| Aflatoxin(AF)is one of the most common and more virulent of mycotoxins.Among the various types of Aflatoxin,aflatoxin B1(AFB1)is more commonly encountered and it is also considered to have most toxicity than others,which mainly contaminate cereals and oils.Aflatoxin M1(aflatoxin M1)is a derivative of AFB1,which mainly contaminate milk and dairy products.As the first important defense line of the body's immune system,the intestinal tract is an important mucosal barrier for the body to fight against pathogens such as bacteria and endotoxin,as well as other antigens such as nutritional antigen and toxin from the blood circulation and tissues.This barrier function depends on the complete intestinal epithelial and mucous layer structure,as well as the stable intestinal microflora.As early exposure cell of mycotoxins,intestinal cells often face a higher exposure dose than other tissue cells,intestinal cell are also targeted cell because of their high efficiency of protein synthesis and metabolic turnover.Therefore,in recent studies,intestinal structure and function have gradually become a new target for the study of toxicity effects of mycotoxins.With the improvement of people's living standard,"milk + cereal" has gradually become a common family meal pattern.Under this mode,the human body will face the risk of taking AFB1 and AFM1 together.But there are fewer studies about the intestinal toxicity of the AFB1 and AFM1,and the molecular mechanisms of the interaction of AFM1 and AFB1 has not been answered.Therefore,in order to reveal the effects of individual and combined intestinal toxicity effects of AFB1 and AFM1 in the animal model,to find molecular mechanisms of the toxicity effects,to screen for biological target,we carried out the experiment.The results of the study will have significant scientific implications for the health hazards of the toxicity assessment of aflatoxins and precaution of new food safety issue.The ICR mice were randomized into four groups of 10 each and received an oral administration of 0.3 mg/kg b.w.AFB1,3.0 mg/kg b.w.AFM1,mixture of AFB1 and AFM1(0.3 mg/kg b.w.AFB1,3.0 mg/kg b.w.AFM1)and vehicle for 28 days(once a day),and the status of the mice and body weight was recorded during the period.Mice were executed after gavage,and blood,intestinal tissue and intestinal contents were collected.Subsequently,the organ index was measured,and the intestinal injury indexes in blood(the activity of diamine oxidase,the content of D-lactic acid,intestinal fatty acid binding protein and citrulline in serum)were determined.And the morphology of intestinal villi was observed,the apoptosis rate of intestinal epithelial cells was measured,and the number of intestinal goblet cells was determined.The changes of bacterial community structure of intestinal contents and the changes of intestinal tissue proteome were analyzed.Finally,combined with the results of our experiment and the literature data,we discuss the intestine damage mechanism of individual or combined effects of AFB1 and AFM1 in mice.discovered the change of the intestinal bacterial community structure,analyzed the molecular mechanisms of individual or combined toxicity effects of AFB1 and AFM1.The main results are as follows:1.Individual or combined of AFB1 and AFM1 did not significantly affect the body weight gain andorgan index in mice.The content of citrulline in the blood of AFB1+AFM1 group was significantly lower than that in the control group and the individual group(p < 0.05).The groups of individual AFB1 or combined with AFM1 had a significantly higher blood diamine oxidase activity in mice in comparison to that in AFM1 group and control group(p < 0.05).The groups of individual AFM1 or combined with AFB1 had a significantly higher content of intestinal fatty acid binding protein in comparison to that in control group(p < 0.05),and combined of AFB1 and AFM1 group was higher than AFM1 group(p < 0.05).The groups of individual or combined of AFB1 and AFM1 had a significantly higher content of D-lactic acid in comparison to that in control group(p < 0.05),and combined of AFB1 and AFM1 group was higher than individual group(p < 0.05).The length of the villus along the whole small intestine segment was reduced in AFB1 and combined of AFB1 and AFM1group(p < 0.05),for AFM1 group,it was just happened in middle and posterior segment of the small intestine.The crypt depth of the villus of middle and posterior segment of the small intestine in AFM1 and combined of AFB1 and AFM1 group.The length and crypt depth ratio of the villus along the whole small intestine segment in individual or combined of AFB1 and AFM1 group were reduced significantly(p < 0.05),and the combined of AFB1 and AFM1 has more obvious effects in posterior segment.The combined of AFB1 and AFM1 could induce apoptosis in the posterior segment of the small intestine,and apoptosis rate in combined group was higher than individual groups.The single and combined treatment of AFB1 and AFM1 did not affect the number of ileal goblet cells,but the combined of AFB1 and AFM1 significantly reduced the number of colonic goblet cells.The Occludin1 protein in the intestinal epithelial cells of AFB1+AFM1 group was dislocated by clustering and internalization,and the distribution of the apical region of the cell top membrane and the lateral membrane of the cell was decreased,while the distribution of cytoplasm was increased.In addition,ZO-1 protein translocated from the apical region to the basal side in the cytoplasm.2.Compared with the control group,there was no significant change in the dominant bacteria microflora in the three treatment groups,regardless of the phylum level,family level or genus level.However,different toxin treatment still resulted in significant changes in the abundance of different bacteria microflora.Among them,pathogenic bacteria or opportunistic pathogens abundance in the AFB1 treatment and its combined treatment group with AFM1,such as Facklamia,Staphylococcus,Corynebacterium,and Trichococcus were significantly increased(p < 0.05).3.(1)Compared with the control group,the abundance of proteins related to intestinal cell vitality,protein transport,cell shape and skeletal control,fungal response and drug metabolism expressed differently in individual AFB1 and AFM1 and their combined treatment group,and combined treatment group had more quantity differentially expressed proteins than individual treatment group.(2)Cell apoptosis signaling pathway,cell carcinogenic correlation signaling pathway in individual AFB1 and AFM1 and their combined treatment group was activated,and the combined treatment group was more significant.(3)It was found that expression amount of the proteins related to toxin metabolism and detoxication,such as UDP-GT1?UDP-GT12?Gstm6 were significantly reduced(p < 0.05),and the expression amount of the protein related to tight junction structure,Claudin7 and IQGAP2 weresignificantly reduced(p < 0.05),and the expression amount of the cell apoptosis-related protein like MAP4K3 was significantly increased(p < 0.05).The results of this experiment indicated that AFB1 and AFM1 combined to damage intestinal cells and improve the permeability of intestinal epithelium.Individual and combined effect of AFB1 with AFM1 resulted in atrophy of villi and the deepening of the crypt,which affect the function of intestinal tract,and the combined effect is stronger than the individual effect.AFB1 combined with AFM1 induced apoptosis of small intestinal epithelial cells in mice,and reduced the number of large intestine goblet cells and damaged the integrity of the intestinal epithelial structure.And it was found that that AFB1 combine with AFM1 to induce the migration of tight junction protein in the epithelial cells,and cause damage to the epithelial barrier function.The combination of AFB1 and AFM1 enhanced the damage to the intestinal structure,cell vitality and barrier function by individual AFB1 and AFM1.AFB1 alone and combined with AFM1 can lead to the increased abundance of pathogenic bacteria or conditioned pathogen in the intestinal tract of mice,disturb the microflora and the function of intestinal biological barrier.The main molecular mechanisms of the intestinal toxicity of individual and combined AFB1 and AFM1 included: influence the abundance of proteins related to intestinal cell vitality,protein transport,cell shape and skeletal control,fungal response and drug metabolism,and the combined effect is more significant;activate the intestinal cell apoptosis signaling pathways,cellular canceration related signaling pathway,and the combined effect is more significant;The main target proteins account for the enhanced intestinal toxicity in mice of combined AFB1 and AFM1 was UDP-GT1,UDP-GT2,Gstm6,MAP4K3,Claudin7 and IQGAP2.The combined of AFB1 and AFM1 down-regulate the expression of UDP-GT1,UDP-GT2 and Gstm6 to inhibit the metabolism and detoxification of toxins,up-regulate the expression of MAP4K3,to promote apoptosis;down-regulate the expression of Claudin7 and IQGAP2 to impair the function of intestinal cell barrier.The results of the study will have significant scientific implications for the health hazards of the toxicity assessment of aflatoxins and precaution of new food safety issue. |
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