Phenethyl isothiocyanate as an anti-nutritional factor attenuates deoxynivalenol-induced IPEC-J2 cell injury through inhibiting ROSmediated autophagy

Deoxynivalenol(DON)is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans.Phenethyl isothiocyanate(PEITC)in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal.In the current study,we aimed to explore the effects of PEITC on DON-induced apoptosis,intestinal tight junction disorder,and its potential molecular mechanism in the porcine jejunum epithelial cell line(IPEC-J2).Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity,decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels,and increasing zonula occludens-1(ZO-1),occludin and claudin-1 mRNA/protein expression.Meanwhile,PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase(iNOS)and cyclooxygenase 2(COX-2)mRNA levels and intracellular reactive oxygen species(ROS)level,and a decrease of glutathione peroxidase 1(GPx1),superoxide dismutase 2(SOD2),catalase(CAT)and heme oxygenase 1(HO-1)mRNA levels.Additionally,PEITC treatment significantly down-regulated autophagy-related protein 5(ATG5),beclin-1 and microtubuleassociated protein 1 light chain 3B(LC3-II)mRNA/protein levels,decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3(GFP-LC3)puncta and phosphatidylinositol 3 kinase(PI3K)protein expression,and up-regulated phospho-protein kinase B(p-Akt)and phospho-mammalian target of rapamycin(p-mTOR)protein expression against DON.However,the activation of autophagy by rapamycin,an autophagy agonist,abolished the protective effects of PEITC against DON-induced cytotoxicity,apoptosis and intestinal tight junction disorder.Collectively,PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROSmediated autophagy.

4-Phenylbutyric acid accelerates rehabilitation of barrier function in IPEC-J2 cell monolayer model

As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier,the intestinal epithelial barrier plays a determinant role in pigs’health and growth.4-Phenylbutyric acid(4-PBA),an aromatic fatty acid,was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis.However,whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown.This study aimed to explore the effects of 4-PBA on the intestinal barrier function,using in vitro models of well-differentiated intestinal porcine epithelial cell(IPEC-J2)monolayers in the transwell plates.Cell monolayers with or without 4-PBA(1.0 mmol/L)treatment were challenged with physical scratch,deoxynivalenol(DON,2.0μg/mL,48 h),and lipopolysaccharide(LPS,5.0μg/mL,48 h),respectively.Transepithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran(FD-4)permeability were measured to indicate barrier integrity and permeability.Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins.As expected,physical scratch,DON,and LPS challenges decreased TEER and increased FD-4 permeability.4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage.However,once 48-h DON and LPS challenges were removed,rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment.Also,the relative gene and protein expressions of zonula occludens-1(ZO-1),occludin,and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation.Taken together,4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch,DON-,and LPS-induced damage,via enhancing cell mitigation and expressions of tight junction proteins.