Abstract
Toll-like receptor 4 (TLR-4) is crucial in maintaining intestinal epithelial homeostasis, participates in a vigorous signaling process and heightens inflammatory cytokine output. The objective of this study was to determine the effects of glutamine (GLN) on TLR-4 signaling in intestinal mucosa during methotrexate (MTX)-induced mucositis in a rat. Male Sprague–Dawley rats were randomly assigned to one of four experimental groups of 8 rats each: 1) control rats; 2) CONTR-GLN animals were treated with oral glutamine given in drinking water (2%) 48 hours before and 72 hours following vehicle injection; 3) MTX-rats were treated with a single IP injection of MTX (20 mg/kg); and 4) MTX-GLN rats were pre-treated with oral glutamine similar to group B, 48 hours before and 72 hours after MTX injection. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. The expression of TLR-4, MyD88 and TRAF6 in the intestinal mucosa was determined using real time PCR, Western blot and immunohistochemistry. MTX-GLN rats demonstrated a greater jejunal and ileal mucosal weight and mucosal DNA, greater villus height in ileum and crypt depth and index of proliferation in jejunum and ileum, compared to MTX animals. The expression of TLR-4 and MyD88 mRNA and protein in the mucosa was significantly lower in MTX rats versus controls animals. The administration of GLN increased significantly the expression of TLR-4 and MyD88 (vs the MTX group). In conclusion, treatment with glutamine was associated with up-regulation of TLR-4 and MyD88 expression and a concomitant decrease in intestinal mucosal injury caused by MTX-induced mucositis in a rat.
Highlights
The family of mammalian Toll-like receptors (TLRs) are homologs of the Drosophila Toll protein, which was originally identified as a receptor that plays a central role in the establishment of the embryonic dorsal-ventral pattern in fly embryos [1]
Recent studies have shown that there is an overexpression of TLR2 and TLR4 in intestinal epithelial cells which is correlated with the severity of mucosal damage, together with an increase of apoptotic cells and markedly impaired proliferation [5]
We showed that treatment with glutamine attenuates alterations in toll-like receptor 4 (TLR-4), myeloid differentiation primary response gene-88 (MyD88) and TRAF6 intestinal expression during LPS endotoxemia in a rat [16]
Summary
The family of mammalian Toll-like receptors (TLRs) are homologs of the Drosophila Toll protein, which was originally identified as a receptor that plays a central role in the establishment of the embryonic dorsal-ventral pattern in fly embryos [1]. Growing evidence suggests that TLRs have a critical role in the recognition of bacterial lipoproteins, major components of bacterial cell walls. Their signaling pathway is known to activate target genes such as nuclear factor-κB (NF-κB) and cytokines that are involved passed onward by a cascade of the receptor-associated kinase (IRAKs), receptor-associated factor 6 (TRAF6), and NF-kappa B inducing kinase (NIK), resulting in activation of NFκB which amplifies the signal, and leads to the induction or suppression of genes that orchestrate the inflammatory response [2,4]. The increased expression of TLRs was found in the intestinal tissues during experimental necrotizing enterocolitis (NEC) which is a well known pediatric intestinal inflammatory disease [6]. TLRs when activated by bacterial ligands signal intracellularly to up-regulate the expression of various cytokines in the intestine, including pro-(IL-1β, IL-6, IL-8) and anti-inflammatory cytokines (IL-10) in human and different NEC models [7,8]
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