Glutamine attenuates the inhibitory effect of methotrexate on TLR signaling during intestinal chemotherapy-induced mucositis in a rat

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.

Chemotherapy Modulates Intestinal Immune Gene Expression Including Surfactant Protein-D and Deleted in Malignant Brain Tumors 1 in Piglets

“…The SAM analysis revealed differences in the expression of several genes previously identified to be associated with chemotherapy-induced mucositis, including MMP1, 2, 9, and 12 and their regulators TIMP-1 and TIMP- 2 and ADAMTS-1, the tight-junction protein OCLN, the proinflammatory cytokine IL-1β, TLR2 and 4, KLF4, MUC4, and ASS1 [7,8,10,11,13,14,15,16,17,18,22]. Furthermore, it revealed differential regulation of several other genes related to innate mucosal immunology and GI mucosal homeostasis [12,20,21,23,35,36].…”

“…Earlier findings in piglets showed no difference in intestinal levels of IL-1β and TNF 11 days after BUCY treatment while IL-6 and IL-8 were elevated compared to control values [9]. The TLR pattern recognition receptors, more specifically TLR2, 4, and 9 [10,11,12,14], may also play an important role in the inflammatory responses and immune tolerance [35]. Regardless, the role of TLR remains controversial and in our study neither TLR9 nor TLR4 was differentially regulated while TLR2 was upregulated only in DOX piglets (table 1).…”

“…Earlier preclinical research in this area has mainly focused on the initiation or early phase after chemotherapy [8,10,11,16,17], although a few studies also discuss later consequences of GI toxicity [7,13,15]. In this study, we were not able to determine drug class-specific overlaps associated with the initiation of mucositis, as the timing of sampling of our two studies differed, and the responses should be viewed as the relatively late consequences of chemotherapy treatment.…”

“…It is now widely accepted that the progression of mucositis involves 5 successive pathobiological stages [6]. Mucositis involves changes in luminal factors, such as mucins and gut microbiota [7,8,9], pattern recognition receptors involved in tissue homeostasis and immune tolerance (toll-like receptors 2, 4, and 9) [10,11,12], and changes in tight-junction proteins with consequences for epithelial cell-to-cell adhesion and intestinal barrier function [13,14]. The matrix metallopeptidases (MMP) and their regulators, a group of endopeptidases participating in extracellular matrix turnover, are likely also involved in mucositis [15,16,17], as are the Krüppel-like factors (KLF), specifically KLF4 [7,8].…”

Preventing and Managing Toxicities of High-Dose Methotrexate

“…Small animal models, mainly rats, have been used to investigate the mechanisms of gastrointestinal methotrexate toxicity [86,87], the effect on the resident microbiome [88], and a variety of approved and experimental interventions. and severity of cytopenias with pharmacokinetically guided leucovorin is low.…”