The Intestinal Toxicity Of OTA Or DON,and The Potential Mechanisms

Ochratoxin A(OTA),a natural occurring mycotoxin produced by fungi of Aspergillus and Penicillium genera,can contaminate feed and food and raw materials.Because of the wide distribution of Aspergillus and Penicillium,animals or humans are at high risk of exposure to OTA through ingestion of contaminated feed and foodstuff.Deoxynivalenol(DON)is a mycotoxin produced by Fusarium graminearum and F.culmorum.DON is commonly detected in cereals and grains.Due to its location and function,the intestinal epithelium may be a potential target for the toxic effect of OTA or DON after intake of OTA or DON contaminated food and feed.The intestinal epithelium allows the absorption of nutrients while providing a selective permeable physical barrier to the permeation of pathogens,toxins.Recently,there is an increasing awareness of the relationship between intestinal barrier dysfuncton and the development of autoimmune diseases,intestinal diseases,and inflammatory diseases.It is thus of importance to study the toxic effects of OTA and DON on the intestinal epithelium.OTA has been found to damage the intestinal epithelium in vivo studies in chicken,dog and rat.However,the research on OTA toxicity in the intestinal epithelium is still relatively lacking,and the mechanisms responsible for the intestinal toxicity are still poorly understood.First,through experiments in vivo and vitro,we study the toxic effect of OTA on intestinal epithelium cells,explore the adaptive protection mechanism of intestinal epithelium cells under OTA explosure,study the effect of OTA on intestinal barrier function,in order to elucidate the intestinal toxicity of OTA and the potential regulation mechanism.The toxicity effect of DON on intestinal epithelial cell lines and the intestinal barrier function have been well studied,and some studies have found that DON possesses indirect pro-inflammatory effect.Therefore,we study the effect of DON on LPS-induced inflammatory reaction of IPEC-J2 cells in vitro;In addition,few studies report the interaction between DON exposure and pathogens in gastrointestinal tract.We,study the effect of DON on S.typhimurium-induced enteritis in mice,to help to provide new idea about the intestinal toxicity of DON and clarify the potential mechanism.Experiment 1.OTA induces intestinal epithelial cells apoptosis and its mechanismFirst,with the purpose to explore the mechanisms associated with the intestinal toxicity of Ochratoxin A(OTA),an intestinal porcine epithelial cell line(IPEC-J2)was applied in this study as in vitro models for intestinal epithelium.MTT analysis results confirmed that OTA induced IPEC-J2 cell toxicity.Hoechst 33258 staining and flow cytometer analysis results showed that OTA could induce apoptosis in IPEC-J2 cells.And then,we observed that OTA induced the mitochondrial reactive oxygen species(ROS)production and mitochondrial permeability increase,Cyt-c release and caspase-3 activaion.Treatment with Mito-TEMPO,the mitochondria-targeted ROS scavenger,blocked OTA-induced mitochondrial ROS generation and apoptosis in IPEC-J2 cells.In vivo,SPF C57BL/6 mouse were fed with OTA(1 mg/kg,2mg/kg)for 2 weeks.OTA treatments did not effect the body weight,the intestine structure of mouse,compared with control groups.2mg/kg OTA significantly reduced antioxidant enzyme activity and increased lipid peroxidation,increased the expression of active caspase-3.Overall,OTA could induce intestinal epithelial cells oxidative stress,lead mitochondrial dysfunction,and finally induce apoptosis.Experiment 2.Autophagy protects intestinal epithelial cells from OTA-inducedapoptosis and potential regulation mechanism The purpose of this study is to explore the adaptive cyto-protection of intestinalepithelium against OTA exposure and relevant regulation mechanisms.In vitro,we observed that OTA induced autophagy in IPEC-J2 cells,as indicated by increase in the levels of LC3-II and decrease in the levles of P62.In vivo,OTA alos induced autophagy in intestine of mouse.Chloroquine(CQ),the autophagy inhibitor,enhanced OTA-induced apoptosis in IPEC-J2 cells.In addition,CQ aggravated the production of mitochondrial reactive oxygen species,the release of cytochrome c release,and the activation of caspase-3.Then,we found that OTA induced extracellular signal-regulated protein kinases 1 and 2(ERK1/2)activation in IPEC-J2 cells.Furthermore,U0126,the ERK1/2 inhibitor,blocked OTA-induced autophagy and aggravated OTA-induced apoptosis.Taken together,these results suggest that ERK1/2-mediated autophagy is required for intestinal epithelial cell survival against OTA toxicity.Experiment 3.OTA induces intestinal epithelial barrier dysfunction and its mechanismIn this study,we used an intestinal porcine epithelial cell line(IPEC-J2)as in vitro model and mice as in vivo model to explore the effect of OTA on intestinal barrier function.In vivo study,a single oral OTA administration(25 mg/kg.bw)in mice induced after 4 h acute intestinal injury,decreased the antioxidant capacity of the intestine.Using oral FITC-dextran gavage,OTA increased the level of FITC-dextran translocation in the serum,indicaring the increased the permeability of intestinal epithelium.Western blot analysis showed that OTA decreased the expression level of tight junction protein ZO-1.Immunofluorescent staining showed that OTA changed the distribution of ZO-1 in intestine tissues.In vitro study,we observed that OTA induced intestinal barrier dysfunction as indicated by the reduction in transepithelial electrical resistance(TEER)and elevation in paracellular permeability to 4 kDa dextran.The barrier dysfunction was accompanied with tight junction disruption including a down-regulation in ZO-1 expression by using western blot analysis and change in distribution of ZO-1 by using immunofluorescent staining.Moreover,OTA exposure increased reactive oxygen species(ROS)generation,activated myosin light chain kinase(MLCK).Furthermore,NAC,a ROS scavenger or inhibition of MLCK with ML-7 markedly prevented OTA-induced barrier dysfunction and tight junction disruption.Taken together,our results indicated that OTA could induce intestinal barrier dysfunction and disrupt tight junction.Experiment 4.DON potentiates LPS-induced inflammatory response and intestinal barrier dysfunction in IPEC-J2 cellsIn this experiment,we used an intestinal porcine epithelial cell line(IPEC-J2)as in vitro model to study the effect of DON on LPS-induced inflammatory response,measure the changes in intestinal barrier function and tight junction,explore the potential regulation mechanism.The results showed that DON enhanced LPS-induced inflammatory response,as indicating by increase in the mRNA levels of TNF-α and IL-6.In addition,DON aggrevated LPS-induced intestinal barrier dysfunction as indicated by the reduction in transepithelial electrical resistance(TEER)and elevation in paracellular permeability to 4 kDa dextran.Meanwhile,DON potentiated LPS-induced down-regulation in ZO-1 expression and change in the distribution of ZO-1.Furhermore,we found that DON enhanced LPS-activated NF-κB.Inhibition of NF-κB with BAY11-7082 could inhibited the effect of DON on LPS-induced inflammatory response,intestinal barrier dysfunction.BAY11-7082 significantly inhibited DON and LPS-induced decrease in ZO-1 expression and change in ZO-1 dsitribution.Overall,DON could potentiate LPS-induced NF-κB activation,and then enhance inflammatory response,intestinal barrier dysfunction and tight junction disruption.Experiment 5.The effect of DON on S.typhimurium-induced enteritis in miceIn SPF C57BL/6 mouse,oral infection with S.typhimurium leads to induce enteritis,and then fed with DON(5 mg/kg)for 1 week.The results showed that DON alone treatment did not effect the body weight.While,S.Typhimurium-infected mice exhibited the the symptoms as including spiritlessness,loss of weight,but without diarrhea and only softened faecal pellets were observed in the colon.DON treatment after oral with S.Typhimurium led mice more spiritlessness and more soft and semisolid feces were observed in the colon.Actually,DON did not significantly effect the colonization of S.typhimurium in the intestine.Histopathological analysis revealed that DON exacerbates the symptoms of intestinal inflammation caused by S.Typhimurium infection;RT-PCR results indicated that DON exposure in the S.Typhimurium-infected mice increased the mRNA levels of pro-inflammatory cytokines,TNF-α and IL-6 compared with other groups,suggesting that DON aggravates S.Typhimurium-induced intestinal inflammation in mice.In addition,DON exposure exacerbates S.typhimurium-induced intestinal barrier dysfunction,exacerbated S.typhimurium-induced down-regulation in ZO-1 expression and change in ZO-1 distribution.Futhermore,DON increased the translocation of Salmonella to liver and spleen,exhibited more marked lesions in liver and spleen compared to mice infected by S.typhimurium alone.Taken together,DON exposure potentiates the intestinal inflammation and intestinal barrier dysfuntion,promotes the dissemination of Salmonella Typhimurium to extraintestinal tissues.