Nontoxic Concentration Of OTA Aggravates Don-induced Barrier Dysfunction In Intestinal Epithelial Cell And Its Mechanism

Ochratoxin A(OTA)is a fungal secondary metabolite produced by Aspergillus and Penicilliums species,which target organ is considered to be kidney.However,inhibition of cellular proliferation,altering intestinal absorption functions and barrier function have been reported in vitro as toxic effects of OTA.In chicken,rat and pig,OTA has also been found to be associated with an inflammatory reaction and necrosis of the intestinal mucosa.Deoxynivalenol(DON)is also known as vomitoxin,which is one of the most prevalent mycotoxins produced by Fusarium species and has been considered to have the most significant enterotoxicity.Studies have shown that exposure to DON is responsible for vomiting,diarrhea and malabsorption of nutrients.At the cellular levels,studies on different intestinal epithelial cells from different sources have shown that DON can inhibit cell viability and immune function,and destroy intestinal barrier function.In addition,co-contamination of different mycotoxins has been frequently demonstrated,being a more common event than single contamination.The interaction between coexisting mycotoxins and their relationships with intestinal tissue make up a complex system when animal intake of diet contaminated by mycotoxin.As two mycotoxins with strong enterotoxicity,the co-occurrence of OTA and DON has been reported many times.However,most current toxicity studies commonly evaluate the effects of individual mycotoxins on the target cell biological functions.Studies on effects of co-contaminating DON and OTA are rarely reported.This study is designed to evaluate the effects of combined exposure of OTA and DON on intestinal barrier function,particularly the effects of nontoxic concentration of OTA on DON-induced enterotoxicity,and involved mechanism by using intestinal epithelial cells derived from the jejunum of pigs(IPEC-J2 cells),which is helpful for understanding the effects of nontoxic concentrations of toxin on coexisting toxin-induced toxicity,and provides new ideas for preventing and controlling the negative effects of combined mycotoxins on animal gut.Experiment Ⅰ Effects of different time and concentration of DON or OTA on intestinal barrier function,membrane structure and cell viabilityIn this study,different concentrations of DON or OTA incubated on IPEC-J2 cells for 6 h,12 h and 24 h.Firstly,by detecting transepithelial electrical resistance(TEER)and paracellular permeability,our study confirmed that DON or OTA can induce barrier dysfunction in IPEC-J2 cells,which was correlated with time and concentration.Among them,the most significant dose-effect relationship was 24 h.After incubated with 24h,4μM DON or 8μM OTA significantly decreased TEER values(P<0.05)and increased paracellular permeability(P<0.05).Moreover,in order to eliminate the interference of cell death and structural damage,which may lead to the destruction of barrier function,the effects of different concentrations of DON or OTA on membrane structure and viability of IPEC-J2 cells were further investigated by lactate dehydrogenase activity(LDH),MTT and neutral red(NR)uptake assay.The results showed that 0～8 μM DON or 0～32 μM OTA had no toxic effects on the membrane structure and cell viability of IPEC-J2 cells after 24 h incubation.Based on the results of this study,we selected the nontoxic concentrations of DON(2 μM)and OTA(4 μM)and the lowest concentrations of DON(4 μM)or OTA(8 μM),to use in subsequent experiments.Experiment Ⅱ Effects of nontoxic concentration of OTA on DON-induced intestinal barrier dysfunction and inflammatory factorsBased on the optimal incubated time(24 h)and appropriate concentrations of OTA and DON selected in experiment Ⅰ this experiment evaluated the combined effects of OTA and DON on the barrier function of IPEC-J2 cells.The results indicated that 4 μM(nontoxic concentration)OTA significantly aggravated the barrier dysfunction of IPEC-J2 cells induced by 4 μM(entero-toxic concentration)DON,as demonstrated by decreased TEER values(P<0.01)and increased paracellular permeability(P<0.01).At the same time,the effects of nontoxic concentration of OTA and entero-toxic concentration of DON on the expression and distribution of tight junction(TJ)proteins Claudin-3 and Claudin-4 were further evaluated by real-time PCR,Western Blot and Immunofluorescence assay.The results showed that the combined treatment of OTA and DON significantly decreased Claudin-3(P<0.01)and Claudin-4 protein levels(P<0.01),but their mRNA expression levels were increased.The results of immunofluorescence showed that combined exposure of OTA and DON disrupted the structure of Claudin-3 and Claudin-4 but had no effect on their cytological localization.In addition,the combined incubation of OTA and DON up-regulated the mRNA expression levels of tumor necrosis factor-α(TNF-α)and interleukin-8(IL-8)by real-time PCR(P<0.01).Experiment Ⅲ Mechanism study of nontoxic concentration of OTA promoting DON-induced intestinal barrier dysfunctionPrevious results of experiment have shown that nontoxic concentration of OTA could aggravate DON-induced harrier dysfunction in IPEC-J2 cells,but which mechanism needs to be further studied.The expression levels of P65 and IKB-α in cytoplasm or nucleus,NFκB signaling pathway-associated proteins,were detected by Western Blot,and the distribution of P65 protein inside or outside the nucleus were analyzed by Immunofluorescence assay.The results showed that the combined OTA and DON significantly decreased the expression of P65 and IKB-α in cytoplasm(P<0.01),while the expression of nuclear P65 increased significantly(P<0.01).In the control group,P65 protein was mainly distributed outside the nucleus,while the P65 protein in the combined treatment group was mainly distributed in the nucleus.In addition,by adding ammonium pyrrolidinedithiocarbamate(PDTC),NFκB specific inhibitor,we investigated the role of this pathway in OTA aggravating DON-induced barrier dysfunction.The results showed that PDTC could inhibit the activation of NFκB signaling pathway induced by OTA and DON,confirmed by the decrease of P65 and IKB-α protein expression in cytoplasm(P<0.05)and the increase of P65 protein expression in nucleus(P<0.01).Moreover,PDTC could reverse the decrease of TJ protein Claudin-3 and Claudin-4 expression(P<0.01),and hinder intestinal barrier dysfunction induced by OTA and DON,which is characterized by elevated TEER(P<0.01)and decreased pericellular permeability in IPEC-J2 cells(P<0.01).At the same time,the results of real-time PCR showed that PDTC could inhibit the increased mRNA expression levels of pro-inflammatory factors TNF-α and IL-8.Thus,NFκB signaling pathway plays an important role in nontoxic concentration of OTA promoting DON-induced intestinal barrier dysfunction and its inflammatory effects.In other word,OTA can aggravate DON-induced intestinal barrier damage and inflammatory response by activating NFκB signaling pathway.