| Five eperiements were conducted to investigate the attenuation of intestinal inflammation by individual supplementation of xylanase and probiotics and the related mechanisms.Exp.l was conducted to investigate the effects of xylanase supplementation to a wheat-based diet on the growth performance and gut health of broilers challenged by Clostridium perfringens. In a 2 x 2 factorial arrangement,336 one-day-old male broiler chicks (Ross 308) were assigned to 4 groups. C. perfringens challenge significantly decreased feed conversion efficiency, which was increased by xylanase addition. Pathogen infection reduced some indices such as nutrient digestibility, the mRNA expression of nutrient transporters and digestive enzyme activities, which was improved by xylanase supplementation. In the challenged birds, xylanase addition tended to decrease intestinal lesion score and significantly suppressed the plasma endotoxin levels. The supplementation of xylanase improved the intestinal morphology and the mRNA expression of tight junctions, and decreased ileal apoptosis and duodenal IL-1β mRNA expression. It also tended to reduce the richness and diversity of ileal microflora. In conclusion, xylanase supplementation of a wheat-based diet alleviated the intestinal lesion, improved intestinal barrier function, modulated the activities of digestive enzymes and the mRNA expression of nutrient transporters, optimized the ileal microbial structure, and thereby enhanced the nutrient digestibility and growth performance of birds challenged by C. perfringens.In Exp.2, an in vitro study was conducted to investigate the inflammatory responses to C. perfringens type A and a-toxin in primary intestinal epithelial cells of chicken embryos and the related mechnisms. C. perfringens as well as commercially available peptidoglycan (PGN) and a-toxin challenge up-regulated mRNA expression of IL-6, IL-8 and iNOS with a dosage-dependent manner at 3 h post infection (p.i.). Time-course effects of three stimulators at high concentration were further examined. C. perfringens infection elevated IL-6, IL-8 andiNOS levels from 1 h to 9 h p.i., while PGN treatment increased IL-6 and IL-8 expression at 1 h and 3 h p.i.. Bacterial and PGN treatments induced NOD1 expression at 6 h p.i. and only bacterial infection boosted NF-κB p65 expression at 6 h and 9 h p.i. a-Toxin treatment up-regulated IL-6 and IL-8 expression along infection, as well as iNOS, TNF-a and NF-κB p65 expression at later hours p.i.. In conclusion, both C. perfringens and a-toxin challenge induced intense cytokine expression associated with NF-κB activation in chicken intestinal epithelial cells. The receptors for the recognition of PGN component of C. perfringens need further investigation.Exp.3 was carried out to study the effects of Lactobacillus acidophilus and Lactobacillus fermentum on the growth, biofilm formation and a-toxin secretion of C. perfringens as well as the alleviation of inflammation by both lactobacillus bacteria in C. perfringens-infected chicken primary intestinal epithelial cells. When co-cultured with lactobacillus bacteria, C. perfringens was significantly decreased and its biofilm formation was suppressed, which was due to the lower pH and antibacterial secretions produced by lactobacillus bacteria. Both lactobacillus bacteria reduced the a-toxin production without affecting the number of C. perfringens, and degraded the established a-toxin. C. perfringens cultured in a lower pH (5.2) MRS broth secreted less a-toxin than that in normal pH (6.2) broth. L. acidophilus suppressed the attachment of C. perfringens to cells, but L. fermentum did not show the similar effect. The pretreatment of both lactobacillus bacteria attenuated the upregulation of cytotoxicity and the mRNA expression of proinflammatory cytokines (IL-6, IL-8, iNOS, TNF-α and IL-1β), NF-κB p65, NOD1 and TLR2.2 induced by C. perfringens infection. In conclusion, both lactobacillus bacteria inhibited the growth and biofilm formation of C. perfringens by competing the substrate, lowing the pH and producing antibacterial substance. The suppression of a-toxin secretion by lactobacillus bacteria was associated with lowing pH. Lactobacillus bacteria alleviated the C. perfringens-induced inflammatory responses by downregulating receptors and NF-κB activation, and thereby protected the intestinal epithelial cells of chicken embryos.In Exp4, transcription profiling analysis of gene expression was analyzed in immature human fetal intestinal epithelial cells (H4 cells) pretreated with conditioned media from Bifidobacterium infantis (BCM) or Lactobacillus acidophilus (LCM) prior to IL-1β stimulation. Compared with control media, the two probiotic-conditioned media (PCM) treatments altered the expression of hundreds of genes involved in the immune response, apoptosis and cell survival, cell adhesion, the cell cycle, development and angiogenesis. In IL-1β-stimulated cells, PCM treatment decreased the upregulation of genes in the NF-κB activation pathway and downregulated genes associated with ECM remodeling. Compared with LCM, BCM showed more significant modulatory effects on ECM remodeling, reflected by a lower p value. IL-6 and IL-8 production was significantly reduced in IL-1β-stimulated cells pretreated with PCM, which was consistent with their altered gene expression. Western blot analysis showed that compared with IL-1β stimulation alone, PCM treatment attenuated the decrease of cytoplasmic IλBα and NF-κB p65 levels as well as the increase of nuclear NF-κB p65 levels in the stimulated cells.In conclusion,PCM treatment exerted anti-inflammatory effects in immature human fetal enterocytes primarily by modulating genes in the NF-κB signaling and ECM remodeling pathways. Additionally, some components of these signaling pathways,particularly the ECM remodeling pathway,were more profoundly affected by BCM than LCM.Exp5. was designed to determine the protective effects of PCM from B. infantis and L. acidophilus on IL-1β-induced intestinal barrier compromise. BCM and LCM induced a dose-dependent increase in Caco-2 TER after 4 and 24 hours of incubation. Preincubation with BCM and LCM for 4 hours significantly prevented the decrease of Caco-2 TER induced by 24 hours of stimulation with 10 ng/mL IL-1β. BCM and LCM decreased paracellular permeability in both stimulated and unstimulated Caco-2 monolayers. IL-1β stimulation decreased occludin expression and increased claudin-1 expression in Caco-2 cells, which was prevented in cells treated with BCM or LCM. The changes of claudin-1 expression in H4 cells were similar to Caco-2 cells in response to PCM treatment and IL-1β stimulation,but a similar response in occludin was not demonstrated. The IL-1β-induced nuclear translocation of NF-κB p65 in Caco-2 cells was prevented by pretreatment with both PCM.In conclusion, BCM and LCM protected the intestinal barrier against IL-1β stimulation by normalizing the protein expression of occludin and claudin-1 and preventing IL-1β-induced NF-κB activation in Caco-2 cells, which may be partly responsible for the preservation of intestinal permeability.In conclusion, xylanase supplementation to a wheat-based diet could improve the intestinal digestion, barrier function, immune function and microflora of birds infected by C. perfringens, and thereby decreased intestinal lesion and enhanced the gut health and growth performance. Incubating chicken primary intestinal epithelial cells with C. perfringens and a-toxin induced strong inflammatory responses by activating NF-κB. However, both L. acidophilus and L. fermentum inhibited the growth and a-toxin secretion of C. perfringens and attenuated the pathogen-induced inflammation. The conditioned media from B. infantis and L. acidophilus exerted anti-inflammatory effects in immature human fetal enterocytes primarily by modulating genes in the NF-κB signaling and ECM remodeling pathways and protected intestinal epithelial barrier function, which was helpful to alleviate the intestinal lesion in necrotizing enterocolitis. |
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