| In the study, we explored the monosaccharides and immunomodulatory activity of unfermented Yupingfeng polysaccharides (UYP) and fermented Yupingfeng polysaccharides synbiotic (FYP) obtained using Rhizopus oligosporus SH via GC-MS, cell assays in vitro and weaning rex rabbit assay in vivo, and the effects of UYP and FYP on intestinal flora and intestinal barrier of weaning rex rabbit were also evaluated. To explore the in vitro immune activity of UYP and FYP, different concentrations of UYP or FYP and normal or LPS-induced immune cells from mices were incubated; Forty-five, weaning rex rabbits were randomly divided into three groups after seven days of pre-feeding. During the entire experimental period of four weeks, rex rabbits were fed with customized fodder with saline, UYP and FYP (1 g/kg) in the normal control (NC) group, UYP group and FYP group, respectively. Results were as follows:1. The monosaccharides of UYP and FYP are primarily consist of fructose and glucopyranoside by GC-MS. The glucopyranoside significantly increased in FYP than that in UYP(P<0.01).2. MTT assay showed that UYP. FYP and LPS significantly increased the proliferations of splenocyte, thymocyte and macrophage (P<0.01), but the activities of UYP and FYP were significant lower than LPS (P<0.01). Compared with UYP, FYP significantly enhanced the proliferation of peritoneal macrophages (P<0.01), and the concentrations of FYP were 0.100,0.200,0.400 and 3.200 mg/mL, the proliferations of lymphocytes were significantly stronger than UYP (P<0.05 or P<0.01). In addition, UYP and FYP significantly reduced the proliferations of LPS-induced lymphocytes (P<0.01). The proliferation of splenocyte in FYP treated with 0.200 mg/mL polysaccharide was not significantly different (P>0.05) than that of in normal splenocyte, while the concentration of polysaccharide was at 3.200 mg/mL in UYP group. When the concentration of polysaccharide was 0.800 mg/mL, the thymocyte proliferation in UYP and FYP was not significantly different than that in normal thymocyte (P>0.05), and the proliferations of FYP-treated LPS-induced thymocyte was closer to normal thymocyte.3. The neutral red test showed that UYP, FYP and LPS significantly promoted murine macrophage phagocytosis and synthetic NO (P<0.01). The phagocytic activities and the amount of NO secretion were higher in FYP than in UYP (P<0.01). However, phagocytosis and NO production in FYP and UYP were lower than those in LPS (P<0.01).4. Effect of UYP and FYP on genes expression in vitro of immune cells in mice by rtPCR, the results showed that LPS significantly upregulated the expression of inflammatory cytokines, TLR4, NF-?B and iNOS (P<0.01), the mRNA levels of TLR4 and NF-?B in splenocytes were significantly improved in UYP (P<0.05), and the expression of iNOS (P<0.01) in macrophages, TLR4 (P<0.01) in splenocytes and NF-?B (P<0.05) in lymphocytes were strongly upregulated in FYP. Simultaneously, the mRNA concentrations of above-described genes were higher in FYP than UYP, but was significant lower than LPS (P<0.01). In addition, UYP and FYP significantly inhibited the expressions of LPS-induced inflammatory cytokines, TLR4, NF-?B (P<0.01) and iNOS (P<0.05); Compared with UYP, FYP significantly reduced the expressions of NF-?B and TLR4 in LPS-induced macrophage and TLR4 in LPS-induced thymocytes (P<0.01).5. Rex rabbit tests in vivo showed that UYP (P<0.05) and FYP (P<0.01) significantly increased the average daily gain of rex rabbits, and the ADG in the FYP was more higher than that of in UYP (P<0.01). UYP and FYP significantly improved the spleen index (P<0.01), and the activity of FYP was significantly higher than that of UYP (P<0.05).6. rtPCR analysis showed that UYP and FYP could promote the expressions of cytokines in spleen of rex rabbit, FYP was more effective regulation the mRNA of IL-2, TNF-? and IL-10 (P<0.0 5). ELISA assay showed that UYP and FYP increased the protein concentrations of IL-2, IL-4, IL-12, TNF-? and IFN-? in serum; Compared with UYP, FYP more effectively elevated the protein concentrations of cytokines in serum, wherein the levels of IL-2, IL-6, TNF-? and IL-10 were significantly increased (P<0.05 or P<0.01).7. rtPCR analysis showed that UYP significantly increased the mRNA expressions of TLR2 in jejunum (P<0.05), ileum and spleen (P<0.01) and TLR4 in ileum, cecum and spleen (P<0.01) of rex rabbit; FYP significantly promoted the mRNA expressions of TLR2 in jejunum, ileum and spleen and TLR4 in jejunum, ileum, cecum and spleen (P<0.01). Compared with UYP, FYP significantly up-regulated TLR2 mRNA expressions in jejunum (P<0.05) and ileum (P<0.01) and TLR4 in jejunum (P<0.05) and spleen (P<0.01).8. The structures of intestinal microflora in rex rabbit were analyzed by PCR-DGGE, the results indicated that the DGGE patterns found in feces from UYP group and FYP group seemed to be more similar than those from NC group in jejunum and ileum. The dendrograms from the total samples illustrated that the samples in FYP group and UYP group were clustered into a large branch in jejunum and ileum. Meanwhile, PCA1 distinguished UYP group and FYP group from NC group in jejunum and ileum. Compared with NC group, shannon index, evenness and richness were significantly higher in UYP group and FYP group in jejunum and ileum (P<0.05 or P<0.01). Compared with UYP. FYP more effectively improved the shannon index, evenness and richness in jejunum (P<0.05) and ileum.9. The abundance of intestinal flora in rex rabbit was analyzed by rtPCR, the results indicated that UYP and FYP significantly improved the abundance of total bacteria, Bacteroides, Firmicutes, P. prophyromonas, B. fibrisolvens, Clostridium cluster IV and Clostridium cluster XIVa (P<0.01) in jejunum and ileum, and the number of lactobacillus (P<0.05) in jejunum and cecum, and significantly reduced the number of Enterococcus spp. in jejunum, ileum and cecum (P<0.01 or P<0.05) and Streptococcus spp. (P<0.01) in ileum and cecum. Compared with UYP, FYP significantly increased the number of total bacteria, Firmicutes, B. fibrisolvens in ileum (P<0.01), F. succinogenes in jejunum (P<0.05) and Bifidobacterium in cecum (P<0.05) and reduced the abundance of Enterobacteriaceae and Enterococcus spp. in ileum and Enterococcus spp. in jejunum (P<0.01).10. rtPCR analysis showed that FYP significantly upregulated the mRNA expressions of ?-defensins, EGF (P<0.05) and TFF (P<0.01) in jejunum and ZO-1, claudin, (3-defensins, TFF (P<0.05), PIGR and EGF (P<0.01) in ileum. Compared with UYP, FYP strongly upregulated the expression of above genes, wherein the expression of EGF (P<0.05) in jejunum and ZO-1, claudin (P<0.05), PIGR and EGF (P<0.01) in ileum were significantly increased.In conclusion, this study elucidated the bidirectional immunomodulatory activity of UYP and FYP in vitro. UYP and FYP could enhance immunity, improve intestinal microflora and intestinal barrier in jejunum and ileum of rex rabbit. The beneficial effects of FYP are superior over that of UYP. |
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