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


CONCLUSIONS
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.
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Érica Martins Ferreira
ÉM
Gotardo
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Gilberto de Almeida
Gde A
Ribeiro
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Thayane Rodrigues Leite
TR
Clemente
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Camila Henrique
CH
Moscato
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Renata Bortolin Guerra
RB
Tomé
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Thalita
T
Rocha
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
José
J
Pedrazzoli
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Marcelo Lima
ML
Ribeiro
Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP 12916-900, Brazil.
Alessandra
A
Gambero
D000818:Animals; D003092:Colitis / Q000139:chemically induced* / Q000378:metabolism*; D003106:Colon / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D064451:Hepcidins / Q000378:metabolism*; D016753:Interleukin-10 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008297:Male; D009195:Peroxidase / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D014302:Trinitrobenzenesulfonic Acid / Q000737:chemistry*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
C578703:Hamp protein, rat; D064451:Hepcidins; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D014302:Trinitrobenzenesulfonic Acid; D007501:Iron; D009195:Peroxidase
Anemia; Hepcidin-25; Interleukin-6; Iron; Stat-3; Toll-like receptor 4