The Mechanism Of Dietary Fiber Regulating Piglet Mucosal Immunity

In this study,we used citrus pectin as a typical dietary fiber,to research the effect of citrus pectin on the structure and predicted function of intestinal microflora,mucosal immunity,"microbiota-mucosal immunity" axis and intestinal epithelial cells of weaned piglets.The purpose of this study was to explore the mechanism of microbial dependent and independent of dietary fiber on intestinal mucosal immunity of piglets It provides theoretical basis for controlling intestinal health and green breeding.In chapter 2,we analyzed the effects of citrus pectin on different intestinal mucosal microbiota in piglets.Sixteen healthy Duroc × Long White × Big White weaned piglets(mean initial weight 6.77±0.92kg)with the same litter size were selected and divided into two treatment groups according to their weight in a completely randomized zone group design,with eight repetitions of one pig each.The two treatment groups were maize-soybean meal base diet+ 5%microcrystalline cellulose(Control,CON)and maize-soybean meal base diet+5%citrus pectin.The adaption was 5 days and experimental period was 21 days.Collection of jejunum,ileum,cecum,and colon mucosal microbes for 16s rDN A high-throughput sequencing to study changes in microbial lineage and function.The results showed that citrus pectin not only altered the microbial zone of the cecum and colon mucosa,but also altered the flora structure and function of the small intestine mucosa.Citrus pectin intake increased the relative abundance of some intestinal probiotics(Lactobacillus,Lactococcus,Bifidobacterium,and Faecalibacterium)and reduced the relative abundance of conditionally pathogenic bacteria such as the genera Clostridium,Fusobacterium and Helicobacter.Microbial prediction of functional metabolic pathways associated with impaired barriers,such as bacterial infestation of epithelial cells,Staphylococcus aureus infection,and lipopolysaccharide biosynthesis,were significantly reduced.It can be seen that citrus pectin not only alters the intestinal lumen microbes,but also significantly alters the mucosal microbes.And the change of position is not limited to the rear gut.Mucosal microbial species and metabolic functions are more altered due to citrus pectin than host immune related.In chapter 3,we investigate the effects of citrus pectin on piglet mucosal immunity and barrier-related genes,cytokines and microbial metabolites.The correlation network analysis and partial least squares regression analysis were used to investigate the mechanisms by which microbially mediated citrus pectin influences pig mucosal immunity.Citrus pectin significantly reduced the levels of the proinflammatory cytokines IFN-?,IL-6,IL-8,IL-12 and IL-18.Suppressed the activation of the NF-?B signaling pathway.The relative expression levels of MUC2 and TFF as well as the sIgA content were increased.Correlation network analysis showed that the effect of citrus pectin on mucosal immunity is related to its altered microorganisms and their metabolites,such as Akkermansia,Faecalibacterium,Oscillibacter,Lawsonia and butyric acid.In chapter 4,we investigate whether citrus pectin affects intestinal IL-22 secretion in piglets and achieves a protective effect on the mucosal barrier through IL-22-mediated signaling pathways.IL-22 plays an important role in the maintenance of intestinal health by increasing tightly linked protein expression,increasing intestinal epithelial regeneration,and decreasing the organisms inflammatory response.Citrus pectin increased the relative expression of AHR and IL-10 and the content of IL-22 in the jejunum mucosa of piglets,and the phosphorylation level of STAT3 protein downstream of IL-22 was elevated.Network analysis revealed that changes in AHR and IL-22 were associated with citrus pectin-induced alterations in intestinal mucosal microorganisms.Elevation of IL-22 also altered changes in its associated Wnt and Notch signaling pathway-related genes,such as the transcription factor Hesl.Moreover,citrus pectin-induced changes in IL-22 were accompanied by expression of the tight junctions protein Claudin-2,but there was a tendency for ZO-1 protein expression to decrease.In chapter 5,we investigated the direct immunomodulatory effects of citrus pectin on the intestinal epithelial cell barrier using the piglet jejunal epithelial cell line IPEC-J2 as an in vitro model.It was found that citrus pectin can reduce the damage of LPS attack on the cell barrier formed by IPEC-J2 in piglets small intestinal epithelial cell line,inhibit the LPS-induced decrease of IPEC-J2 transmembrane resistance and maintain the stability of transmembrane resistance value.Relative mRNA expression of ZO-1 in tightly linked proteins was significantly decreased after LPS attack compared to controls,whereas citrus pectin could inhibit the decrease in ZO-1 expression but had no significant effect on oclaudin and claudin-2,suggesting its selective regulatory effect.Pectin reduced the levels of proinflammatory cytokines IL-8 and IL-18 and increased the levels of anti-inflammatory cytokines IL-10,which may be related to the inhibition of the activation of downstream cytokine production by increasing the expression of negatively regulating factor Tollip.