Effects And Mechanism Of Low-Dose DON On The Mice Colitis Model And RAW264.7 Cells Inflammatory Model

Vomitoxin exists widely in cereals,and its toxicology is widely studied,causing acute or chronic poisoning.The toxicity of vomitoxin is mainly immunotoxicity and enterotoxicity.Inflammation is a clinical manifestation of the immune response.Chronic colitis is an autoinflammatory disease with congenital and adaptive immune dysregulation,characterized by an abnormally elevated inflammatory response of the gut.There is no study on the effect of vomitoxin on the body under the inflammatory state at present.Although strict limits have been set for vomitoxin in cereals,low levels of vomitoxin contamination are still widespread in cereals.A new study shows that low-dose DON can cause mild intestinal inflammation in mice.Therefore,we constructed inflammatory models,then the effect of low-dose DON on DSS-induced chronic colitis in mice was investigated in vivo,and its mechanism was preliminarily discussed.In order to deepen the study of the mechanism,the inflammatory cell model was constructed in vitro by knockdown the expression of IL-10 gene,so as to explore the effect of low-dose DON on it and its mechanism.The purpose of this study was to explore the effects of low level of vomitoxin pollution on the body under inflammatory state.The specific content includes the following two parts:Experiment 1: Effect of low-dose vomitoxin on DSS-induced murine colitis modelDifferent concentrations of DSS added to the mice’s drinking water can induce acute or chronic colitis models.In this study,48 6-week-old C57BL/6J female mice were randomly divided into the Control group,DSS group,DSS+DON(25 μg/kg bw/day)group,and DSS+DON(50 μg/kg bw/day)group.All mice except the Control group were periodically libitum to drink 3% DSS solution.The experiment lasted 28 d,and at 18 d the DON group was fed different concentrations of DON to study the effects of low-doses DON on murine chronic colitis models.After the experiment,the immune organs index,levels of immunoglobulin,histological changes in colon were detected,and DAI scoring was performed on mice.At the same time,the protein secretion levels of IgG,IgA and inflammatory factors in the colon of mice were determined by ELISA.Fluorescent quantitative PCR was used to detect m RNA expression levels of inflammatory factors(IL-1β,IL-6,TNF-α,IL-10 m RNA),inflammatory mediators(iNOS,COX-2,NO),intestinal barrier-related proteins(Occludin,MUC2),and JAK2/STAT3/SOCS3 signal pathway related proteins.The experimental results showed that,compared with the Control group,DSS could cause colon injury in mice,activate immunity,promote inflammatory response and oxidative stress,and cause diarrhea,blood in stool and weight loss in mice.Compared with mice in DSS group,low-dose DON can aggravate colon injury,further increase spleen index,serum immunoglobulin level,expression of inflammatory factors and inflammatory mediators,and further increase DAI score.These results suggest that low-dose DON can aggravate colon injury,immune activation,inflammatory response and clinical symptoms in DSS colitis mice.To study the mechanism of low-dose DON on murine colitis model,we found that the expression of genes related to the JAK2/STAT3/SOCS3 signaling pathway was increased in the colon of mice in DSS groups,and compared with mice in the DSS group,Gene expression of JAK2/STAT3/SOCS3 signaling pathway was significantly increased in DSS+DON group(50 μg/kg bw /day).The above results indicate that the effects of low-dose of DON on colitis mice may be related to JAK2/STAT3/SOCS3 signaling pathway.Experiment 2: The influences of low-dose vomitoxin on IL-10-silencing RAW264.7Macrophages in the gut lamina propria is the most,function as devouring antigens and secreting inflammatory factors,which are often used in immune-related clinical studies.This experiment constructed an in vitro inflammatory model by interfering IL-10 gene of RAW264.7 cell to explore the effects of different concentrations of Vomitoxin on IL-10-silencing RAW264.7 cells.First,the effects of DON on the cell activity and cell damage of RAW264.7 cells were detected by MTT assay and LDH assay,and DON concentrations with no obvious toxicity to cells were screened out after 24 h treatment.The concentration of 6.25,12.5 and 25 ng/mL DON was finally determined for subsequent experiments.Subsequently,IL-10-silencing RAW264.7 cells were treated with the above concentration of DON,and the inflammatory responses,cell migration and phagocytosis were detected.The results showed that IL-10 silencing induced the inflammatory response of RAW264.7 cells and enhanced the migration and phagocytosis ability compared with the control group.Compared with IL-10-silencing RAW264.7 cells,25 ng/mL DON further enhanced the inflammatory response of cells and enhanced the ability of cell migration and phagocytosis.These results suggested that low-dose DON further enhanced the inflammatory response of IL-10-silencing RAW264.7 cells.To investigate the mechanism by which low-dose DON promotes IL-10-silencing RAW264.7 cells’ inflammatory response,we found that IL-10 can increased expression of proteins related to the JAK2/STAT3/SOCS3 signaling pathway,and low-dose DON can further increase the expression of related proteins.The addition of AG490,a JAK2-specific antagonistic inhibitor,could block the promotion of low-dose DON on the inflammatory response of IL-10-silencing RAW264.7 cells,suggesting that the JAK2/STAT3/SOCS3 signaling pathway was involved in the effect of low-dose DON on RAW264.7 cells.In summary,low-dose DON can promote the inflammatory response and immune activation of murine inflammatory model,and exacerbate the clinical symptoms of the model in vivo.At the same time,studies demonstrated that it may be related to JAK2/STAT3/SOCS3 signaling pathway.The results of this study not only provide a reference for the limit standard of vomitoxin,but also provide healthy dietary advice for animals suffering from autoinflammatory diseases such as chronic colitis.