Protective effect of Dulaglutide, a GLP1 agonist, on acetic acid-induced ulcerative colitis in rats: involvement of GLP-1, TFF-3, and TGF-β/PI3K/NF-κB signaling pathway

Abstract

A chronic inflammatory condition of the colon called ulcerative colitis (UC) is characterized by mucosal surface irritation that extends from the rectum to the near proximal colon portions. The rationale of this work was to conclude if dulaglutide (Dula) could protect rats from developing colitis caused by exposure to acetic acid (AA). Rats were randomly divided into seven groups (each with eight rats): Normal control, Dula control, AA (received 2 milliliters of 3% v/v AA through the rectum), Sulfasalazine (SLZ); given SLZ (100 mg/kg) orally from day 11 to day 21 then AA intrarectally on day 22 and Dula groups ( pretreated with 50, 100 or 150 μg/kg subcutaneous injection of Dula - once weekly for three weeks and AA on day 22 to induce ulcerative colitis, colon tissues and blood samples were taken on day 23. By generating colonic histological deviations such as inflammatory processes, goblet cell death, glandular hyperplasia, and mucosa ulcers, Dula dropped AA-induced colitis. Additionally, these modifications diminished blood lactate dehydrogenase (LDH), C-reactive protein (CRP), colon weight, and the weight/length ratio of the colon. In addition, Dula decreased the oxidative stress biomarker malondialdehyde (MDA) and increased the antioxidant enzymes (total antioxidant capacity (TAC), reduced glutathione (GSH), and superoxide dismutase (SOD) concentrations). Dula also significantly reduced the expression of transforming growth factor-1 (TGF-β1), phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT) signaling pathway, and the inflammatory cytokines: nuclear factor kappa B (NF-κB), interleukin-6 (IL-6), and interferon-γ (IFN-γ) in colonic cellular structures. In addition, Dula enforced the levels of glucagon-like peptide-1 (GLP-1) and trefoil factor-3 (TFF-3) that were crucial to intestinal mucosa regeneration and healing of wounds. By modulating TGF-β1 in conjunction with other inflammatory pathways like PI3K/AKT and NF-κB, regulating the oxidant/antioxidant balance, and improving the integrity of the intestinal barrier, Dula prevented AA-induced colitis in rats.

Keywords: Dulaglutide; Phosphatidylinositol-3-kinase; Protein kinase B; Transforming growth factor-1; Trefoil factor-3; Ulcerative colitis.

Fig. 1

Schematic presentation of the experimental design. AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide

Fig. 2

Effect of Dula (50, 100, and 150 μg/kg) on macroscopic scores. A Macroscopic scores and B photographed colon tissues. Data were expressed as median ± IQR. AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide. ^@,$p<0.05, significantly different from the control and AA-injected groups, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 3

Effect of Dula (50, 100, and 150 μg/kg) on colon histopathological changes (H&E Bar size: 100 μm). A Sections of the normal colon showed normal colonic mucosal glands, average in size and shape lined with columnar cells and goblet cells separated by fibroblastic stroma. B Section of colon with only Dula 150 μg/kg protection (control) average-sized colonic mucosa (red arrow) with underlying submucosa with mild congested vessels (blue arrows). C Section of the diseased colon (AA) showed sub-mucosal large infiltrate of chronic inflammatory cells with lymphoid follicle formation score 3 (blue arrow) with hyperplasia of glands score 2 and loss of goblet cells score 2 (red arrows). D Section of the colon SLZ-treated group showed individual chronic inflammatory cells infiltrate score 1 with hyperplastic glands score 1 with no loss of goblet cells score 0. E Section of the colon of Dula 50 μg/kg-treated group showed focal ulceration score 3 (black arrow) with heavy inflammation score 3 (blue arrow), glandular hyperplasia score 2 with goblet cells loss score 2 (red arrow). F Section of the colon of Dula 100 μg/kg-treated group showed healed ulcer (red arrow) with mild inflammation score 2 (blue arrow), glandular hyperplasia score of 1 with goblet cells loss score 1 (black arrows). G Section of colon of Dula 150 μg/kg-treated group showed no ulceration with individual cell infiltration score 1 (red arrows), moderate glandular hyperplasia score 2 (blue arrows) with no goblet cells loss score 0

Fig. 4

Histopathology-based scatter dot plots of mucosal ulceration grades. A colonic inflammation; B goblet cell loss; C glandular hyperplasia; D ulceration. Data were expressed as median ± IQR. ^{@, +}p<0.05, significantly different from the control and AA-injected groups, respectively, applying the multiple comparison test of Dunn after the Kruskal-Wallis test

Fig. 5

Effect of Dula (50, 100, and 150 μg/kg) on colonic levels of GLP-1. Data were expressed as mean ± SEM (n = 8). AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide; GLP-1: glucagon like peptide-1. Data were expressed as mean ± SEM (n = 8). ^$,λ,ϕ,∇p<0.05, significantly different from the control group, AA-injected group, SLZ-treated group, and Dula 50 μg/kg-injected group, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 6

Effect of Dula (50, 100, and 150 μg/kg) on the colonic expression level of TFF-3. Data are expressed as mean ± SEM (n = 8). AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide; TFF-3: trefoil factor-3. ^$,λ,ϕ,∇p<0.05, significantly different from the control, AA-injected group, SLZ-treated group, and Dula 50 μg/kg-injected group, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 7

Effect of Dula (50, 100, and 150 μg/kg) on colonic level of TGF-β1. Data were expressed as mean ± SEM (n = 8). TGF-β1: transforming growth factor-β; AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide. ^$,λ,ϕ,∇p<0.05, significantly different from the control group, AA-injected group, SLZ-treated group, and Dula 50 μg/kg-injected group, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 8

Effect of Dula (50, 100, and 150 μg/kg) on colonic levels of PI3K and AKT. Data were expressed as mean ± SEM (n = 8). PI3K: phosphatidylinositol-3-kinase; AKT: protein kinase B; AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide. ^$,λ,ϕ,∇p<0.05, significantly different from the control group, AA-injected group, SLZ-treated group, and Dula 50 μg/kg-injected group, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 9

Effect of Dula (50, 100, and 150 μg/kg) on colonic expression level of NF-κB (high magnification X: 400 bar 50. IHC counterstained with Mayer’s hematoxylin). Microscopic pictures of immunostained colonic sections against NF-κB showed the following: A, B mild positive brown intensity in very few crypts (black arrows) in the control group and Dula control group. C a marked increase in positively stained crypts appears (black arrows) in AA-treated group. D Mild positive brown staining is seen in a few crypts (black arrows) in SLZ group. E Higher numbers of positively stained crypts (black arrows) are seen in Dula (50 μg/kg) more than the SLZ group. F Numbers of positively stained crypts (black arrows) are still seen (black arrows) in Dula (100 μg/kg). G The positive brown intensity markedly lowered and was observed in a few crypts (black arrows) in Dula (150 μg/kg). H NF-κB scores. Data were expressed as median ± IQR. AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide; NF-κB: nuclear factor kappa B. ^@,+p<0.05, significantly different from the control and AA-injected groups, respectively, applying the multiple comparison test of Dunn after the Kruskal-Wallis test

Fig. 10

Dula (50, 100, and 150 μg/kg) affects the level of IL-6 expression in the colon (high magnification X: 400 bar 50. IHC counterstained with Mayer’s hematoxylin). Microscopic pictures of immunostained colonic sections against IL-6 showed the following: A, B no positive cells in submucosa (black arrows) in the control group and Dula control group. C Many positive cells appear in submucosa in the AA group. D Numbers of positive cells markedly reduced in submucosa (black arrows) in the SLZ-treated group. E Few positive cells appear in submucosa (black arrows) in the Dula (50 μg/kg) group. F Very few positive cells appear in submucosa (black arrows) in the Dula (100 μg/kg) group. G The IL-6 positive cells are accidentally seen in submucosa (black arrows) in the Dula (150 μg/kg) group. H IL-6 scores. Data were expressed as median ± IQR. AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide. ^@,+p<0.05, significantly different from the control and AA-injected groups, respectively, applying the multiple comparison test of Dunn after the Kruskal-Wallis test

Fig. 11

The impact of Dula at 50, 100, and 150 μg/kg on the level of IFN-γ expression in the colon. Data were expressed as mean ± SEM (n = 8). IFN-γ: interferon-γ, AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide. ^$,λ,ϕ,∇p<0.05, significantly different from the control group, AA-injected group, SLZ-treated group, and Dula 50 μg/kg-injected group, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 12

Effect of Dula (50, 100, and 150 μg/kg) on serum levels of LDH and CRP. A LDH; B CRP. Data were expressed as mean ± SEM (n = 8). AA: acetic acid; SLZ: sulfasalazine; Dula: dulaglutide; LDH: lactate dehydrogenase; CRP: C-reactive protein. ^$,λ,ϕ,∇p<0.05, significantly different from the control group, AA-injected group, SLZ-treated group, and Dula 50 μg/kg-injected group, respectively, employing Tukey-Kramer multiple comparisons post hoc test after one-way ANOVA

Fig. 13

An illustration of the potential cascades that could be involved in Dula’s protective actions against AA-induced UC in rats. TAC is for total antioxidant capacity; Dula stands for dulaglutide; MDA is for malondialdehyde; SOD is for superoxide dismutase; and GSH is for reduced glutathione. Glucagon-like peptide-1 (GLP-1), trefoil Factor-3 (TFF-3), nuclear factor kappa B (NF-κB), interleukin-6 (IL-6), interferon-γ (INF-γ), transforming growth factor-1 (TGF-β1), phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), lactate dehydrogenase (LDH), and C-reactive protein (CRP)