Mechanisms Of Deoxynivalenol-Induced Immunological Stress And Anorexia

Deoxynivalenol?DON?is a kind of type B trichothecene mycotoxin produced by Fusarium.DON is one of the most common fungal toxins in grain and grain products and is one of the major pollutants detected in feed and feed materials worldwide.This mycotoxin has been associated with a spectrum of adverse effects,including anorexia,nausea,emesis,growth suppression,weight loss,neuroendocrine changes,and immunotoxicity.Anorexia and consequent growth suppression induced by DON are of particular concern for children and young animals.However,the mechanism of the immunological stress and anorexia induced by DON is not fully known.DNA methylation and miRNA are the two most common epigenetic modifications,and play an important role in regulating gene expression on the level of transcription or after transcription,which affect the growth of animals in turn.DON was studied in this experiment,on the one hand,the epigenetic mechanism of the immune stress of weaning piglets was explored by DNA methylation sequencing and miRNA sequencing?miRNA-sequencing,miRNA-seq?;On the other hand,we further explored the mechanism of the anorexia induced by DON in the regulation of the gut satiety hormones glucagon-like peptide 1?GLP-1?and gastric inhibitory peptide?GIP?.1.Culture of Fusarium and preparation of DON contaminated feedConventional recovery fusarium graminearum strain preserved in our laboratory to preparation for DON contaminated feed of piglet.High performance liquid chromatography?HPLC?was used to detect the level of DON in contaminated feed,the levels of other common mycotoxin in the feed were also tested.The results showed that the level of DON in contaminated feed was 9.93 mg/kg,the level of AFB1,FB1,OTA was 0 mg/kg,and the level of ZEA was 27?g/kg,which is lower than that of the maximum dose?40 ?g/kg bw per day?for pigs.So,this contaminated feed can be used for subsequent experiments on pigs.2.Effects of DON on the performance,physiology and immune function of piglets18 21-days old Duroc × Landrace × Large?DLY?weaning piglets were randomly divided into 3 groups,control group?NC?feed health diet,DON low dose group?DON-L?contains 1.0 mg/kg DON contaminated feed,DON high dose group?DON-H?contains 4.0 mg/kg DON contaminated feed,the trial lasted 28 d.The results showed that:1)compared with the NC group,the average daily gain and average daily intake of piglets in the DON-H group decreased significantly.The average daily weight gain and average daily intake of piglets in the DON-L group showed a decreasing trend,but the difference was not significant.2)Compared with NC group,the number of white blood cells in the DON-H group increased significantly,the number of red blood cells and hemoglobin decreased,but the difference was not significant;the number of white blood cells in the DON-L group showed a increasing trend,but the difference was not significant after 7 d.3)Compared with NC group,the levels of total protein and globulinlevel of DON-H group increased significantly,the levels of total cholesterol,triglyceride and alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase showed a increasing trend,but the difference was not significant;the levels of total protein,albumin,globulin,creatinine,total cholesterol and alanine aminotransferase of the DON-L group showed a decreasing trend,but the difference was not significant after 7 d.4)Compared with the NC group,the proportion of CD3+T lymphocytes increased significantly in the peripheral blood of DON-L and DON-H group after 14 d;the proportion of CD3+T lymphocytes in the peripheral blood of DON-H group was still significantly higher than that in the NC group after 21 d and 28 d.5)the level of IgG in the serum of DON-L and DON-H group was significantly increased compared with that of the NC group,but the level of IgA and IgM in the serum showed a increasing trend,but the difference was not significant after 7 d.6)the level of IL-1??IL-6 and TNF-? in the serum of DON-H group was significantly increased compared with that of the NC group,but the level of IL-1??IL-6 and TNF-? in the serum of DON-L group showed a increasing trend,but the difference was not significant after 7 d.3.Effect of immune stress induced by DON on the methylation level of DNA of piglets'spleenDNA in the spleen of piglets was extracted,simplify representative bisulfite Sequencing?Reduced representation bisulfite Sequencing,RRBS?library was established and Sequencing,the Sequencing result is analyzed,the results showed that:1)compared with NC group,DON-L group and DON-H group have 13 and 14 differences methylation area respectively.2)The chromosome CGIs and Promoter methylation mappings were mapped.3)The enrichment of GO function and KEGG pathway analysis showed no significant difference of enrichment between differentially expressed genes,but it was found that the differentially expressed genes mainly related to the biological processes such as cell catabolism regulation,negative regulation of cell proliferation,cerebellar development,and mitotic cell cycle regulation.4.Effect of immune stress induced by DON on the level of miRNA of piglets' spleenTotal RNA in the spleen was extracted,the miRNA-Sequencing?microRNAs-Seq?library was established and Sequencing.The sequencing results were analyzed and 415 novel miRNAs were found.Three groups of comparisons?DON-L_VS_NC,DON-H_VS_NC,DON-L_VS_DON-H?found 120,73 and 120 differentially expressed miRNAs which were already known respectively.Some miRNA target was projected.GO function enrichment analysis and KEGG pathway analysis were performed and top 10 GO items and pathways were selected.GO items mainly include metabolic process,organic substance metabolic process,cellular metabolic process,primary metabolic process,cell,binding,protein binding,ion binding and catalytic activity.KEGG pathway mainly include AGE-RAGE signaling pathway in diabetic complications,Sphingolipid signaling pathway,Inositol phosphate metabolism,Epstein-Barr virus infection,Proteoglycans in cancer,Galactose metabolism,Pancreatic cancer,MAPK-signaling pathway,RIG-I-like receptor signaling pathway,TNF signaling pathway,NF-kappa B signaling pathway,Apoptosis,HTLV-I infection,Hepatitis B,Chronic myeloid leukemia,VEGF signaling pathway,Cell cycle and Endocytosis.5.Role of Glucagon-Like Peptide-1 and Gastric Inhibitory Peptide in AnorexiaThe role of GLP-1 and GIP in DON-induced anorexia was determined by a mouse food consumption model.The mice were orally gavaged with 1.0 or 2.5 mg/kg bw of DON.Blood was collected 0.5,1,2,and 6 h after exposure.A direct competition ELISA method was used to determine the concentration of satiety and GLP-1 and GIP.The results showed that the elevation of plasma GLP-1 and GIP concentrations markedly corresponded to anorexia induction by DON within 30min.The mice were injected IP with 0,1.0 or 2.5 mg/kg bw of DON,and their food intake was measured under red light conditions during the dark cycle at 0.5 h,1 h,2 h,3 h and 6 h post-exposure.To assess hormone-induced anorexia,food intake was measured at the same time points after the administration of 0.1 and 0.125 mg/kg bw of GLP-1 and GIP.Acute IP exposure of mice to 1.0 or 2.5 mg/kg bw doses of DON caused a significant reduction in food intake after 0.5,1,2 and 3 h compared to that of the control.Mice were treated with exogenous GLP-1 and GIP to confirm that these hormones led to food refusal in this mouse model.GLP-1 significantly reduced food intake by 48%,49%,40%and 48%relative to the control after 0.5,1,2 and 3 h,respectively.Relative to the control,GIP significantly reduced food intake within 2 h.In addition,the mice were injected IP with the antagonist Exendin9-39 or Pro3GIP 30 min before IP injection of DON at a dose of 1.0 mg/kg bw,then food consumption was determined.The results showed that the elevation of plasma GLP-1 and GIP concentrations markedly corresponded to anorexia induction by DON within 30min,the GLP-1 receptor antagonist Exendin9₃9 and the GIP receptor antagonist Pro3GIP induced attenuation of DON-induced anorexia.