Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats

Abstract

Aim: To investigate hepcidin expression, interleukin-6 (IL-6) production and iron levels in the rat colon in the presence of trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Methods: In rats, we evaluated the severity of colitis induced by repeated TNBS administration using macroscopic and microscopic scoring systems and myeloperoxidase activity measurements. The colonic levels of hepcidin, tumor necrosis factor alpha (TNF-α), IL-10 and IL-6 were measured by Enzyme-Linked Immunosorbent Assay, and hepcidin-25 expression and iron deposition were analyzed by immunohistochemistry and the Prussian blue reaction, respectively. Stat-3 phosphorylation was assessed by Western blot analysis. Hematological parameters, iron and transferrin levels, and transferrin saturation were also measured. Additionally, the ability of iron, pathogen-derived molecules and IL-6 to induce hepcidin expression in HT-29 cells was evaluated.

Results: Repeated TNBS administration to rats resulted in macroscopically and microscopically detectable colon lesions and elevated colonic myeloperoxidase activity. Hepcidin-25 protein levels were increased in colonic surface epithelia in colitic rats (10.2 ± 4.0 pg/mg protein vs 71.0 ± 8.4 pg/mg protein, P < 0.01). Elevated IL-6 levels (8.2 ± 1.7 pg/mg protein vs 14.7 ± 0.7 pg/mg protein, P < 0.05), TNF-α levels (1.8 ± 1.2 pg/mg protein vs 7.4 ± 2.1 pg/mg protein, P < 0.05) and Stat-3 phosphorylation were also observed. Systemic alterations in iron homeostasis, hepcidin levels and anemia were not detected in colitic rats. Iron deposition in the colon was only observed during colitis. Hepcidin gene expression was increased in HT-29 cells after IL-6 and lipopolysaccharide [a toll-like receptor 4 (TLR-4) ligand] treatment. Deferoxamine, ferric citrate and peptidoglycan (a TLR-2 ligand) were unable to alter the in vitro expression of hepcidin in HT-29 cells.

Conclusion: Colitis increased local hepcidin-25 expression, which was associated with the IL-6/Stat-3 signaling pathway. An increase in local iron sequestration was also observed, but additional studies are needed to determine whether this sequestration is a defensive or pathological response to intestinal inflammation.

Keywords: Anemia; Hepcidin-25; Interleukin-6; Iron; Stat-3; Toll-like receptor 4.

Figure 1

Colonic hepcidin expression in control and colitis rats. A: Immunohistochemistry of control rat tissue (x 100); B: Immunohistochemistry of an adjacent colitic area (x 100); C: Immunohistochemistry of a colitic area (x 100); D: Immunohistochemistry of control rat tissue (x 400); E: Immunohistochemistry of an adjacent colitic area (x 400); F: Immunohistochemistry of a colitic area (x 400); G: Enzymatic assay detection of hepcidin expression levels. Data are expressed as the means ± SE (n = 4 per group), and ^bP < 0.01 compared with control.

Figure 2

Stat-3 phosphorylation levels in control and colitic rats. A: Representative Western blot images (from one individual experiment); B: Bar graph densitometry quantifications of phospho-Stat-3 expression levels that were normalized to colonic Stat-3 expression levels (n = 4 per group). Data are expressed as the means ± SE, and ^aP < 0.05 compared with control.

Figure 3

Prussian blue reaction assessing the presence of ferric iron and ferritin in colons. A: Control (× 200); B: Colitis (× 200). Arrows denote areas of Prussian blue reactivity.

Figure 4

Analysis of hepcidin mRNA expression (hepcidin to β-actin ratio) in HT-29 cells. Untreated control cells were compared with cells treated with interleukin-6 (IL-6) (40 ng/mL), iron citrate (20 μmol/L), deferoxamine (30 mmol/L), lipopolysaccharide (LPS) (1 μg/mL) or peptidoglycan (PGN) (10 μg/mL). Data are expressed as the means ± SE of 2 experiments performed in triplicate. ^aP < 0.05 and ^bP < 0.01 compared with control.