The alleviation of skin wound-induced intestinal barrier dysfunction via modulation of TLR signalling using arginine in gilthead seabream (Sparus aurata L)

Highlights

• The effect of arginine on toll-like receptors (TLRs) in skin wound-induced intestinal barrier dysfunction was studied.

• 1% arginine inhibited intestinal inflammatory response and tight junction disruption in skin-wounded gilthead seabream.

• Dietary 1% arginine prevent the intestinal dysfunction after suffering skin wounds.

• Arginine displayed anti-inflammatory action via the TLR-mediated NF-κB signalling pathway.

• TLR gene expression correlates with the gene expression of tight junction proteins in fish fed arginine.

Abstract

The present study sought to investigate the effect of arginine on the involvement of toll-like receptors (TLRs) in skin wound-induced intestinal barrier dysfunction in gilthead seabream (Sparus aurata L.). Two replicates of fish (n = 8) were fed a commercial diet (CON, total 2.75% arginine), CON diet enriched with 1% arginine (ARG1, total 3.65% arginine) and 2% arginine (ARG2, total 4.53% arginine) for 30 days. Half of the fish were sampled, whereas the others were injured and sampled 7 days post-wounding. The intestinal histology results showed that a more intense infiltration of mixed leucocytes was evident in the wounded fish, which was remarkably reduced in fish that were fed the ARG1 diet. Serum IgM levels were significantly higher in the ARG1 group than levels in the CON group at 7 days post-wounding. Compared with the fish in the CON group after wounding, dietary administration of 1% arginine markedly downregulated the gene expression of TLRs (TLR2 and TLR5), MyD88, and proinflammatory cytokines (CSF1R, IL-1β, and TNFα), but significantly enhanced the gene expression of IκBα, the anti-inflammatory cytokine TGF-β1, and tight junction proteins (tricellulin and occludin) in wounded fish. Furthermore, the ARG2 diet demonstrated no additional benefits on intestinal cells, compared to both the ARG1 and the CON diets, and it even appeared to induce negative effects. In summary, dietary administration of 1% arginine significantly inhibited intestinal inflammatory response and tight junction disruption in skin-wounded gilthead seabream by modulating TLR signalling in the intestine.

Introduction

The intestine is the main organ involved in the absorbing of nutrients, water, and electrolytes from food. Furthermore, it provides a tight barrier against pathogenic infections and coexists with a myriad of commensal organisms [1,2]. Toll-like receptors (TLRs) prompt proinflammatory responses in reaction to the detection of components of foreign pathogens referred to as pathogen-associated molecular patterns (PAMP), thereby triggering the host defense response [3,4]. Besides the regulation of inflammatory responses in the intestine, TLR signalling also plays a crucial role in epithelial cell proliferation, tight junction assembly, and antimicrobial peptide expression, contributing to an epithelial barrier [5]. Recent investigations on mammals have increasingly suggested that inflammatory responses triggered by TLR signalling might be critical in the development of intestinal dysfunction [[6], [7], [8]]. However, only a few studies on fish have indicated the role of TLRs in the progression of intestinal dysfunction. One study on Atlantic salmon (Salmo salar L.) pointed that the TLR downstream-regulated gene myeloid differentiation primary response gene 88 (MyD88) was significantly upregulated during the early and late inflammation stages of soybean meal-induced enteropathy (SBMIE) [9]. Furthermore, another SBMIE-related study on turbot (Scophthalmus maximus L.) demonstrated that TLR signalling in the intestine was activated [10]. Lastly, a previous study conducted by our research group showed that skin wounds in gilthead seabream induced intestinal dysfunction, which included inflammatory response, changes in the mucus layer, and tight junction disruptions [11]. However, the role of TLR signalling has not been well defined.

In mammals, many studies have indicated the positive effect of arginine on intestinal dysfunction [12,13]. For example, a study on mice with dextran sulphate sodium colitis showed that arginine preserved intestinal mucosa and tended to decrease inflammation [14]. Additional research on rats have revealed that oral l-arginine could significantly decrease the serum levels of TNF-α, inhibit the colonic expression of iNOS, NF-κB, and cytochrome c, and increase the expression of HSP70 in trinitrobenzene-sulfonic acid (TNBS)-induced colitis [15].

Arginine is an essential amino acid for fish [16]. Arginine could improve intestinal enzyme activities and maintain intestinal microbial balance by decreasing the growth of harmful bacteria in juvenile Jian carp (Cyprinus carpio var. Jian) [17]. One study on juvenile Jian carp showed that dietary arginine deficiency induced intestinal inflammation and disrupted intestinal tight junction [18]. Furthermore, an increasing number of studies have been conducted on fish to study the beneficial effects of arginine on enteropathy. A study on turbot demonstrated that dietary arginine activated the innate and adaptive immune system and suppressed the intestinal inflammation response in SBMIE [19]. In addition, a coincident study on turbot suggested that dietary administration of arginine could significantly alleviate soybean meal-induced tight junction disruption and decrease intestinal inflammatory response by suppressing the activation of NF-κB [20]. One study on juvenile Jian carp treated with lipopolysaccharide (LPS) also provided evidence that dietary arginine alleviated overexpression of proinflammatory cytokines in the intestine [21]. In line with previously mention research, the present study aims to investigate the effects of dietary arginine supplementation on skin wound-induced intestinal dysfunction and the potential regulatory role of TLR signalling involved in this process. The present study was conducted with gilthead seabream, which is a representative species of Mediterranean aquaculture. Therefore, the effects of arginine on intestinal histology, the gene expression of intestinal barrier function-related genes, and the molecules associated with TLR signalling were investigated to elucidate regulation by arginine on fish intestinal function. The results of the present study will help to uncover novel coping mechanisms or strategies for traumatic injury-induced intestinal dysfunction during intensive farming.