Dual Role of Endogenous Serotonin in 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis

Abstract

Background and aims: Changes in gut serotonin (5-HT) content have been described in Inflammatory Bowel Disease (IBD) and in different experimental models of colitis: the critical role of this monoamine in the pathogenesis of chronic gastrointestinal inflammation is gradually emerging. Aim of the present study was to evaluate the contribution of endogenous 5-HT through the activation of its specific receptor subtypes to the local and systemic inflammatory responses in an experimental model of IBD.

Materials and methods: Colitis was induced by intrarectal 2,4,6-TriNitroBenzene Sulfonic acid in mice subacutely treated with selective antagonists of 5-HT1A (WAY100135), 5-HT2A (Ketanserin), 5-HT3 (Ondansetron), 5-HT4 (GR125487), 5-HT7 (SB269970) receptors and with 5-HT1A agonist 8-Hydroxy-2-(di-n-propylamino)tetralin.

Results: Blockade of 5-HT1A receptors worsened TNBS-induced local and systemic neutrophil recruitment while 5-HT1A agonist delayed and mitigated the severity of colitis, counteracting the increase in colonic 5-HT content. On the contrary, blockade of 5-HT2A receptors improved global health conditions, reduced colonic morphological alterations, down-regulated neutrophil recruitment, inflammatory cytokines levels and colonic apoptosis. Antagonism of 5-HT3, 5-HT4, and 5-HT7 receptor sites did not remarkably affect the progression and outcome of the pathology or only slightly improved it.

Conclusion: The prevailing deleterious contribution given by endogenous 5-HT to inflammation in TNBS-induced colitis is seemingly mediated by 5-HT2A and, to a lesser extent, by 5-HT4 receptors and coexists with the weak beneficial effect elicited by 5-HT1A stimulation. These findings suggest how only a selective interference with 5-HT pro-inflammatory actions may represent an additional potential therapeutic option for intestinal inflammatory disorders.

Keywords: 5-HT1A receptor; 5-HT2A receptor; apoptosis; inflammation; intestine.

FIGURE 1

Effects of 5-HT receptor antagonists on TNBS-induced disease severity. DAI at day 4 (A), MS (B), colonic length (C), and colonic thickness (D) assessed in normal mice (N) and in TNBS-treated mice administered with vehicle (C), WAY100135 5 mg/kg (W), Ketanserin 5 mg/kg (K), Ondansetron 10mg/kg (O), GR125487 10 mg/kg (G) and SB269970 10 mg/kg (S) (n = 6–12 data per group). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. N mice; ^#P < 0.05, ^##P < 0.01 vs. C mice; one-way ANOVA followed by Bonferroni’s post-test. Kruskal–Wallis analysis followed by Dunn’s post-test was applied for statistical comparison of DAI and MS.

FIGURE 2

Histology. Representative hematoxylin-eosin stained sections of colonic specimens harvested from normal mice (A) and from TNBS-treated mice administered with vehicle (B), Ketanserin 5 mg/kg (C) or 8-OH-DPAT 1 mg/kg (D). TNBS colonic instillation caused epithelial degeneration, neutrophilic infiltration, and submucosal edema (indicated by arrows) in vehicle-treated animals (B), not overtly modified either by Ketanserin (C) or 8-OH-DPAT (D) treatment.

FIGURE 3

Effects of 5-HT receptor antagonists on TNBS-induced neutrophil infiltration in colon and lung. MPO activity in colonic (A) and lung (B) tissues excised from normal mice (N) and from TNBS-treated mice administered with vehicle (C), WAY100135 5mg/kg (W), Ketanserin 5mg/kg (K), Ondansetron 10mg/kg (O), GR125487 10mg/kg (G), and SB269970 10mg/kg (S) (n = 6–12 data per group). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. N mice; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. C mice; one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 4

Effects of 5-HT receptor antagonists on cytokines levels. Colonic concentrations of TNFα (A), IL-1β (B), IFNγ (C), and IL-10 (D) and plasmatic levels of TNFα (E) and IL-1β (F) in normal mice (N) and in TNBS-treated mice administered with vehicle (C), WAY100135 5 mg/kg (W), Ketanserin 5 mg/kg (K), Ondansetron 10 mg/kg (O), GR125487 10 mg/kg (G) and SB269970 10 mg/kg (S) (n = 6–12 data per group). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. N mice; ^#P < 0.05 vs. C mice; Kruskal–Wallis analysis followed by Dunn’s post-test.

FIGURE 5

Effect of Ketanserin on colonic caspase-3 and HSP70 levels. Colonic concentrations of caspase-3 (A) and HSP70 (B) in normal mice (N) and in TNBS-treated mice administered with vehicle (C) or Ketanserin 5 mg/kg (K) (n = 6–12 data per group). ^∗P < 0.05, ^∗∗P < 0.01 vs. N mice; ^#P < 0.05 vs. C mice; one-way ANOVA followed by Bonferroni’s post-test.

FIGURE 6

Effects of 5-HT_1A ligands on disease progression. Colitis severity, expressed as DAI, assessed on all four days following TNBS i.r. instillation in normal mice (black circles) and in mice administered with vehicle (black squares), WAY100135 5 mg/kg (black triangles) and 8-OH-DPAT 1mg/kg (white triangles) (n = 6–12 data per group). ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. N mice; ^#P < 0.05, ^###P < 0.001 vs. C mice; Kruskal–Wallis analysis followed by Dunn’s post-test.

FIGURE 7

Effects of 8-OH-DPAT on cytokines levels. Colonic concentrations (pg/mg protein) of TNFα, IL-1β, IFNγ, and IL-10 and plasmatic levels (pg/ml) of TNFα and IL-1β in normal mice (white bars) and in TNBS-treated mice administered with vehicle (black bars) and 8-OH-DPAT 1mg/kg (gray bars) (n = 6–12 data per group). ^∗P < 0.05, ^∗∗P < 0.01 vs. N mice; Kruskal–Wallis analysis followed by Dunn’s post-test.