Effects Of Deoxynivalenol Exposure On Intestinal Health Of Piglets And Its Mechanism

Deoxynivalenol(Deoxynivalenol,DON)is a common mycotoxin in feed production,which is very harmful to the animal husbandry.Piglets are particularly sensitive to DON,and low dose exposure may cause great damage to it.Trefoil peptides(Trefoil factor family,TFF)play an important regulatory role in the repair of gastrointestinal mucosa and a variety of gastrointestinal injuries.Abnormal expression of TFFs will affect intestinal health.SAM pointed domain cintaining Ets transcription factor(SAM pointed domain cintaining Ets transcription factor,SPDEF)may have a regulatory role in the expression of TFFs.Therefore,the experiment set up the DON level of the diet which is close to the DON limit in the current Feed Hygiene Standard(GB 13078-2017),and studied the effects of DON exposure on the intestinal health and the expression of TFFs of weaned piglets.The effects of DON exposure on the intestinal health of weaned piglets,the expression of TFFs,and the regulation of SPDEF in the process were studied by constructing DON stress model of weaned piglets and porcine intestinal epithelial cells(IPEC-J2).It was preliminarily discussed that the effects of DON exposure on intestinal health of piglets and its mechanism,which provided a reference for the prevention and control of DON harm in piglets.The main results are as follows:1 The effects of DON exposure on intestinal health and TFFs expression of weaned piglets24 28-day-old castrated piglets(Duroc × Landrace × Large white)with similar body weight were randomly divided into 3 groups,8 replicates in each group,1 pig in each replicate.They were fed diets containing 0.61 mg/kg,1.28 mg/kg and 2.89mg/kg DON respectively,and slaughtered 28 days later.These were studied that the effects of DON exposure on growth performance,diarrhea,intestinal histology,liver histology and the expression of TFFs,SPDEF and Claudin-4 in intestine of weaned piglets.The main results are as follows:1.28 mg/kg DON exposure would have adverse effects on the growth performance and health,reduce the body weight,average daily gain and average daily feed intake of weaned piglets,increase the incidence of diarrhea of weaned piglets.The weight of weaned piglets in 1.28 mg/kg DON treatment group was significantly lower than that in control group on the 14 th and 28 th day(P < 0.05);the body weight of weaned piglets decreased significantly on the 28 th day(P < 0.01).The average daily gain of weaned piglets were significantly decreased in 1.28 mg/kg and2.89 mg/kg DON treatment groups at 1-14 days,15-28 days and 1-28 days(P < 0.05).The average daily feed intake of weaned piglets in 1.28 mg/kg and 2.89 mg/kg DON treatment groups were significantly lower than that in control group at 1-14 days,15-28 days and 1-28 days(P < 0.05).There was no significant difference in meat to feed ratio among the three treatment groups.In addition,the fecal morphology score and diarrhea rate of weaned piglets in 1.28 mg/kg and 2.89 mg/kg DON treatment groups were higher than those in the control group.DON exposure would damage the intestine and liver of weaned piglets.The villus height and villus height/crypt depth in duodenum and jejunum were significantly decreased of weaned piglets in 1.28 mg/kg and 2.89 mg/kg DON treatment groups(P < 0.001),while the villus height and villus height/crypt depth were significantly lower in ileum than those of control group of weaned piglets(P <0.05).In the control group,the morphology of liver cells and the liver tissue structure were normal;in the 1.28 mg/kg DON treatment group,the liver began to appear abnormal,the disorder of hepatic cords and vacuolation of liver cells could be seen;in the 2.89 mg/kg DON treatment group,the disorder of hepatic cord and vacuolation of liver cells were more serious.DON exposure would inhibit the expression of TFFs,SPDEF and Claudin-4 in intestine.The mRNA expressions of TFF1,TFF2,TFF3 and SPDEF were significantly decreased in jejunum of weaned piglets in 1.28 mg/kg and 2.89 mg/kg DON treatment groups(P < 0.05).1.28 mg/kg and 2.89 mg/kg DON exposure significantly reduced the mRNA expression of TFF2,TFF3 and SPDEF in ileum of weaned piglets(P < 0.05).2.89 mg/kg DON exposure reduced the mRNA expression of TFF1,TFF2,TFF3 and SPDEF in cecum of weaned piglets(P < 0.05).However,the effect of DON exposure was limited on the expression of TFFs and SPDEF mRNA.The expression of Claudin-4 mRNA was significantly decreased in jejunum,ileum,cecum and colon in 2.89 mg/kg DON treatment group(P < 0.05).The protein expressions of TFF2,TFF3 and SPDEF were significantly lower in jejunum in 1.28mg/kg and 2.89 mg/kg DON treatment groups than those in control group of weaned piglets(P < 0.05).DON exposure would affect the jejunal gene expression of weaned piglets.The effect of DON on the production performance of weaned piglets may be related to PI3K-Akt,PPAR and other signal pathways.Exposure to 2.89 mg / kg DON resulted in significant changes in the expression of 498 genes in the jejunum of weaned piglets,of which 304 genes were up-regulated(P < 0.05)and 194 genes were down regulated(P < 0.05).Through the COG classification statistics and molecular function results of and GO enrichment analysis,it was found that the differentially expressed genes(Differentially expressed gene,DEG)were mainly distributed in intracellular trafficking et al.The biological process results of go enrichment analysis showed that the changes of regulation of cell population proliferation et al were obvious.The results of cell components enriched and analyzed in go showed that extracellular region par et al were significantly affected.Through the analysis of KEGG signal pathway it was found that PI3K-Akt,PPAR and other signaling pathway changed significantly.2 The effect of SPDEF on TFFs expressionIn this study,IPEC-J2 cells were transfected with SPDEF empty vector and SPDEF overexpression vector respectively,and then the transfected cells were were treated with(0.5 μmol/L)or without DON exposure.The cells were divided into four groups by two factor and two-level crossover design: SPDEF empty vector group,SPDEF overexpression group,SPDEF empty vector + DON group and SPDEF overexpression + DON group.These were studied that the effect of SPDEF on the expression of TFFs and the inhibitory effect of SPDEF on the expression of TFFs with DON exposure.The main results are as follows:DON exposure would reduce the expression of TFF1,TFF2,TFF3,SPDEF and Claudin-4 mRNA in IPEC-J2 cells,and the effect and degree of influence were related to the exposure time;SPDEF overexpression would increase the mRNA expression of TFFs in IPEC-J2 cells.The expression of TFF1 increased significantly(P < 0.001),TFF2 decreased significantly(P < 0.001),TFF3 decreased significantly(P < 0.05),but Claudin-4 showed no difference after 6 h 0.5 μmol/L DON exposure;the expression of TFF2,TFF3,SPDEF and Claudin-4 decreased significantly(P < 0.05),but there was no difference in TFF1 expression after 12 h 0.5 μmol/L DON exposure.When SPDEF was overexpressed,the expression of TFF1 was significantly increased(P < 0.01);the expression of TFF2 and TFF3 were also significantly higher than those in the control group(P < 0.05).SPDEF overexpression could improve or even reverse the inhibitory effect of DON on TFFs expression in IPEC-J2 cells.The protein expression of SPDEF was decreased by DON exposure,and SPDEF overexpression could significantly reverse the inhibitory effect of DON(P < 0.01).The mRNA expression levels of TFF1 and TFF2 in SPDEF-overexpressed cells were significantly higher than those in normal cells under DON exposure(P < 0.05).In summary,this study drews the following conclusions:(1)Dietary DON exposure of 1.28 mg/kg would reduce the growth performance,cause damage to intestinal tract and liver,and increase the diarrhea rate of weaned piglets;(2)Dietary DON exposure of 1.28 mg/kg would inhibit the expression of TFFs,SPDEF and Claudin-4 in intestine of weaned piglets,affect jejunal transcriptome expression;(3)SPDEF would regulate the expression of TFFs.The dysregulation of TFFs induced by DON exposure is related to SPDEF,a positive regulator of TFFs.