Full length articleThe alleviation of skin wound-induced intestinal barrier dysfunction via modulation of TLR signalling using arginine in gilthead seabream (Sparus aurata L)
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.
Section snippets
Ethics statement
This experiment was conducted at the Marine Fish Facilities at the University of Murcia, Spain. Animal care and treatment procedures were approved by the Ethical Committee of the University of Murcia and followed the European Union guidelines for experimental animal handling (2010/63/EU).
Diets and fish husbandry
A total of 48 gilthead seabream were purchased from a local farm (Murcia, Spain). Before the start of the feeding trial, the fish were maintained for two weeks to acclimate them to the experimental conditions.
Intestinal morphology
The indications of an intact epithelial barrier include well-arranged nucleus position in the enterocytes, evenly distributed goblet cells, and a slight infiltration of admixed leucocytes in the thin mucosal fold and the lamina propria. No apparent infiltration of admixed leucocytes was evident in the lamina propria of fish from the ARG1 group compared with those from the CON group sampled at 30 days of feeding. However, dietary administration of 2% arginine seemed to provoke the infiltration
Discussion
Our previous study confirmed a bidirectional connection between skin and intestine, in which skin wounds led to mild enteropathy in the intestine of gilthead seabream [11]. Similarly, in the present study, the experimental skin wounds made on fish led to deeper infiltration of admixed leucocytes into the lamina propria, which led to an increase in the thickness of the lamina propria. The recruitment of leucocytes is a biomarker of the local intestinal inflammation in fish, which plays a
CRediT authorship contribution statement
Zhichu Chen: Methodology, Software, Investigation, Data curation, Writing - review & editing. Diana Ceballos-Francisco: Methodology, Investigation, Data curation, Writing - review & editing. Francisco A. Guardiola: Methodology, Software, Validation, Formal analysis, Data curation, Supervision, Writing - review & editing. Dong Huang: Validation, Formal analysis, Supervision, Writing - review & editing. M. Ángeles Esteban: Conceptualization, Formal analysis, Resources, Visualization, Supervision,
Acknowledgments
Z. C. appreciated the financial support from the China Scholarship Council (CSC) by a State Scholarship Fund (No.201806330100). This work was supported by the MINECO co-funded by the European Regional Development Funds (ERDF, FEDER) (grant no. AGL2017-88370-C3-1-R) and Fundación Seneca de la Región de Murcia (Grupo de Excelencia grant no. 19883/GERM/15).
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