Melatonin reduces inflammatory injury through inhibiting NF-kappaB activation in rats with colitis

Jun-Hua Li, Jie-Ping Yu, Hong-Gang Yu, Xi-Ming Xu, Liang-Liang Yu, Jin Liu, He-Sheng Luo, Jun-Hua Li, Jie-Ping Yu, Hong-Gang Yu, Xi-Ming Xu, Liang-Liang Yu, Jin Liu, He-Sheng Luo

Abstract

Proinflammatory mediators are important in the pathogenesis of IBD, which are regulated by activation of NF-kappaB. The aim of this study was to investigate whether melatonin reduces inflammatory injury and inhibits proinflammatory molecule and NF-kappaB in rats with colitis. Rat colitis model was established by TNBS enema. NF-kappaB p65, TNF-alpha, ICAM-1, and IkappaBalpha in colon tissue were examined by immunohistochemistry, EMSA, RT-PCR, and Western blot analysis. Expression of proinflammatory molecule and activation of NF-kappaB were upregulated and IkappaB level decreased in rats with colitis. Melatonin reduces colonic inflammatory injury through downregulating proinflammatory molecule mediated by NF-kappaB inhibition and blockade of IkappaBalpha degradation.

Figures

Figure 1
Figure 1
Immunohistochemical staining of RelA in colon tissues of (a) model rats and (b) rats with melatonin treatment (10 mg.kg−1). SP×200. RelA protein is mainly expressed in the nucleus and cytoplasmic accumulation of RelA is also detected. RelA expression decreases dramatically in melatonin group.
Figure 2
Figure 2
Immunohistochemical staining for TNF-α protein expression in (a) the model group and (b) melatonin group (10 mg.kg−1). SP×200. The number of TNF-α immunoreactive positive cells in melatonin group is significantly less than that of model group.
Figure 3
Figure 3
Immunohistochemical staining for ICAM-1 protein expression. SP×200. Expression of ICAM-1 in (a) the normal group, (b) melatonin group (10 mg.kg−1). The ICAM-1 expression was downregulated markedly in melatonin group.
Figure 4
Figure 4
The mRNA expressions of (a) TNF-α and (b) ICAM-1 were assessed using RT-PCR standardized by coamplifying the housekeeping gene β-actin. Lanes 1–7: model, normal, melatonin (2.5, 5.0, 10.0 mg.kg−1), 5-ASA, marker.
Figure 5
Figure 5
Electrophoretic mobility shift assay demonstrating nuclear translocation and DNA binding of NF-κB. (a) Lanes 1–6: model, melatonin(2.5, 5.0, 10.0 mg.kg−1), 5-ASA, normal. (b) Lanes 1–4: the model group, lanes 2–3: specific competitor (using excess of unlabeled oligonucleotide), lanes 1 and 4: supershift (addition of p65 antibodies to the nuclear extracts).
Figure 6
Figure 6
Western blotting showed levels of IκBα in colon tissue of rats. Lanes 1–6: normal, model, melatonin (2.5, 5.0, 10.0 mg.kg−1), 5-ASA.

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