Telmisartan attenuates colon inflammation, oxidative perturbations and apoptosis in a rat model of experimental inflammatory bowel disease

Abstract

Accumulating evidence has indicated the implication of angiotensin II in the pathogenesis of inflammatory bowel diseases (IBD) via its proinflammatory features. Telmisartan (TLM) is an angiotensin II receptor antagonist with marked anti-inflammatory and antioxidant actions that mediated its cardio-, reno- and hepatoprotective actions. However, its impact on IBD has not been previously explored. Thus, we aimed to investigate the potential alleviating effects of TLM in tri-nitrobenezene sulphonic acid (TNBS)-induced colitis in rats. Pretreatment with TLM (10 mg/kg p.o.) attenuated the severity of colitis as evidenced by decrease of disease activity index (DAI), colon weight/length ratio, macroscopic damage, histopathological findings and leukocyte migration. TLM suppressed the inflammatory response via attenuation of tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2) and myeloperoxidase (MPO) activity as a marker of neutrophil infiltration besides restoration of interleukin-10 (IL-10). TLM also suppressed mRNA and protein expression of nuclear factor kappa B (NF-κB) p65 and mRNA of cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proinflammatory genes with concomitant upregulation of PPAR-γ. The alleviation of TLM to colon injury was also associated with inhibition of oxidative stress as evidenced by suppression of lipid peroxides and nitric oxide (NO) besides boosting glutathione (GSH), total anti-oxidant capacity (TAC) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). With respect to apoptosis, TLM downregulated the increased mRNA, protein expression and activity of caspase-3. It also suppressed the elevation of cytochrome c and Bax mRNA besides the upregulation of Bcl-2. Together, these findings highlight evidences for the beneficial effects of TLM in IBD which are mediated through modulation of colonic inflammation, oxidative stress and apoptosis.

Figure 1. Effect of telmisartan on the severity of TNBS-induced colitis in rats.

(A) Change of Body weight (%) (B) Disease activity index. (C) Colon weight/length ratio. (D) Colon macroscopic damage. Colon injury was induced by a single intrarectal instillation of TNBS (50 mg/kg) in 50% ethanol solution whereas the control group received the same volume of physiological saline solution rectally. Telmisartan was orally administered (10 mg/kg/day), starting 1 week before TNBS instillation and was continued till the 4^th day post TNBS insult. On the 5^th day, rats were euthanized and the colons were immediately excised. Values of body weight changes and colon weight/length ratio (parametric data) are expressed as mean ± SEM (n =  8) while the scores of disease activity index and macroscopic damage (non-parametric) are expressed as median; n = 8. *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 2. Telmisartan ameliorates histopathological damage and myeloperoxidase activity in colonic tissues of rats with TNBS colitis.

Representative photomicrographs of sections from colonic samples taken on the 5^th day post TNBS-rectal instillation. (A) Control rats receiving saline rectally showed normal architecture of mucosa (mu) with intact epithelial surface, submucosa and muscularis (ml) layer (open arrows). (B) Control rats receiving saline rectally + TLM (10 mg/kg/day p.o.) displayed no histological modifications. (C-E) TNBS-treated group (50 mg/kg) was characterized by focal necrosis of mucosa (nmu) and submucosa (nsmu) and diffuse infiltration of leukocytes (m) and edema (o) in the submucosal layer. (F) TNBS + TLM administration (10 mg/kg/day p.o.) revealed mucosal preservation, diminished inflammatory cell invasion and edema. Hematoxylin and eosin staining. (G) Microscopic damage scores (expressed as median; n = 8). (H) TLM inhibits colon myeloperoxidase (MPO) activity (mean ± SEM; n = 8). Histological analysis was performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 3. Telmisartan modulates inflammatory cytokines and PGE₂.in colon of rats with TNBS colitis.

Levels of tumor necrosis factor-α; TNF-α (A), interleukin-10; IL-10 (B) and prostaglandin E₂; PGE₂ (C) were determined by ELISA. Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Data are expressed as mean ± SEM (n =  8) *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 4. Effect of telmisartan on the mRNA expression of PPAR-γ and mRNA/ protein expression of NF-κB in the colon of rats with TNBS colitis.

(A) mRNA expression of peroxisome proliferator activated receptor-gamma; PPAR-γ. (B) mRNA expression of nuclear factor kappa B; NF-κB. The mRNA expression was detected by quantitative real-time RT-PCR. Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Data are expressed as mean ± SD (n =  6). *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. (C) Immunohistochemical detection of NF-κB p65 expression. Representative images for the detection of NF-κBp65 expression from colon samples harvested on the 5^th day post TNBS (magnification: × 200). Control and control + TLM gps: minimal expression; TNBS gp: extensive expression (brown color); TNBS+ TLM gp: attenuated expression. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 5. Effect of telmisartan on the mRNA expression of COX-2 and iNOS proinflammatory genes in colon of rats with TNBS colitis.

(A) Cyclo-oxygenase-2; COX-2. (B) Inducible nitric oxide synthase; iNOS. The mRNA expression was detected by quantitative real-time RT-PCR. Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Data are expressed as mean ± SD (n = 6). *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 6. Telmisartan ameliorates oxidative stress and enhances antioxidant defenses in the colon of rats subjected to TNBS-induced colitis.

(A) Lipid peroxides expressed as malondialdehyde; MDA. (B) Nitric oxide; NO. (C) Reduced glutathione; GSH. (D) Total antioxidant capacity; TAC. Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Data are expressed as mean ± SEM (n =  8) *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 7. Telmisartan enhances activites of superoxide dismutase; SOD (A) and glutathione peroxidase; GPx (B) antioxidant enzymes in colon of rats with TNBS colitis.

Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Data are expressed as mean ± SEM (n =  8) *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 8. Effect of telmisartan on the mRNA expression, activity and protein expression of caspase-3 in the colon of rats with TNBS colitis.

(A) Caspase-3 mRNA expression. (B) Caspase-3 activity. Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Caspase-3 mRNA expression was detected by quantitative real-time RT-PCR (data are expressed as mean ± SD; n =  6) and the activity was measured using ELISA (results are expressed as mean ± SEM; n = 8). *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. (C) Immunohistochemical detection of caspase-3 protein expression. Representative images of caspase-3 expression from colon samples harvested on the 5^th day post TNBS (magnification: × 200). Control and control + TLM gps: minimal expression; TNBS gp: extensive expression (brown color); TNBS+ TLM gp: attenuated expression. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 9. Effect of telmisartan on mRNA expression of Cyt c, Bax and Bcl-2 apoptotic genes in colon of rats with TNBS colitis.

(A) Cytochrome c; Cyt c. (B) Bcl-2 associated x protein; Bax. (C) B cell lymphoma-2; Bcl-2. mRNA expression was detected by quantitative real-time RT-PCR. Measurements were performed 5 days post TNBS instillation and TLM was administered for 12 days starting 1 week before colitis induction. Data are expressed as mean ± SD (n =  6). *Significant difference from control gp at p<0.05, # Significant difference from TNBS colitis gp at p<0.05. TLM; telmisartan, TNBS; tri-nitrobenzene sulfonic acid.

Figure 10. Diagram depicting the alleviating actions of telmisartan in TNBS-induced colitis.