Using Cross-omics To Explore The Mechanism Of Compromised Intestinal Barrier Induced By Aflatxoin M1 And Ochratoxin A

Aflatoxin M1(AFM1)is the only mycotoxin with maximum residue limits in milk products worldwide.Ochratoxin A(OTA)not only in milk,is one of the major sources of contaminated cereals.With the current improvement in living level,“milk + cereal(bread)” has gradually become a common diet.Given that,the potential risk of the co-occurrence of AFM1 and OTA needed to be paid attention.The intestine is the first barrier against the invasion of external contaminations.It is therefore necessary to determine the effect induced by the combined AFM1 and OTA on intestinal barrier.First,the results showed that AFM1(0.12 and 12 ?M)and OTA(0.2 and 20 ?M)individually or collectively decreased the expression levels of tight junction(TJ)proteins and disrupted their structures,and then increased epithelial permeability.The inhibitor experiment comfirmed that p44/42 mitogenactivated protein kinase(MAPK)partially involved in the mycotoxins-induced disruption of intestinal barrier.In addition,AFM1 and OTA yield additive or synergistic effects,which was analyzed by comparing the measured and expected values.Second,transcriptional and proteomic analysis revealed the mechanisms of a synergistic effect induced by AFM1 and OTA on the intestinal epithelial integrity.The results showed that a synergistic effect was behaved in the combination of 12 ?M AFM1 and 9.9 ?M OTA on disrupting intestinal integrity.The integrated transcriptome and proteome analysis demonstrated that this synergistic effect was mechanistically linked to intestinal integrity-related pathways enriched by down-regulated genes and proteins,associated with focal adhesion,adheren junction,and gap junction pathways.And then knockdown of the protein kinase C-?(PKC-?)gene was performed by siRNA assay,suggesting a role of PKC-? in modulating gap junctions with subsequent impact on intestinal integrity.Thirdly,the results showed that a synergistic effect on the intestinal immunotoxicity was performed in the combination of 12 ?M AFM1 and 9.9 ?M OTA.A transcriptome and proteome cross-omics analysis identified several mechanisms underlying this synergy:(i)compared with individual mycotoxins,combined mycotoxins resulted in stronger induction of proteins involved in immunity-related pathways,(ii)the combination targeted different points in the same pathways,and(iii)the combination activated specific inflammation-related pathways.Fourthly,in the present study,we established an ICR mouse model for 35 days of continuous oral administration of individual AFM1,OTA(3.5 mg/kg b.w.)and combined AFM1 + OTA(3.5 mg/kg b.w.+ 3.5 mg/kg b.w.).In mice of mycotoxins treatment,the number of inflammatory cells in the intestine increased,the number of goblet cells decreased,and the localization of TJ protein changed,the indicating the disrupted intestinal physical,chemical and immunologic barrier.In addition,AFM1 and OTA treatment resulted in the increased Ruminococcaceae,Enterobacteriaceae and Clostridium genus,as well as the decreased Lactobacillus and Alloprevotella,suggesting the damaged intestinal microbial barrier.In conclusion,in this study,we focused on the hazard assessment of the combined AFM1 and OTA,and regarded the intestinal barrier as the research objective,as well as constructed an experimental model combining in vitro and in vivo.It was found that the combination of AFM1 and OTA increased the intestinal toxicity.Cross-omics was used to analyze the underlying molecular mechanisms,and the related pathways and target proteins related to toxic effects were screened.The results of these studies played an important scientific and practical significance in establishing a risk assessment method for the interaction of multiple mycotoxins,and shed light on the importance of systematically analyzing the harm of multimycotoxins to human health.