EPA and DHA attenuate deoxynivalenol‐induced intestinal porcine epithelial cell injury and protect barrier function integrity by inhibiting necroptosis signaling pathway

Abstract

Abbreviations: DON, deoxynivalenol; Drp1, dynamin-related protein 1; FD4, fluorescein isothiocyanate (FITC)-labeled dextran; HMGB1, high mobility group box-1 protein; IPEC-1, intestinal porcine epithelial cell line 1; LDH, lactate dehydrogenase; MLKL, mixed lineage kinase-like protein; PGAM5, phosphoglycerate mutase family 5; RIP1, receptor interacting protein kinase 1; RIPK3, receptor interacting protein kinase 3; TEER, transepithelial electrical resistance; TNFR1, tumor necrosis factor receptor 1. Abstract Deoxynivalenol (DON) is one of the most common mycotoxins that contaminates food or feed and cause intestinal damage. Long-chain n-3 polyunsaturated fatty acids (PUFA) such as EPA and DHA exert beneficial effects on intestinal integrity in animal models and clinical trials. Necroptosis signaling pathway plays a critical role in intestinal cell injury. This study tested the hypothesis that EPA and DHA could alleviate DON-induced injury to intestinal porcine epithelial cells through modulation of the necroptosis signaling pathway. Intestinal porcine epithelial cell 1 (IPEC-1) cells were cultured with or without EPA or DHA (6.25-25 μg/mL) in the presence or absence of 0.5 μg/mL DON for indicated time points. Cell viability, cell number, lactate dehydrogenase (LDH) activity, cell necrosis, transepithelial electrical resistance (TEER), fluorescein isothiocyanate-labeled dextran 4kDa (FD4) flux, tight junction protein distribution, and protein abundance of necroptosis related signals were determined. EPA and DHA promoted cell growth indicated by higher cell viability and cell number, and inhibited cell injury indicated by lower LDH activity in the media. EPA and DHA also improved intestinal barrier function, indicated by higher TEER and lower permeability of FD4 flux as well as increased proportions of tight junction proteins located in the plasma membrane. Moreover, EPA and DHA decreased cell necrosis demonstrated by live cell imaging and transmission electron microscopy. Finally, EPA and DHA downregulated protein expressions of necroptosis related signals including tumor necrosis factor receptor (TNFR1), receptor interacting protein kinase 1 (RIP1), RIP3, phosphorylated mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase family 5 (PGAM5), dynamin-related protein 1 (Drp1), and high mobility group box-1 protein (HMGB1). EPA and DHA also inhibited protein expression of caspase-3 and caspase-8. These results suggest that EPA and DHA 2484 | XIAO et Al.

Omega-3 Polyunsaturated Fatty Acids Counteract Inflammatory and Oxidative Damage of Non-Transformed Porcine Enterocytes

“…In particular, intestinal cell models are widely utilized for the validation of bioactive compounds in food and feed analysis [ 15 , 26 , 27 , 28 ]. Earlier, the anti-inflammatory and anti-necroptosis activity of ω3-PUFAs were demonstrated in normal and cancer enterocyte models [ 12 , 13 , 17 ]. However, cancer cells face certain limitations as they exhibit aberrant cell surface sugars, glycoproteins, glycosylation and metabolic pathways [ 14 , 15 , 16 ].…”

“…However, cancer cells are not a realistic representative of normal physiology [ 14 , 15 , 16 ]. Recently, ω-3 PUFAs were shown to reverse mycotoxin damage of non-transformed IPEC-1 cells [ 17 ]. However, there has been limited research investigating the ω-3 PUFA effects on IEL injury in various stress conditions.…”

Supplementation of Diet With Different n-3/n-6 PUFA Ratios Ameliorates Autistic Behavior, Reduces Serotonin, and Improves Intestinal Barrier Impairments in a Valproic Acid Rat Model of Autism

“…Dietary supplementation of n-3 PUFAs can improve the intestinal barrier function in rats. It has been reported that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can prevent deoxynivalenol-induced intestinal cell injury and reinforce barrier function, which is associated with the inhibition of necroptosis signaling pathway (14). Furthermore, Zhu et al found that marine oil supplementation was beneficial for the improvement of intestinal barrier function and inhibition of CRH/CRHR1 signaling pathway and mast cell density (15).…”

Dietary Carbohydrates and Lipids in the Pathogenesis of Leaky Gut Syndrome: An Overview

“…Furthermore, Willemsen et al [92] showed that AA, EPA, DHA and (to a lower extent) GLA enhanced basal TEER and strongly reduced IL-4-mediated permeability suggesting that those PUFAs support barrier integrity. Moreover, recent studies showed that EPA and DHA improve intestinal barrier function as indicated by higher TEER and lower FITC-dextran flux as well as increased proportions of TJ proteins located in the plasma membrane [93] (Table 2). In fact, the addition of lipase, which was used to simulate in vivo digestion and release fatty acids from the glycerol backbone, increased basal TEER in T84 cells in a dose-dependent manner.…”

“…It was showed that PKC antagonists facilitate the changes mediated by GLA and DHA suggesting that PKC also may be engaged in the mechanism of leaky gut [91]. [91] Willemsen et al [92] Xiao et al [93] Onuki et al [96] Abbreviations: AA-arachidonic acid, ALA-α-linolenic acid, DHA-decosahexaenoic acid, EPA-eicosapentaenoic acid, 51 Cr-EDTA- 51 chromium-labeled ethylenediamine tetraacetic acid, GLA-γ-linolenic acid, LA-linolenic acid, NS-not significant/no change, OA-oleic acid, ↑-increased, ↓-decreased.…”