammation and mucosal inflammatory cell infiltrates; (E & F): mCPBG and mCPBG + ondansetron, respectively: destruction of mucosal architecture and infiltration of neutrophils. TNBS= 2,4,6-trinitrobenzenesulfonic acid; mCPG= meta-chlorophenylbiguanide

Figure 4. Changes in MPO activity of rat colon with TNBS-induced colitis. All treatments were made daily for 6 days. Ond.= ondansetron (2 mg/kg); Dex.= dexamethasone (1 mg/kg); mCPBG= meta chlorophenylbiguanide (5 mg/ kg); TNBS= 2,4,6-trinitrobenzenesulfonic acid (50 mg/kg); MPO= myeloperoxidase. Values are presented as mean ± SEM, n=6. ^*P<0.05 vs. TNBS-control group

Figure 5. Colonic TNF-α level of rats with TNBS-induced colitis quantified by ELISA. Ond.= ondansetron (2 mg/kg); Dex.= dexamethasone (1 mg/kg); mCPBG= metachlorophenylbiguanide (5 mg/kg); TNBS= 2,4,6-trinitrobenzenesulfonic acid (50 mg/kg); TNF-α= tumor necrosis factor-alpha. Values are presented as mean ± SEM, n=6. ^*P<0.05 vs. TNBS-control

Figure 6. Colonic IL-1β level of rats with TNBS-induced colitis quantified by ELISA. Ond.= ondansetron (2 mg/kg); Dex.= dexamethasone (1 mg/kg); mCPBG= metachlorophenylbiguanide (5 mg/kg); TNBS= 2,4,6-trinitrobenzenesulfonic acid (50 mg/kg); IL-1β= interleukin-1 beta. Values are presented as mean ± SEM, n=6. ^*P<0.01 vs. Normal group; ^#P<0.01 vs. TNBS- control group

Figure 7. Colonic IL-6 level of rats with TNBS-induced colitis quantified by ELISA. Ond.= ondansetron (2 mg/kg); Dex.= dexamethasone (1 mg/kg); mCPBG= metachlorophenylbiguanide (5 mg/kg); TNBS= 2,4,6-trinitrobenzenesulfonic acid (50 mg/kg); IL-6= interleukin-6 .Values are presented as mean ± SEM, n=6. ^*P<0.05 vs. TNBS-control