Effects And The Regulatory Mechanism Of Dietary Fiber On Intestinal Barrier Function In Piglets

Dietary fiber(DF)can be alternatives of antibiotic growth promoters,zinc or copper to promote growth of piglets.However,extensive sources and physicochemical properties variations of DFs result in the difficulty in selecting appropriate DF to improve gut health of piglets,which restricted the application of DF in piglet diet.In this study,5% DFs with different physicochemical properties was added to the commonly used corn-soybean meal in piglet diet.The effects of DFs with different properties on growth performance,apparent nutrient digestibility,mucosal physiology and innate immunity,the structure and function of microbiota and microbial metabolites in the intestine of piglets were investigated.Through the combination of in vitro and in vivo experiment,the mechanism of DF on regulating the intestinal innate immunity was emphatically discussed to establish the relationship between physicochemical properties of DFs and intestinal health of piglets.Part 1: In vivo animal experimentTwenty-four piglets(11.05 ± 0.11 kg)were randomly allotted to one of four diets,i.e.a corn-soybean meal basal diet(95 %)with 5 % corn starch(CON),and diets in which 5 % corn starch was replaced by 5 % inulin(INU),5 % Pectin(PEC)or 5 % Microcrystalline Cellulose(MCC),the trial period was for 72 days.The results were as follows:1.Inclusion of 5% different DF types in piglets diet didn’t induced significant differences in growth performance,but INU group reduced pig feed conversion ratio by 0.11 and PEC group increased the body weight by 6 ~ 14.5 % of piglets than that of the CON group.MCC group significantly reduced the digestibility of DM,GE,CP and carbohydrates in piglets by 2~5% in comparison with the CON group(P < 0.05).Furthermore,INU or PEC group significantly increased the ratio of villus height to crypt depth at the jejunum(~70%,~65%)and the numbers of colonic goblet cells(~ 40%)than that in the MCC group,respectively.INU or PEC group significantly increased the ZO1 mRNA expression in the colon.MCC group significantly reduced the MUC2 and ZO-1 mRNA expression in the cecum.These results indicated that inclusion of 5% MCC have a negative impact on potential productivity and nutrient digestibility,while inclusion of 5% INU or PEC can improve the intestinal mucosal physiology.2.Alpha and beta-diversities of gut microbiome revealed distinct differences in microbial compositions between the ileum and cecum and colon,as well as between the lumen and mucosa.Coabundance was uncovered among Prevotellaceae,Lachnospiraceae and Veillonellaceae in the luminal and mucosal microbiome,while opportunistic pathogens from γ-Proteobacteria in the mucosa.Strong co-exclusion was shown between Enterobacteriaceae with Prevotellaceae-centered microbial groups in the lumen.Redundancy analysis found bile acids(37.1%)and short-chain fatty acids(SCFAs,41%)explained 51.6% of variations in the luminal microbial composition.Functional analyses demonstrated that Prevotella,Veillonellaceae,Lachnospiraceae and Ruminococcaceae were positively correlated with gene functions related to amino acids,energy,cofactors and vitamins metabolism,which are indispensable for the hosts.These results suggested site specific colonization and co-occurrence of swine gut microbiome closely relate to the microenvironment in each niche.Interactions of core gut microbiome greatly contributed to metabolism and/or immunity in the swine intestine.3.The effects of DFs with different viscosity and fermentability on the composition and function of microbiota resulted in the differences between the ileum and large intestine(LG)as well as between the lumen and the corresponding mucosa.Inclusion of 5% DFs in piglets significantly reduced the colonization of mucosal microbiota of ileum(especially some pathogens),which resulted in limited changes in the corresponding lumen.The addition of 5% different DFs achieved the goal of optimizing the microbial community of LG via increasing the proportion of different core microbial population,with increase in the abundance of Bifidobacteria in the mucosa of LG in piglets fed INU and the abundance of Prevotella in piglets fed PEC.The results of KEGG classification system showed that the number of metabolic pathways affected by the DFs in the ileum mucosa was more than that in the corresponding lumen,while the number of metabolic pathways involved in the mucosa of LG was less than that of the corresponding lumen.Based on the analysis of KEGG metabolic pathway,functional gene changes related to the inclusion of DFs in the mucosal microbiome of ileum mainly involved in the cell growth,proliferation,differentiation-related metabolic pathways.Functional gene changes related to inclusion of DFs in the luminal and mucosal microbiome of LG mainly enriched in the metabolism of amino acid and energy.Those results indicated that the effects of DFs on the microbial communities were diverse in various intestinal niches.4.This study showed that DF with different types had diverse effects on microbial metabolites: bile acids(BAs),SCFAs and the related receptors.INU increased the production of SCFAs in the LG and affected the composition of BAs in the intestine(P < 0.05).PEC reduced SCFAs in the ileum,increased LG SCFAs,and up-regulated the expression of bile acid receptors FXR and EGF in the cecum.MCC had little effect on SCFAs and BAs,whereas the expression of membrane receptor TGR5 in the ileum was upregulated.The functional properties are not yet clear.5.INU significantly down-regulated TNF-α mRNA in the ileum.Pro-inflammatory cytokine of IL-1β mRNA in the ileum(P < 0.05)and TNF-α mRNA in the cecum(P < 0.001)were also significantly reduced in piglets fed INU or PEC.Both INU or PEC groups tended to decrease the expression of TNF-α mRNA in the cecum.The expression of anti-inflammatory cytokine TGF-β mRNA was significantly up-regulated in the cecum of PEC group than that of CON and INU group.The effects of DFs on the cytokine profiles of ileum mucosa were determined using liquid-phase suspension chips.Inclusion of INU or PEC tended to decrease the IFN-γ content in the cecum.INU tended to decrease IL-8 content and increase IL-10 content in the cecum.Those results suggested that both DFs have different immunomodulatory effects in piglets.Part 2: In vitro cell experimentThe mechanism of DFs modulating innate immunity of intestinal epithelial cells was investigated by stimulating IPEC-J2 cells with INU or PEC directly,which was divided into 2 parts.1.The effects of DFs and LPS at different time points(0.5,1,2,4,12,24 h)on the cytokine profiles of IPEC-J2 cells were investigated respectively to determine stimulation/inflammatory time of fibers/LPS.Only IL-8 stimulated with LPS(LPS100 ug/ml)showed a time-dependent increase,which was significantly raised in all determined time points and greatly increased from 2 to 4 h.IL-18 was upregulated by INU from 0.5 to 12 h and reached a peak after 1 h incubation with INU.Furthermore,major pro-inflammatory cytokines(IL-6,IL-2,GM-CSF)and anti-inflammatory cytokine(IL-10)were all increased dramatically by INU from 0.5 to 24 h and achieved a peak value at 0.5~1h.Besides,the secretion of IL-18 and IL-8 also increased in IPEC-J2 cells incubated with PEC for 24 h.Those results indicated that DF has a direct immunomodulatory effect.2.To investigate whether there are interactions between INU or PEC and LPS on the cytokine response of IPEC-J2 cells,three interaction trials were conducted.Each trial included four treatments: CON(complete cell culture medium),LPS(complete cell culture medium + LPS),INU(inulin + LPS)and PEC(pectin + LPS).Under the stimulation of those four treatments,similar results were observed in the 3 trials.IL-10 content was increased after stimulation with INU or PEC than that in the CON or LPS.However,IL-8 content induced by LPS wasn’t downregulated by INU or PEC.In addition,the overall cytokine secretion was similar to that of 24 h after treatment in the first part when only fiber was added.The pro-inflammatory and anti-inflammatory cytokines were also up-regulated as results shown in the first part of this cell experiment.Those results indicated that INU or PEC had different immunomodulatory effects on IPEC-J2.No interactions between INU or PEC and LPS on the cytokine response of IPEC-J2 cells were observed.Anti-inflammatory cytokine IL-10 was upregulated by INU or PEC than that of CON.PEC had a three-fold increase in IL-10 content than that of INU,suggested that PEC have a better anti-inflammatory effect than INU in in vitro.