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

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 prevent DON-induced intestinal cell injury and enhance barrier function, which is associated with inhibition of the necroptosis signaling pathway.