Dipeptidyl peptidase 4 inhibitor sitagliptin protected against dextran sulfate sodium-induced experimental colitis by potentiating the action of GLP-2

Abstract

Dipeptidyl peptidase 4 (DPP4), a ubiquitously expressed protease that cleaves off the N-terminal dipeptide from proline and alanine on the penultimate position, has important roles in many physiological processes. In the present study, experimental colitis was induced in mice receiving 3% dextran sulfate sodium (DSS) in drinking water. We found that mice with DSS-induced colitis had significantly increased intestinal DPP activity and decreased serum DPP activity, suggesting a probable correlation of DPP4 with experimental colitis. Then, we investigated whether sitagliptin, a specific DPP4 inhibitor could protect against DSS-induced colitis. We showed that oral administration of single dose of sitagliptin (30 mg/kg) on D7 remarkably inhibited DPP enzyme activity in both serum and intestine of DSS-induced colitic mice. Repeated administration of sitagliptin (10, 30 mg/kg, bid, from D0 to D8) significantly ameliorated DSS-induced colitis, including reduction of disease activity index (DAI) and body weight loss, improvement of histological score and colon length. Sitagliptin administration dose-dependently increased plasma concentrations of active form of GLP-1 and colonic expression of GLP-2R. Co-administration of GLP-2R antagonist GLP-2^3-33 (500 μg/kg, bid, sc) abolished the protective effects of sitagliptin in DSS-induced colitic mice. Moreover, sitagliptin administration significantly decreased the ratio of apoptotic cells and increased the ratio of proliferative cells in colon epithelium of DSS-induced colitic mice, and this effect was also blocked by GLP-2^3-33. Taken together, our results demonstrate that sitagliptin could attenuate DSS-induced experimental colitis and the effects can be attributed to the enhancement of GLP-2 action and the subsequent protective effects on intestinal barrier by inhibiting epithelial cells apoptosis and promoting their proliferation. These findings suggest sitagliptin as a novel therapeutic approach for the treatment of ulcerative colitis.

Keywords: GLP-23-33; apoptosis; dextran sulfate sodium; dipeptidyl peptidase 4; glucagon-like peptide-2; proliferation; sitagliptin; ulcerative colitis.

Fig. 1. DPP enzyme activity in serum and intestinal tissues of normal control mice and DSS-induced colitis mice.

Mice were treated with 3% DSS in drinking water for 5 days to induce experimental colitis. a Relative enzyme activity of intestinal tissues (n = 6–10). b Relative enzyme activity of serum (n = 10–12). Data are expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs the relative normal control.

Fig. 2. Inhibitory effects of sitagliptin on DPP enzyme activity in serum and intestine.

Mice received 3% DSS in drinking water for 7 days to induce experimental colitis. A single dose of sitagliptin (30 mg/kg) or vehicle (distilled water) was orally administered on day 7. The DPP enzyme activity of serum was determined at different time points after dosing. a 0 h, b 4 h, c 8 h. DPP enzyme activity of different parts of the intestine was determined at 8 h after dosing. d Jejunum, e Ileum, f Colon. Data are expressed as the mean ± SEM (n = 10). ^**P < 0.01 vs the relative vehicle control.

Fig. 3. Sitagliptin improved the symptoms and pathological changes in DSS-induced colitis.

DSS (3%) was given to mice for 7 days to induce experimental colitis in mice. Sitagliptin (10, 30 mg/kg) or vehicle (distilled water) was orally administered to the relevant groups from day 0 to day 8 twice daily. a DAI assessment. b Representative images of colons on day 8. c Colon length. d Colon weight/length ratio. e Histological score of colons assigned according to the criteria described in the methods. f Representative micrographs (magnification ×100) and the partially enlarged detail of H&E-stained sections of colon tissues. Data are expressed as the mean ± SEM (n = 10–12). ^*P < 0.05, ^**P < 0.01 vs the DSS control group.

Fig. 4. Effect of sitagliptin on the mRNA expression of proinflammatory mediators and junction-associated components in colons.

a TNFα, b IL-1β, c IL-6, d ZO-1, e occludin, f claudin. Data are expressed as the mean ± SEM (n = 10–12). ^*P < 0.05, ^**P < 0.01 vs the DSS control group.

Fig. 5. Influence of sitagliptin on incretin hormones and correlated genes in DSS-induced colitis mice.

a Colonic expression of proglucagon mRNA. b Colonic expression of PC1/3 mRNA. c Colonic expression of GLP-2R mRNA. d Colonic expression of DPP4 mRNA. e Levels of plasma total GLP-2. f Levels of plasma active GLP-1. Data are expressed as the mean ± SEM (n = 10–12). ^*P < 0.05, ^**P < 0.01 vs the DSS control group.

Fig. 6. GLP-2^3-33 repressed the protective effect of sitagliptin on the DAI, colon length, colon weight/length ratio, and colon histological injury in DSS-induced colitis mice.

DSS (3%) was given to mice for 6 days to induce experimental colitis in mice. Sitagliptin (30 mg/kg) or vehicle (distilled water) was orally administered to the relevant groups from day 0 to day 8 twice daily. GLP-2^3-33 or vehicle (PBS) was administered subcutaneously to mice from day 0 to day 8 twice daily. a DAI assessment. b Colon length. c Colon weight/length ratio. d Histological score of colons assigned according to the criteria described in the methods. Representative micrographs (magnification ×100) and the partially enlarged detail of different kinds of staining sections of colon tissues. e H&E. f Alcian blue. Data are expressed as the mean ± SEM (n = 9–10). ^*P < 0.05, ^**P < 0.01 vs the DSS control group; ^#P < 0.05, ^##P < 0.01 vs the DSS + sitagliptin (30 mg/kg) group.

Fig. 7. The modulatory effects of sitagliptin on the apoptosis and proliferation of colonic epithelial cells were repressed by GLP-2^3-33 in DSS-induced colitis mice.

a Representative micrographs (magnification ×400) and the partially enlarged detail of TUNEL-stained sections. b Representative micrographs (magnification ×200) and the partially enlarged detail of Ki-67 immunostained sections. c Quantification of TUNEL-positive cells in each group. d Quantitative analysis of Ki-67-positive cells in each group. Data are expressed as the mean ± SEM (n = 9–10). ^*P < 0.05, ^**P < 0.01 vs the DSS control group; ^#P < 0.05, ^##P < 0.01 vs the DSS + sitagliptin (30 mg/kg) group.