Effects Of Aflatoxin B₁ Exposure On Ovalbumin Sensitization

Although our living environment and the productivity level are well,the cases of food biosafety occurred sometimes in the world,a causing huge economic losses and great health risks every year.Mycotoxins as a kind of biotoxin will be harmful to human health if human eat some contaminated plant foods（cereals）or animal foods（meat,milk and eggs）.At the same time,the incidence of food allergy is also increasing year by year,which can affect the health and quality of life of patients.Eggs,milk,peanuts and other foods contain major food allergens and are also easily contaminated by mycotoxins.The two food safety risk factors have immunotoxicity and overlap in these foods.Therefore,It is very essential to study the relationship between food mycotoxin contamination and food allergy occurrence,and which can contribute to more scientifically assessment of the risk factors of food allergy.In this study,Ovalbumin（OVA）from egg white was as allergen,the effects of Aflatoxin B₁（AFB₁）exposure on OVA food allergy were investigated by animal model and cell in vivo and vitro respectively.BALB/c mice were used as the allergic model animal and KU812 cell was as model cell.The effects of AFB₁ exposure on OVA-sensitized mice were evaluated by their some immunological indices,including serum specific antibody,mast cell protease,splenocyte secretion cytokine and food allergy related gene expression in jejunum,ect.The effects of AFB₁ exposure on the intestinal mucosal immunity and intestinal flora of OVA-sensitized mice were evaluated through observation the damages of intestinal sections by hematoxylin-eosin（HE）staining method,and intestinal contents flora by the OTUs analysis.Lastly,the effects of AFB₁ exposure on KU812 cell degranulation induced by OVA was evaluated to further elaborated its effect of AFB₁ exposure on food allergy at the cellular level.The main research methods,contents and results are as follows.1.Enzyme-linked immunosorbent assay（ELISA）was for measuring the levels of specific immunoglobulin E（Ig E）,Ig G,Ig G1 and Ig G2a and mast cell protease-1（m MCP-1）in the serum of OVA-sensitized mice.Commercial ELISA kits was used for determination of interleukin 4（IL-4）,IL-5,IL-13 and interferon-gamma（IFN-γ）in the spleen supernatant of OVA-sensitized mice.Real-time fluorescence quantitative PCR（Q-PCR）was used to detect the expression of relevant genes.Flow cytometry was used to detect the number of Th1/Th2/Treg cells.The results showed that the serum levels of specific Ig E,m MCP-1 and cytokines（IL-4,IL-5 and IL-13）produced by Th2 cells in OVA-sensitized mice were significantly increased,indicating that the OVA-sensitization model was successfully constructed.In mice exposed to low dose of AFB₁,the levels of OVA-specific Ig E,IL-4 and IL-13 were significantly increased by 68%,41%and 75%,respectively;in mice exposed to medium dose of AFB₁,the levels of m MCP-1 and IL-13 were significantly increased;in mice exposed to high dose of AFB₁,the levels of m MCP-1 and IL-5 were significant increase,with 107%and 22%increases,respectively;the above-mentioned allergic indexes were not significantly changed in mice exposed to continuous high doses of AFB₁.In all four groups of AFB₁-exposed sensitized mice,the levels of Th1-related cytokines（IFN-γ）were decreased,but not statistically significant.These results were also further validated by Q-PCR and flow cytometric analysis.2.Pathological analysis of the jejunum of OVA-sensitized and AFB₁-exposed sensitized BALB/c mice jejunal tissues was performed by HE staining,while the structure,diversity and differential species of the intestinal flora in mice were analyzed the Illumina Mi Seq high-throughput sequencing platform when the amplification operation was finished,which the bacterial 16S r RNA V3-V4 region in the cecum contentsin mice was amplificated by PCR.The results showed that OVA sensitization caused damage to the intestinal barrier,villi breakage and a significant widening of intestinal gland spacing in mice;AFB₁ exposure could aggravate the damages of the intestinal barrier in OVA-sensitized mice.The results of OTUs clustering analysis（species accumulation curve,RANK analysis,petal plot）showed that the number of OTUs in each groups ranged from 514～521,their OTUs number common rate was 94.6%,and the effect of AFB₁ on the number and uniformity of OTUs of mouse intestinal flora were small.Alpha diversity analysis showed that AFB₁ exposure did not have a significant effect on the diversity and abundance of the intestinal flora in mice.Beta diversity analysis showed that AFB₁ exposure caused significant changes in the structure of intestinal microflora in OVA-sensitized mice,the boxplot of Beta diversity distance showed that there was significant difference in Beta diversity distance between intestinal flora of mice in OVA group and medium/high dose AFB₁ exposure group.At the phylum level,the relative abundance of Firmicutes in gut of OVA sensitized mice was significantly decreased,while the relative abundance of Bacteroidetes and Proteobacteria was significantly increased.AFB₁ decreased the relative abundance of Firmicutes and increased the relative abundance of Bacteroidetes and Proteobacteria in the intestinal tract of OVA-sensitized mice,and the effect was most significant at high dose of AFB₁.3.The effects of AFB₁ on the release of bioactive mediatorsβ-amino-hexossidase（β-HEX）and histamine,as well as cytokines（IL-4,IL-6 and IL-1β）were detected by ELISA.Then,Q-PCR was used to analyze the effects on m RNA expression levels of IL-4 and IL-6.The results showed that AFB₁ had little effect on the release ofβ-HEX and histamine in KU812 cells degranulated by OVA,but it could promote the secretion of cytokines IL-4,IL-6 and IL-1βby 59%,30%and 84%,respectively.Meanwhile,m RNA expression levels of IL-4 and IL-6 were also increased by 60%and 23%,respectively.AFB₁ exposure of 10000 ng/m L will aggravate OVA to stimulate the degranulation of KU812 cells to release more active mediators and cytokines related to allergy and enhance allergic reactionIn summary,the results from the overall animal model,intestinal flora analysis and in vitro cellular experiments demonstrate that AFB₁ exposure can aggravate OVA sensitization.