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.

Calcium overload and reactive oxygen species accumulation induced by selenium deficiency promote autophagy in swine small intestine

Selenium(Se)deficiency can seriously affect the small intestine of swine,and cause diarrhea in swine.However,the specific mechanism of Se deficiency-induced swine diarrhea has rarely been reported.Here,to explore the damage of Se deficiency on the calcium homeostasis and autophagy mechanism of swine,in vivo and in vitro models of swine intestinal Se deficiency were established.Twenty-four pure line castrated male Yorkshire pigs(45 d old,12.50±1.32 kg,12 full-sibling pairs)were divided into 2 equal groups and fed Se-deficient diet(0.007 mg Se/kg)as the Se-deficiency group,or fed Se-adequate diet(0.3 mg Se/kg)as the control group for 16 weeks.The intestinal porcine enterocyte cell line(IPEC-J2)was divided into 2 groups,and cultured by Se-deficient medium as the Se-deficient group,or cultured by normal medium as the control group.Morphological observations showed that compared with the control group,intestinal cells in the Se-deficiency group were significantly damaged,and autophagosomes increased.Autophagy staining and cytoplasmic calcium staining results showed that in the Sedeficiency group,autophagy increased and calcium homeostasis was destroyed.According to the reactive oxygen species(ROS)staining results,the percentage of ROS in the Se-deficiency group was higher than that in the control group in the in vitro model.Compared with the control group,the protein and mRNA expressions of autophagy-calcium-related genes including Beclin 1,microtubule-associated proteins 1 A(LC3-1),microtubule-associated proteins 1 B(LC3-2),autophagy-related protein 5(ATG5),autophagy-related protein 12(ATG12),autophagy-related protein 16(ATG16),mammalian target of rapamycin(mTOR),calmodulin-dependent protein kinase kinaseβ(CAMKK-β),adenosine 5’-monophosphate-activated protein kinase(AMPK),sarco(endo)plasmic reticulum Ca2+-ATPase(SERCA),and calpain in the Se-deficiency group were significantly increased which was consistent in vivo and in vitro(P<0.05).Altogether,our results indicated that Se deficiency could destroy the calcium homeostasis of the swine small intestine to trigger cell autophagy and oxidative stress,which was helpful to explain the mechanism of Se deficiency-induced diarrhea in swine.