Protective Effects And Mechanisms Of Sialyllactose On Intestinal Barrier In Weaned Pigs Infected With Enterotoxigenic Escherichia Coli

Weaning is one of the most important challenges for neonatal pigs,as weaning deprives their protections from maternal passive immunity and may contribute to intestinal and immune system dysfunctions that lead to reduced growth and feed intake.In the past decades,antibiotics have been widely used to prevent weaning-induced diarrhea and intestinal barrier damage in piglets.However,under the background of banning antibiotics,how to protect the intestinal health of weaned piglets is a major challenge for the industry.Sialyllactose（SL）,which is the most abundant components of milk oligosaccharides and is a compound where the N-acetyl-D-neuraminic acid（Neu5Ac or SA）unit is connected to the galactose unit of lactose.SL has antibacterial,antiviral,anti-inflammatory and other biological activities.An enterotoxigenic Escherichia coli（ETEC）-infected pig model was used to study the protective effects of SL on the intestinal barrier of pathogens-infected pigs,and then explored the mechanisms by which SL alleviates the intestinal barrier injury using in vitro model.Experiment 1:Effect of SL supplementation on the growth performance,immune function and intestinal barrier of weaned pigs infected with ETEC.A total of 32 male Duroc×Landrace×Yorkshire pigs weaned at 21 days（with an average body weight of 6.66±0.14 kg）were selected and randomly allotted into a 2（SL）×2（ETEC）factorial experiment of four treatments composed of CON（Pigs were fed with a basal diet）,CSL（Pigs were fed with basal diet containing 5 g/kg SL product）,ECON（Pigs were fed with a basal diet and challenged by ETEC）,ESL（Pigs were fed with basal diet containing 5 g/kg SL product and challenged by ETEC）.Pigs were received the same parental nutrition and management（e.g.sows were fed with the same diet,synchronization estrous）.The trial last for 28 d.On 26 d,the challenge groups were orally treated with 100m L of LB culture containing 1×10¹⁰ CFU/m L of ETEC by using an orogastric tube last for3 d,whereas the non-challenge groups were orally treated with equivalent amount of culture medium.On day 15 of the experiment,pigs were orally dosed with 10%D-xylose at 1 m L per kg BW,and a blood sample and intestinal tissues were collected 1 h later.Results show that:SL supplementation elevated the Average daily gain（ADG）and feed efficiency of the weaned piglets（P<0.05）.SL also improved the digestibilities of Dry matter（DM）,Gross energy（GE）,and ash in non-challenged pigs（P<0.05）.SL not only elevated serum concentrations of immunoglobulins（Ig A,Ig G,and Ig M）,but also significantly decreased the serum concentrations of inflammatory cytokines（TNF-α,IL-1β,and IL-6）upon ETEC challenge（P<0.05）.Moreover,SL significantly decreased serum D-lactate and DAO concentrations increased by ETEC-challenging（P<0.05）.SL increased the villus height,the ratio of Villus height to crypt depth（V/C）,and the activities of mucosal sucrase and maltase in the jejunum and ileum（P<0.05）.SL supplementation sinnificantly increased the abundance of ZO-1 protein in the duodenal and jejunal epithelium of the weaning piglets.SL supplementation prominently increased the ration of G₂ phase cells and decreased the early,late and total apoptosis rate in the ETEC-challenging pigs.SL supplementation significantly elevated the abundance of s Ig A positive cells in the jejunal epithelium of ETEC-challenged piglets.SL supplementation significantly down-regulated the expression level of inflammatory factors（TNF-α,IL-1β,IL-6）and Key molecules of inflammatory signaling pathways（My D88,TLR4,and NF-κB）（P<0.05）.In addition,SL also elevated the abundance of Lactobacillus,Bifidobacterium,and Bacillus and the concentrations of microbial metabolites（e.g.acetic acid,propanoic acid,and butyric acid）in the cecum and colon（P<0.05）.The above results indicate that SL can alleviate the intestinal barrier damage caused by ETEC infection.The mechanism may be related to the inhibition of inflammatory cytokine secretion,increasing of serum immunoglobulin level,improvement of intestinal epithelial function and microbiota.Experiment 2:SL alleviates LPS-induced IPEC-J2 cell damage via the NF-κB signalling pathwayThe NF-κB signaling pathway plays an important role in regulating the intestinal inflammatory response,and Experiment 1 found that SL alleviated the inflammatory damage of the intestinal barrier caused by ETEC infection.However,it is unclear whether SL alleviates inflammatory damage to the intestinal barrier by inhibiting the NF-κB signaling pathway.Therefore,an in vitro model of LPS-induced inflammation was used to investigate the mechanisms by which SL alleviates the inflammatory injury to intestinal epithelial cells.The experiment was divided into four treatment groups:control group（CON）;SL treatment group（5 mg/m L SL）;LPS challenge group（5μg/m L LPS）;SL plus LPS treatment group（5 mg/m L SL+5μg/m L LPS）.IPEC-J2 cells were pretreated with SL for 12 h and then treated with LPS for 6 h.Results have shown that:LPS stimulation significantly decreased the expression level of tight junction protein-related genes ZO-1and Occludin and increased the expression level of inflammatory factors（TNF-α,IL-1β,IL-6）and Key molecules of inflammatory signaling pathways（My D88,TLR4,and NF-κB）（P<0.05）.However,SL pretreatment significantly inhibited LPS binding to IPEC-J2 cells and significantly increased the expression level of tight junction protein-related genes ZO-1 and Occludin and decreased the expession level of genes involved in inflammatory responses（TNF-α,IL-1β,IL-6,My D88 and TLR4）（P<0.05）.LPS stimulation significantly increased the early-stage apoptotic cells,late–stage apoptotic cells,total apoptotic cells and the m RNA expressions of apoptosis-related genes（Caspase 8,Caspase9）in IPEC-J2 cells（P<0.05）.However,exposure to SL reversed the increase in LPS-mediated cell apoptosis and the expression level of Caspase 9（P<0.05）.SL pretreatment significantly decreased the IκBαand NF-κB p65 phosphorylation levels in the LPS-stimulated cells（P<0.05）.The above results suggest that SL can alleviate inflammatory damage in intestinal epithelial cells by inhibiting NF-κB activation,reducing inflammatory cytokine secretion and cell apoptosis.In summary,dietary supplementation with SL improved the growth performance and intestinal barrier function in ETEC-infected pigs.The protective effects of SL on the intestinal barrier in ETEC-infected pigs are related to the inhibition of the activation of key inflammatory signalling molecules（IκBα,NF-κB）,the decreased secretion of inflammatory factors and reduced cell apoptosis.