The Mechanism Of Compromised Intestinal Physical Barrier Induced By Ochratoxin A Using Omics Approaches

Ochratoxin A（OTA）is an important mycotoxin in food and animal feed.Its harm to human beings and animals is a public health problem of global concern,which has attracted people’s attention.Previous studies have shown that OTA has nephrotoxicity,hepatotoxicity,genotoxicity,immunotoxicity,cytotoxicity and neurotoxicity.In recent years,more and more researches have pointed out the intestinal toxicity of OTA,but its mechanism has not been clarified,which makes the research on the mechanism of intestinal toxicity become more and more important.Intestinal barrier is the first barrier against foreign pollutants,which undertakes 70%of the immune defense function of the body.Intestinal epithelial cells and tight junction（TJ）play an important role in maintaining intestinal physical barrier function.The mechanism of intestinal physical barrier in vitro model Caco-2 cells and Balb/c mice in vivo was revealed by using transcriptomics and whole transcriptomics.1.After treated with different concentrations of OTA（0.0005,0.005 and 4μg/m L）for 48 h,differentiated Caco-2 cells showed a dose-dependent apoptosis.Transcriptome analysis showed that a total of 10090 differential expression genes（DEGs）were clustered in two down-regulation patterns.GO and KEGG enrichment analysis showed that these DEGs were mainly enriched in p53 signaling pathway,cell cycle and MAPK signaling pathway.OTA induced apoptosis of differentiated Caco-2 cells by down regulating MDM2 and up regulating CASP3 protein,activating p53 mediated apoptosis signal pathway.2.After gavage OTA（3 mg/kg b.w.）to Balb/c mice for 28 days,the body weight of Balb/c mice significantly decreased from the 7^th day after intragastric administration,and the levels of D-lactate and intestinal fatty acid-binding protein related to intestinal permeability and intestinal injury increased significantly.We found that the villi height and the number of goblet cells in OTA group were significantly lower than those in CTL group after staining the villi and goblet cells of jejunum.According to the whole transcriptome analysis,a total of 619 differentially expressed（DE）m RNA,DEmi RNA71,DElnc RNA493and 144DEcirc RNA were identified.Bioinformatics analysis showed that OTA changed the expression of TJ protein related m RNAs（FZD4,Wnt5a,Axin2,Cav1,ACTG1 and CCND1）,thus activating Wnt/Ca²⁺signaling pathway,which may be regulated by some lnc RNAs（Zeb1,Stk36,Phkb,Prss23,Inpp5e and Gm28588）and mi RNAs（mi R-1258-x,mmu-mi R-1258-3p）.Through the analysis of competitive endogenous RNA（ce RNA）,the key RNAs involved in the regulation of TJ was identified.Lnc RNA Zeb1 targeted binding with mi R-1258-x:1)regulated the binding of FZD4 and Wnt5a,2)regulated the expression of Cav1,control the influx of extracellular Ca²⁺,which reducing TJ protein expression and damaging intestinal physical barrier function.In vitro Caco-2 cell test confirmed that the increase of Ca²⁺induced by OTA did reduce the expression of TJ protein.To sum up,for the purpose of studying the intestinal physical barrier compromised of OTA,the experimental models of Caco-2 cells in vitro and Balb/c mice in vivo were constructed.The results showed that OTA damaged the intestinal physical barrier function.By means of transcriptome and whole transcriptome techniques,the potential mechanism of compromised intestinal physical barrier induced by OTA was successfully explored.These results provide strong in vivo and in vitro toxicity data for the intestinal toxicological mechanism of OTA,which will help us to find the target of compromised intestinal physical barrier induced by OTA and provide a basis for the prevention of intestinal toxicity of OTA.