Protective Effects Of β-Glucan On Intestinal Barrier In Weaned Pigs Challenged By Enterotoxigenic Escherichia Coli

β-glucan（BGL）is a functional polysaccharide formed by D-glucose monomer linked byβ-glycosidic bonds,which has various biological activities such as anti-inflammatory,hypoglycemic lipids,and antioxidant.BGL is widely found in bacteria,fungi and cereal cell walls.In addition,secondary metabolites produced by microbial fermentation are also important sources of BGL.Agrobacterium sp.ZX09 is a salt-tolerant strain isolated from saline soil that can exocytosis large amounts ofβ-（1,3）-glucan.In this experiment,the protective effect of BGL derived Agrobacterium sp.ZX09 on Enterotoxigenic Escherichia coli-induced（ETEC-induced）IPEC-J2 cells damage was first studied by in vitro.On this basis,the ETEC challenge was used as a model to further study the effects of BGL on piglet growth performance,intestinal inflammatory response and the barrier function,and the possible mechanism of alleviating intestinal inflammatory injury in piglets was preliminarily revealed.In vitro experiment was designed as a 2（BGL）×2（ETEC）factorial arrangement of four treatments that composed of control group（CON）,BGL（50μg/m L）,ECON（1×10⁶CFU/m L ETEC）,EBGL（50μg/m L BGL+1×10⁶CFU/m L ETEC）.BGL pretreated IPEC-J2 cells for 6 h and then challenged with ETEC,and cells were collected after 2 h.In vivo experiment,thirty-two weaned barrow pigs at 21 days old（Duroc×Landrace×Yorkshire）were assigned into four groups according to body weight,CON group（Pigs fed with a basal diet）,BGL（Pigs fed with a basal diet containing 500 mg/kg BGL）,ETEC（Pigs fed with a basal diet and challenged with ETEC）and EBGL（Pigs fed with a basal diet containing 500 mg/kg BGL and challenged with ETEC）.The experiment lasted for 28d,on 26 d,the ETEC challenge groups were orally dosed with 100 m L Luria Bertani（LB）medium containing 1×10¹⁰CFU/m L ETEC every day,other groups were equal volume LB mediums.On the morning of 29 d,piglets were given D-xylose（0.1 g/kg BW）and slaughtered 1 h later for analysis of intestinal morphology and cell apoptosis.The results are as follows:（1）ETEC challenge increased the total apoptosis rates IPEC-J2 cells（P<0.05）,but BGL pretreatment significantly reduced the early apoptosis,late apoptosis and total apoptosis of ETEC-induced IPEC-J2 cells（P<0.05）,and significantly down-regulated the expression levels of apoptosis key molecules cysteinyl aspartate specific proteinase 3（Caspase 3）and Caspase 9（P<0.05）;In addition,BGL pretreatment significantly down-regulated the expression levels of tumour necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）and toll-like receptor 4（TLR4）and nuclear factor kappa B（NF-κB）,which were key transcription factors in the inflammatory response in ETEC-induced IPEC-J2 cells（P<0.05）.（2）BGL supplementation had no significant effect on the apparent digestibility of average daily feed intake（ADFI）,average daily gain（ADG）and major nutrients in the unchallenged piglets（P>0.05）.ETEC challenge reduced ADG in piglets（P<0.05）,but BGL supplementation had a trend to improve the ADG of piglets（0.05<P<0.10）.（3）ETEC challenge decreased pig serum concentration of immunoglobulin A（Ig A）,but BGL supplementation significantly increased the serum concentrations of Ig A and Ig G in the ETEC-challenged piglets（P<0.05）,and reduced the concentrations of inflammatory factors TNF-αand interleukin-6（IL-6）（P<0.05）;In addition,BGL supplementation significantly reduced the content of serum D-lactic and the activity of diamine oxidase（DAO）in pigs（P<0.05）,improved the content of serum D-xylose in pigs（P<0.05）.（4）ETEC challenge decreased the jejunal villi height in pigs（P<0.05）,but BGL significantly improved the duodenum,jejunum and ileal villi height and the jejunal ratio villi height to crypt depth（V/C）in the ETEC-challenged pigs（P<0.05）.In addition,BGL significantly supplementation improved the abundance of zonula occludens-1 ZO-1 in the jejunal epithelium upon ETEC challenge（P<0.05）.（5）BGL supplementation increased the duodenal and ileal maltase activities in unchallenged piglets（P<0.05）.ETEC challenge reduced the activities of maltase,lactase and sucrase in the duodenal and jejunal mucosa of piglets,but BGL supplementation significantly increased the duodenal and ileal maltase and sucrase activities,and ileal maltase activities upon ETEC challenge.（6）ETEC challenge increased the ileal content of malondialdehyde（MDA）,but it decreased the activities of duodenal glutathione peroxidase（GSH-Px）,jejunal total antioxidant capacity（T-AOC）and total superoxide dismutase（T-SOD）,and ileal catalase（CAT）（P<0.05）,however,BGL supplementation significantly reduced the production of MDA in small intestinal mucosa and it improved activities of duodenal CAT,ileal GSH-Px and T-SOD in ETEC-challedged pigs（P<0.05）.（7）ETEC challenge increased the jejunal rates of apoptosis,but BGL supplementation significantly decreased the jejunal rates of early apoptosis and total apoptosis in the ETEC-challenged pigs（P<0.05）,and down-regulated the jejunal expression of Caspase 9 and Bax,and ileum expression of Caspase 9 and Caspase 3 in the ETEC-challenged pigs（P<0.05）.In addition,BGL significantly decreased the number of G1 phase cells and increased the number of S phase（P<0.05）.（8）ETEC challenge decreased the jejunal epithelium number of secretory immunoglobulin A（s Ig A）positive cells,but BGL supplementation significantly improved it upon ETEC challenge（P<0.05）,and down-regulated the jejunal mucosa expression levels of TLR4,myeloid differentiation factor 88（My D88）and NF-κB,and jejunal and ileal mucosa expression levels of TNF-αand IL-6 upon ETEC challenge（P<0.05）.（9）BGL supplementation increased the abundance of Lactobacillus in cecum and Lactobacillus and Bacillus in colonic upon ETEC challenge（P<0.05）and increased the colon concentration of propionic acid in pigs（P<0.05）.These results suggested that BGL supplementation could improve the production performance,and attenuate intestinal damage in weaned pigs upon ETEC challenge,and the mechanism may be related to inhibition of intestinal epithelial inflammatory response,improvement of intestinal immunity and antioxidant capacity,and improvement of the intestinal microbiota of piglets.