Study On DON-induced Small Intestinal Injury And NF-κB Inflammatory Signaling Pathway In Piglets

Deoxynivalenol(DON)is a common my cotoxin in feed and has many toxic effects.In this study,we investigated the relationship between DON-induced small intestinal injury and activation mechanism of NF-κB inflammatory signaling pathway in piglets by DON exposure in weaned piglets and in vitro culture of IPEC-J2(small intestinal epithelial cells).On the one hand,30 healthy "DurocxLandracexYorshire" weaned piglets were randomly selected and randomly divided into 3 groups,10 in each group,fed with basic diet(control group),1 mg·kg-1 DON diet(low dose group),and 2 mg kg-1 DON diet(high dose group),the trial was 60 days.On the other hand,IPEC-J2 cells were cultured in vitro,and different concentrations of DON(0,125,250,500,1000,2000 ng·mL-1)were added to the culture solution for 24 h,and the PDTC was used as a reference.At the end of the experiment,serum,IPEC-J2 cells,and cell culture supernatant were collected for detection of inflammatory mediators and intestinal permeability index activity;5 piglets were selected for each group,the duodenum,jejunum,and ileum were taken.Ultrastructural and morphological changes were observed in the tissues and IPEC-J2 cells;the expression and distribution of ZO-1 protein in small intestinal mucosa were detected;the survival rate of IPEC-J2 cells was detected;the relative expression of inflammatory mediators and NF-κB pathway-related genes and proteins in tissues and IPEC-J2 cells were detected;the NF-κB p65 protein distribution and expression in IPEC-J2 cells were detected.The test results are as follows:1.The ultrastructure of intestinal epithelial cells in the experimental groups were destroyed.DON could change the morphology of the small intestine mucosa and tissue damage.The activity of DAO and D-LA in serum increased with the increase of DON dose in the feed(P<0.05 or P<0.01);the distribution and average optical density of ZO-1 protein of the piglets in experimental groups were decreased compared with the control group(P<0.05 or P<0.01).2.DON abnormally regulated the activities of IL-6,NO and TNF-α in serum(P<0.05 or P<0.01),and increased the relative mRNA expression of IL-1β,IL-6 and TNF-α in small intestinal tissues(P<0.05 or P<0.01);the relative expression of NF-κB p65,IKKα/β,iNOS and COX-2 mRNA in tissues increased with the increase of DON concentration(P<0.05 or P<0.01),IκB-α showed a downward trend(P<0.05 or P<0.01).The NF-κB p65,p-NF-κB p65,COX-2 protein relative expression and IκB-α phosphorylation and unphosphorylation The ratio was significantly increased with the increase of DON content in the diet(P<0.05 or P<0.01).3.DON decreased the density of IPEC-J2 cells,and induced cell organ expansion and deformation,nuclear membrane shrinkage,nuclear chromatin accumulation;cell survival rate decreased(P<0.05 or P<0.01);with the increase of challenge DON dose,the content of DAO in the supernatant also increased significantly(P<0.01),and the content of DAO decreased after adding PDTC(P<0.01).4.With the increase of DON concentration,the activities of IL-6,TNF-α,NO and the relative expression of IL-1β and IL-6 mRNA in cells increased with the increase of DON concentration(P<0.01).After the addition of PDTC,the up-regulation trend of some detection factors decreased(P<0.05 or P<0.01).The relative expression levels of NF-κB p65,IKKα/β in the cells were adjusted to different degrees with the increase of DON concentration(P<0.01),while IKB-a showed a downward trend(P<0.01).At the same time,PDTC inhibited the change trend.Increased expression of NF-κB p65 protein;when the concentration of DON reached 250 ng·ml-1 and above,the nuclear expression of NF-κB p65 was significantly increased compared with the control group(P<0.01).After the addition of the pathway inhibitor,the expression decreased(P<0.01).In summary,DON exposure can cause damage to intestinal epithelial cells of weaned piglets,destroy intestinal barrier,increase intestinal permeability,induce inflammation of small intestine,and have certain effects on NF-κB signaling pathway.At the same time,DON can change the morphology of IPEC-J2 cells,destroy its ultrastructure,enhance the permeability of cell membrane.Up-regulation of gene transcription of a part of inflammatory factors in cells and alteration of expression of NF-κB-related gene proteins in cells activates the inflammatory pathway,resulting in an increase in intracellular inflammatory response.