Alpha-Melanocyte Stimulating Hormone Protects Against Cytokine-Induced Barrier Damage In Caco-2 Intestinal Epithelial Monolayers

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of alpha-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of alpha-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-alpha and interleukin-1 beta. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-kappa B) was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. alpha MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-kappa B p65 subunit into Caco-2 cell nuclei, which was inhibited by alpha-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of alpha-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and alpha-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-alpha and interleukin-1 beta cytokines.