Hyperbaric oxygen therapy ameliorates TNBS-induced acute distal colitis in rats

Abstract

Background: This study investigated the therapeutic effects of hyperbaric oxygen in experimental acute distal colitis focusing on its effect on the production of pro-inflammatory cytokines, nitric oxide and hypoxia-inducible factor 1alpha.

Methods: Colitis was induced with a rectal infusion of 150 mg/kg of TNBS under anesthesia with Ketamine (50 mg/kg) and Xylazine (10 mg/kg). Control animals received only rectal saline. After colitis induction, animals were subjected to two sessions of hyperbaric oxygen and were then euthanized. The distal intestine was resected for macroscopic analysis, determination of myeloperoxidase activity, western-blotting analyses of inducible nitric oxide synthase and cyclooxygenase-2 expression and immunohistochemical analysis of hypoxia-inducible factor 1alpha and cyclooxygenase-2. Cytokines levels in the distal intestine were measured using an enzyme-linked immunosorbent assay.

Results: Hyperbaric oxygen therapy attenuated the severity of acute distal colitis, with reduced macroscopic damage score. This effect was associated with prevention in the increase of pro-inflammatory cytokine production; myeloperoxidase activity, in the expression of inducible nitric oxide synthase and cyclooxygenase-2. Finally, hyperbaric oxygen inhibited the acute distal colitis-induced up-regulation of hypoxia-inducible factor 1alpha.

Conclusions: The results indicate that hyperbaric oxygen attenuates the severity of acute distal colitis through the down-regulation of pro-inflammatory events.

Keywords: Cytokines; Experimental colitis; Hyperbaric oxygen; Hypoxia; Inflammatory bowel diseases.

Figure 1

Flow-chart of experiment. All animals (n = 28) were submitted to intramuscular anesthisia (Ketamine 50 mg/kg and Xylazin 10 mg/kg), then divided to four groups: Saline rats (n = 7): submitted to intracolonic infusion of saline solution, no chamber pressurization; Saline/HBO rats: submitted to intracolonic infusion of saline solution plus two sessions of HBO (n = 7); TNBS rats, submitted to intracolonic infusion of TNBS and no chamber pressurization (n = 7); TNBS/HBO rats, submitted to intracolonic infusion of TNBS plus two sessions of HBO (n = 7). After the second HBO session all animals were submitted to intramuscular anesthesia, laparotomy, colon extraction, macroscopic damage score and the colon was divided into four segments, of 1 cm each (First segment: MPO; Second: Cytokines; Third: Western Blot (COX-2 and iNOS); Fourth: Immunohistochemistry (COX-2 and HIF-1α).

Figure 2

Hyperbaric oxygen therapy ameliorates acute distal colitis by diminishing the macroscopic damage scores. (A) Macroscopic damage score. The results are presented as: − Normal Appearance: Score 0; − Focal ulcer: Score 1; − Multifocal ulcer: Score 2; − Diffuse ulcer and necrosis: Score 3. (B) Intestine of control rat (Saline), normal appearance (score zero); (C) Intestine of Saline/HBO rat, normal appearance (score zero); (D) Intestine of TNBS rat, diffuse ulceration and necrosis (Score three); (E) Intestine of rat with colitis treated with hyperbaric oxygen (TNBS/HBO) had reduced macroscopic damage score (Score two). Values expressed as mean ± SEM. (n = 7 per group). * P < 0.02 compared to colitis group (Mann–Whitney).

Figure 3

Hyperbaric oxygen reduces tissue myeloperoxidase activity (MPO) and pro-inflammatory cytokine expression in acute distal colitis. (A) Acute distal colitis was induced by an intracolonic injection of TNBS (150 mg/kg). After two sessions of hyperbaric oxygen (HBO) the rats were killed, the distal 6 cm colonic segment was removed, and the MPO activity was measured. The anti-inflammatory effect of HBO was detected as a decrease in the number of tissue neutrophils. (B) Colitis by TNBS increases the IL-1β tissue expression and HBO decreases its expression. (C) Colitis by TNBS increases the TNF-α tissue expression. There was not a significant decrease in expression after HBO. (D) Colitis by TNBS increases the CINC-1 tissue expression and HBO decreases its expression. (E) Colitis by TNBS increases the IL-10 tissue expression and HBO decreases its expression. Data are expressed as the mean ± S.E.M. (n = 6–7). Data are expressed as the mean ± S.E.M. (n = 6–7). # P < 0.05 compared to the saline-injected group; *P < 0.05 compared to colitis (TNBS) group; (one-way ANOVA followed by the Bonferroni’s test).

Figure 4

Hyperbaric oxygen reduces tissue COX-2 and iNOS expression. (A) Acute distal colitis was induced by the intracolonic injection of TNBS (150 mg/kg). After two sessions of hyperbaric oxygen, the rats were killed, the distal 6 cm colonic segment was removed, and the expression of COX-2 was determined by western blot. The β-Actin level was used as a control. Data are presented as representative blots. (B) Densitometry of the pixel intensity of COX-2 bands relative to β-Actin is present. (C) Expression of iNOS was determined by western blot. The b-actin level was used as a control. (D) Densitometry of the pixel intensity of iNOS bands relative to β-Actin is present. Data are presented as representative blots. Data are expressed as the mean ± S.E.M. (n = 5–7). # P < 0.01 compared to the saline-injected group; * P < 0.01 compared to colitis (TNBS) group (one-way ANOVA followed by the Bonferroni’s test).

Figure 5

Hyperbaric oxygen reduces COX-2 expression in acute distal colitis. Acute distal colitis was induced by intracolonic injection of TNBS (150 mg/kg). After two sessions of hyperbaric oxygen, the rats were killed, the distal 6 cm colonic segment was removed, and the expression of COX-2 in sub mucosa was determined by immunohistochemical analysis. (A) Group I (Saline), (B) Group II (Saline/HBO), (C) Group III (TNBS), (D) Group IV (TNBS/HBO), (E) Rats with colitis (TNBS) presented substantial up-regulation of immunofluorescence of COX-2 in the colon. The HBO treatment (TNBS/HBO) remarkably reduces COX-2 expression. Data are expressed as the mean ± S.E.M. (n = 7). # P < 0.001 compared to the saline-injected group; *P < 0.001 compared to colitis (TNBS) group (one-way ANOVA followed by the Bonferroni’s test).

Figure 6

Hyperbaric oxygen reduces HIF-1α expression in acute distal colitis. Acute distal colitis was induced by intracolonic injection of TNBS (150 mg/kg). After two sessions of hyperbaric oxygen, the rats were killed, the distal 6 cm colonic segment was removed, and the expression of HIF-1α was determined by immunohistochemical analysis. (A) Group I (Saline), (B) Group II (Saline/HBO), (C) Group III (TNBS), (D) Group IV (TNBS/HBO), and (E) Rats with colitis (TNBS) presented substantial up-regulation of immunofluorescence of HIF-1α in the colon. The HBO treatment (TNBS/HBO) remarkably reduces HIF-1α expression. Data are expressed as the mean ± S.E.M. (n = 7). # P < 0.001 compared to the saline-injected group; *P < 0.001 compared to colitis (TNBS) group (one-way ANOVA followed by the Bonferroni’s test).