Protective Effects And Mechanisms Of Alginate Oligosaccharide On The Intestinal Epithelial Barrier In Weaned Pigs

Early weaning piglets are vulnerable to the invasion of various pathogens as well as the nutritional,physiological and environmental stresses as they have immature immune and digestive systems.For a long time,in-feed antibiotic additives have been used to alleviate the diarrhoea and intestinal barrier damage caused by the weaning of piglets and have provided good results.However,under the current background of limited or banned use of antibiotics,ensuring the intestinal health of piglets is a major challenge faced by the livestock industry.Alginate oligosaccharide(AOS)is a linear block compound composed of?-D-mannuronic acid and?-L-guluronic acid.It has anti-microbial,anti-inflammatory and anti-apoptotic properties.To investigate the effects of AOS supplementation on weaned pigs and the mechanisms by which AOS regulates the intestinal barrier function in weaned pigs,five experiments were conducted in this study.The appropriate dosage of AOS was determined by a growth test.Then,the effects of AOS on the intestinal barrier function in weaned pigs and enterotoxigenic Escherichia coli(ETEC)-infected pigs were studied.Finally,a porcine small intestinal epithelial cell line(IPEC-J2)was used to further explore the mechanisms by which AOS protects the intestinal barrier and to identify its key regulatory targets.The main results are as follows:Experiment 1:AOS preparation and its effects on growth performance in weaned pigsAOS was prepared by the degradation of sodium alginate by alginate lyase,and the enzymatic hydrolysates were analysed by electrospray ionisation mass spectrometry.The results showed that AOS is mainly composed of di-,tri-,tetra-,penta-and hexasaccharides.To evaluate the effects of dietary supplementation with AOS on growth performance in weaned pigs,200 weaned pigs(Duroc×Landrace×Yorkshire,weaned at 21 days),with an initial body weight of 6.17(±0.16)kg,were equally assigned to four groups,with five replicate pens per group and 10 pigs per pen.The trial period was 14 days.The groups were as follows:(1)basal diet(control,CON),(2)basal diet+50 mg/kg AOS,(3)basal diet+100 mg/kg AOS and(4)basal diet+200 mg/kg AOS.The average daily gain(ADG)and average daily feed intake of the pigs supplemented with 100 or 200 mg/kg AOS were higher than those of the pigs in the CON group(P<0.05).The growth-promoting effects of 100 mg/kg AOS were better than those of 200 mg/kg AOS.These results demonstrate that AOS is mainly composed of di-to hexasaccharides and inclusion of AOS in the diet can lead to improved growth performance in weaned pigs.The appropriate dosage was found to be 100 mg/kg under the test conditions.Experiment 2:Effects of AOS on growth performance and intestinal barrier function in weaned pigsExperiment 1 revealed that AOS leads to improved growth performance in weaned pigs.This experiment further investigated the effects of AOS on the intestinal barrier function in weaned pigs.A total of 24 weaned pigs(Duroc×Landrace×Yorkshire,weaned at 21 days),with an initial body weight of 6.21(±0.09)kg,were equally divided into two groups for 2 weeks.The groups were as follows:(1)basal diet(control,CON)and(2)basal diet+AOS(100 mg/kg).On days 11–14,a digestion test was performed using endogenous indicator method to evaluate the apparent digestibility of nutrients.Serum and intestinal samples were obtained on day 15.AOS supplementation increased the ADG and the apparent digestibility of crude protein,crude ash,ether extract and gross energy and reduced the serum _D-lactic acid content and diamine oxidase activity in weaned pigs(P<0.05).AOS increased the duodenal and jejunal villus height,digestive enzyme(maltase and sucrase)activities and occludin content and decreased the duodenal and jejunal inflammatory mediator[tumour necrosis factor-?(TNF-?)and tryptase]contents(P<0.05).Moreover,AOS increased the percentage of S-phase epithelial cells and decreased the percentage of apoptotic epithelial cells in the jejunum(P<0.05).In addition,AOS up-regulated the jejunal expression levels of nutrient transport-related genes[Na⁺/glucose co-transporter 1(SGLT1)and divalent metal transporter 1]and cell cycle regulation-related genes[cyclin E1 and cyclin-dependent kinase 2(CDK2)]and down-regulated the jejunal expression levels of inflammatory reaction-related genes(nucleotide-binding oligomerisation domain protein 1 and receptor-interacting serine/threonineprotein kinase 2)and apoptosis-related genes[Bcl-2 associated X protein and cysteinyl aspartate-specific protease-3(Caspase-3)](P<0.05).Thus,dietary supplementation with AOS can improve the intestinal morphology and barrier function,thereby improving the nutrient digestibility in weaned pigs and improving their growth performance.Experiment 3:Protective effects of AOS on intestinal barrier in ETEC-infected weaned pigsETEC is one of the common pathogens in the pig production chain.It colonises the epithelial cells of the host intestine via adhesion factors and causes intestinal damage.Experiment 2 showed that AOS helps maintain the intestinal health of weaned pigs.This experiment was designed to evaluate whether AOS could attenuate ETEC-induced intestinal barrier injury in weaned pigs.A total of 24 weaned pigs were equally allocated to three groups:(1)non-challenged control,(2)ETEC-challenged control and(3)ETEC challenge+AOS treatment(100 mg/kg).On day 12,pigs in the non-challenged control group were orally infused with sterilised Luria–Bertani culture,while pigs in the other two groups were orally infused with ETEC(2.6×10¹¹ colony-forming units).At 3 days after the challenge,all the pigs were orally administered _D-xylose at 0.1 g per kg body weight and then euthanised 1 h later to collect serum and intestinal samples.ETEC infection decreased the ADG,serum _D-xylose content,jejunal and ileal villus height,sucrase activity and occludin abundance(P<0.05).On the other hand,AOS increased the serum _D-xylose content,jejunal and ileal villus height,sucrase activity and occludin abundance in ETEC-infected pigs(P<0.05).AOS decreased the jejunal and ileal E.coli counts,lipopolysaccharide(LPS)and inflammatory mediator[TNF-?,interferon-?(IFN-?)and tryptase]contents and apoptotic epithelial cell percentage and increased the jejunal and ileal proportions of epithelial cells in the S-phase in ETEC-infected pigs(P<0.05).Moreover,AOS up-regulated the jejunal and ileal expression levels of nutrient transport-related gene(SGLT1)and cell cycle regulation-related gene(cyclin E1 and CDK2)and down-regulated the jejunal and ileal expression levels of inflammatory reaction-related gene(Toll-like receptor 4 and myeloid differentiation factor 88)and apoptosis-related gene(Caspase-3,-8 and-9)in ETEC-infected pigs(P<0.05).These results indicate that AOS can reduce the E.coli count and LPS content in the intestinal mucosa as well as reduce the intestinal secretion of inflammatory mediators and epithelial cell apoptosis,thereby alleviating ETEC-induced inflammatory injury of the intestinal barrier in weaned pigs.Experiment 4:AOS alleviates LPS-induced inflammatory injury of intestinal epithelial cells through nuclear factor-?B(NF-?B)signalling pathwayNF-?B is a transcription factor found in various cells.It plays an important role in inflammatory responses.Pathogenic microorganisms and their endotoxins,such as LPS,can activate the NF-?B signalling pathway,promote the overexpression of inflammatory factors and cause inflammatory damage to cells.Experiment 3 revealed that AOS can significantly reduce the expression of intestinal inflammatory factors in ETEC-infected pigs,thereby attenuating intestinal inflammatory injury.However,whether the mitigation effects of AOS on intestinal inflammatory injury are mediated by the aforementioned signalling pathway remains unclear.Therefore,in this experiment,an LPS-induced inflammatory injury model of porcine intestinal epithelial cells was used to explore the effects of AOS on tight junction protein expression,cytokine secretion,cell apoptosis and the NF-?B signalling pathway in intestinal epithelial cells with inflammatory injury.AOS decreased LPS binding to the IPEC-J2 cell surface;it also reduced the TNF-?and IFN-?contents and the apoptosis rate in LPS-treated IPEC-J2 cells(P<0.05).On the other hand,AOS increased the abundance of occludin in these cells(P<0.05).LPS increased the abundance of p-I?B?and p-ERK1/2 proteins and the abundance of the intra-nuclear NF-?B p65 protein in IPEC-J2 cells(P<0.05),while either AOS or BAY 11-7082(a NF-?B signalling pathway inhibitor)decreased abundance of the p-I?B?protein and increased the intra-nuclear abundance of the NF-?B p65 protein in LPS-treated IPEC-J2cells(P<0.05).These results indicate that AOS blocks LPS binding to intestinal epithelial cells,inhibits the activation of the NF-?B signalling pathway and decreases the expression of pro-inflammatory cytokines and the cell apoptosis rate,thereby alleviating LPS-induced inflammatory injury of intestinal epithelial cells.Experiment 5:Protective effect of AOS against intestinal epithelial barrier injury caused by the pro-inflammatory cytokine TNF-?TNF-?,a multi-functional pro-inflammatory cytokine,can recognise TNF receptor 1(TNFR1)on the cell membranes,leading to cell apoptosis.Experiments 3 and 4 revealed that both ETEC infection and LPS stimulation significantly increase the TNF-?content;however,whether AOS has a regulatory effect on TNF-?-induced intestinal epithelial cell apoptosis remains unknown.Hence,a cell apoptosis model of porcine intestinal epithelial cells was constructed using TNF-?to further investigate the mechanisms by which AOS alleviates the inflammatory injury of the intestinal epithelial barrier.TNF-?treatment increased the apoptosis rate and Caspase-3 and-8 activities in IPEC-J2 cells(P<0.05),while either AOS or Z-VAD-FMK(a pan-caspase inhibitor)decreased the apoptosis rate and Caspase-3 and-8 activities in TNF-?-treated IPEC-J2 cells(P<0.05).Moreover,AOS increased the contents of occludin,cellular inhibitor of apoptosis protein 2 and Fas-associated death domain protein(FADD)-like interleukin(IL)-1?-converting enzyme-inhibitory protein and decreased the contents of IL-6 and TNF-?and the expression levels of TNFR1,TNFR1-associated death domain protein and FADD in TNF-?-treated IPEC-J2 cells(P<0.05).These results indicate that AOS can reduce cell apoptosis by down-regulating the expression of key molecules in the apoptosis signalling pathway,thereby alleviating TNF-?-induced inflammatory injury of the intestinal epithelial barrier.In summary,dietary supplementation with AOS can improve the growth performance and intestinal health of weaned pigs.AOS can reduce the expression of pro-inflammatory cytokines and cell apoptosis by inhibiting the activation of the NF-?B signalling pathway,thereby relieving intestinal epithelial barrier injury in ETEC-infected pigs.These results not only reveal the mechanisms by which AOS improves the intestinal barrier function in weaned pigs but also provide new ideas for nutritional modulation of the intestinal health of piglets.