| This study aimed to investigate the effects of different levels of zearalenone（ZEA）on the intestinal barrier of the jejunum and cecum in weaned piglets,and to explore possible mechanism by which ZEA damages intestinal barrier in weaned piglets by using HE staining,immunohistochemistry,q RT-PCR,Western Blot and 16 s r RNA sequencing.The experiment selected 32 healthy ternary hybrid（Duroc × Landrace × Yorkshire）weaned gilts（35 days,17.39 ± 0.24 kg）and randomly divided them into four treatments,namely Control,ZEA0.15,ZEA1.5 and ZEA3.0 treatments which had 8 replicates each.The Control treatment was fed the basal diet.And the ZEA0.15,ZEA1.5 and ZEA3.0 treatments were fed the basal diet supplemented with 0.15,1.5 and 3.0 mg/kg ZEA,respectively.The pre-feeding period was 7 days,and the formal test period was 32 days.At the end of the feeding trial,fixed and cryopreserved samples of the small intestine and cecum,and cecal contents were collected.The main results of this study are as follows:（1）As the level of ZEA in the feed increased,the levels of diamine oxidase（DAO）and Dlactate in the serum of piglets increased linearly（P < 0.001）.The levels of DAO and D-lactate in ZEA1.5 and ZEA3.0 treatments were significantly higher than those of the Control and the ZEA0.15 treatments（P < 0.05）.The D-xylose concentration of serum in ZEA1.5 treatment was significantly higher than that of the other three treatments（P < 0.05）.（2）The small intestinal villi of the duodenum,jejunum,and ileum in the ZEA1.5 and ZEA3.0 treatments were broken,and the epithelium was shed.The injure in ZEA3.0 treatment was more serious.The numbers of goblet cells in jejunal villi and crypt in the ZEA1.5 and ZEA3.0 treatments were significantly lower than those in the Control and ZEA0.15 treatments（P < 0.05）.TFF3 was mainly localized in the goblet cells of villi and crypts,and the protein expressions of TFF3 in the jejunum of the ZEA1.5 and ZEA3.0 treatments were significantly lower than those in the Control and ZEA0.15 treatments（P < 0.05）.The m RNA and protein expressions of jejunal occludin,claudin-1 and ZO-1 in ZEA1.5 and ZEA3.0 treatments were significantly lower than those in Control and ZEA0.15 treatments（P < 0.05）.The expression levels of TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3 and Smad7 in the ZEA1.5 and ZEA3.0treatments were significantly higher than those in the Control and ZEA0.15 treatments（P <0.05）,while the expression levels of Smad4 in ZEA1.5 and ZEA3.0 treatments were significantly lower than those in the Control and ZEA0.15 treatments（P < 0.05）.（3）Under the light microscope,the mucosal epitheliums of the cecum in the ZEA1.5 and the ZEA3.0 treatments were sloughed off.The M cells in cecal mucosa epithelial in the ZEA1.5and ZEA3.0 treatments showed a protruding shape and dense distribution.And the surrounding microvilli became short.The numbers of cecal goblet cells in the ZEA1.5 and ZEA3.0treatments were significantly lower than those in the Control and ZEA0.15 treatments（P <0.05）.TFF3 was mainly localized in goblet cells in the cecal gland,and the protein expressions of TFF3 in the cecum of the ZEA1.5 and ZEA3.0 treatments were significantly lower than those of the Control and ZEA0.15 treatments（P < 0.05）.The m RNA and protein expressions of occludin,claudin-1 and ZO-1 in the cecum in the ZEA1.5 and ZEA3.0 treatments were significantly lower than those in the Control and ZEA0.15 treatments（P < 0.05）.The expression levels of TGF-β1,Smad2 and p-Smad2 in the ZEA3.0 treatment were significantly lower than those in the Control,ZEA0.15 and ZEA1.5 treatments（P < 0.05）,and the expression levels of Smad3 and p-Smad3 in the ZEA0.15,ZEA1.5 and ZEA3.0 treatments were the significantly lower than those in the Control treatment（P < 0.05）.The expression levels of Smad4 in the ZEA1.5 and ZEA3.0 treatments were significantly lower than those in the Control and ZEA0.15treatments（P < 0.05）.The expression level of Smad7 in ZEA0.15 treatment was significantly lower than that in Control,ZEA1.5 and ZEA3.0 treatments（P < 0.05）.（4）The differences in cecal microbiota were detected by 16 s r RNA sequencing.The results showed that: at the phylum level,the relative abundance of Firmicutes in the ZEA3.0 treatment was significantly lower than that in the Control treatment（P < 0.05）.The relative abundance of Bacteroidetes in the ZEA3.0 treatment was significantly higher than those in the Control and ZEA1.5 treatments（P < 0.05）.The relative abundance of Actinobacteria in the ZEA1.5treatment was the highest,and was significantly higher than that in the ZEA3.0 treatment（P <0.05）.At the genus level,the relative abundance of Prevotella＿9 in the ZEA3.0 treatment was the highest and significantly higher than that in the Control,ZEA0.15 and ZEA1.5 treatments（P < 0.05）.The relative abundance of Agathobacter in the ZEA1.5 treatment was the lowest and significantly lower than that in Control and ZEA3.0 treatments（P < 0.05）.The relative abundance of Prevotella in ZEA3.0 treatment was significantly higher than that in Control and ZEA1.5 treatments（P < 0.05）.The relative abundance of Blautia in ZEA1.5 treatment was the highest and significantly higher than that in Control,ZEA0.15 and ZEA3.0 treatments（P <0.05）.In conclusion,under our experimental conditions,diets supplemented with 1.5 mg/kg and3 mg/kg of ZEA damaged the jejunum and cecum barrier by impairing intestinal morphology,reducing the number of goblet cells and the expressions of TFF3 and tight junction proteins.The additions of 1.5 mg/kg and 3.0 mg/kg of ZEA to the diet both decreased the expressions of Smad4 in the jejunum and cecum,inhibited the TGF-β1/Smads pathway,thus inhibited the repair of intestinal mucosal epithelium.The additions of 1.5 mg/kg and 3.0 mg/kg of ZEA to the diet changed the abundance of some cecal flora. |
