| Spore-forming Bacillus species have been widely used as probiotics for the prevention and treatment of a broad variety of gastrointestinal disorders, due to their functioning ability to improve intestinal health and enhance the immunity of host. In recent years, researchers have paid much attention to the immunomodulating properties of lactic acid bacteria. However, little information is available on the effects of Bacillus species on the immune system of host. Therefore, the present studies were performed to investigate the adhesive properties of three Bacillus strains and also their effects on modulating immune response of Caco-2cells, RAW264.7cells, normal and immunosuppressive BALB/c mice.The main results are listed as follows:(1) This experiment was carried out to investigate the adhesive properties of different Bacillus strains, Escherichia coli K88and Salmonella choleraesuis to Caco-2cells, and the effects of Bacillus strains, E. coli K88and S. choleraesuis on cell vitality, cytokines, and the expression of gene involved in innate and adaptive immune response in Caco-2cells.The results showed that the adhesion rate of the tested Bacillus strains to Caco-2cells was less than E. coli K88and S. choleraesuis, and Bacillus strains had no significant effect on the adhesion of E. coli K88and S. choleraesuis to Caco-2cells. The cell survival rate and cell membrane integrity of Caco-2cells were significantly decreased in both E. coli K88and5. choleraesuis groups, whereas Bacillus strains did not affect cell survival rate and cell membrane integrity. Bacillus strains, E. coli K88and S. choleraesuis significantly increased the level of pro-inflammatory cytokine a proliferation inducing ligand (APRIL)(P<0.01), and had no influence on the levels of interleukin-6(IL-6) and IL-8(P>0.05). Bacillus B4, E. coli K88and S. choleraesuis significantly reduced the level of anti-inflammatory cytokine IL-10(P<0.01), whereas the level of IL-10were unaffected in both Bacillus B1and B10groups (P>0.05). Bacillus strains played a dual role in modulating expression of gene involved in innate and adaptive immune response by gene chip technology. The mRNA levels of27,10and19genes were up-regulated in Bacillus B1, B4and B10group, respectively. While the mRNA levels of3,9and5genes were significantly reduced in Bacillus B1, B4and B10group, respectively. These results manifested that the adhesion rate of the tested Bacillus strains to Caco-2cells was less than E. coli K88and5. choleraesuis, that Bacillus strains had no effect on the adhesion of E. coli K88and S. choleraesuis to Caco-2cells and no cytoxicity towards Caco-2cells, and could modulate the levels of cytokines and the expression of genes involved in innate and adaptive immune response in Caco-2cells.(2) This experiment was conducted to investigate the effects of Bacillus spores, E. coli K88and5. choleraesuis on cell vitality, cytokines, nitric oxide (NO) and enzymes involved in immune response of RAW264.7cells. The results showed that Bacillus spores had no influence on cell survival rate and cell membrane integrity, whereas cell survival rate and cell membrane integrity of RAW264.7cells were significantly reduced in both E. coli K88and S. choleraesuis groups. Furthermore, Bacillus spores, E. coli K88and S. choleraesuis significantly increased the extracellular production of NO and superoxide dismutase (SOD), and the intracellular activities of inducible nitric oxide synthase (iNOS)(P<0.01). The intracellular activities of acid phosphatase (ACP) and lactate dehydrogenase (LDH) were significantly increased by Bacillus spores (P<0.01). However, E. coli K88and S. choleraesuis significantly decreased the intracellular activities of ACP and LDH (P<0.01). Spores of Bacillus B1significantly increased intracellular SOD activities (P<0.01), whereas intracellular SOD activities was unaffected by spores of Bacillus B4and B10(P>0.05). Bacillus spores, E. coli K88and S. choleraesuis significantly increased the levels of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN-γ), IL-1β, IL-6and IL-12(P<0.01). The secretion of anti-inflammatory cytokine IL-10was significantly increased by Bacillus B1and B4spores, E. coli K88and S. choleraesuis (P<0.01), whereas spores of B10significantly decreased the production of IL-10(P<0.01). These results indicated that Bacillus spores showed no cytoxicity to RAW264.7cells, and could modulate the activities of enzymes and the levels of cytokines and NO involved in immune responses of RAW264.7cells.(3) The experiment was designed to determine whether dietary Bacillus could affect bacterial enzyme activities of intestinal digesta, intestinal, humoral and cellular immune function of mice. A total of120female BALB/c mice (4weeks old) with similar weight, were randomly assigned to group A, group B, group C and group D fed the control die (basal diet), and the basal diets containing Bacillus B1, B4and B10, respectively, with6replicates per group,5mice per replicate. The results showed that dietary Bacillus could improve intestinal health through affecting bacterial enzyme activities of caecal and small intestinal digesta. Dietary Bacillus significantly increased the production of cytokines, β-defensins (β-DF) and the activities of myeloperoxidase (MPO) and secretory phospholipase A2(sPLA2)(P<0.01), and decreased the activity of iNOS (P<0.01). However, dietary Bacillus have no influence on the level of secretory immunoglobulin A (slgA) and the activity of diamine oxidase (DAO)(P>0.05). Dietary Bacillus B1and B10significantly increased serum antibody titers and the number of plaque-forming cells (PFC)(P<0.01). However, dietary Bacillus had no significant effects on serum antibody titers and the number of PFC (P>0.05). Bacillus significantly increased respiratory burst activity of blood phagocytes, the phagocytic function of the mononuclear phagocyte system (MPS), NK cell-mediated cytotoxicity, the percentage of IFN-γ-producing CD4+and CD8+T cells (P<0.01), the percentages of CD3+and CD4+cell (P<0.05). However Bacillus had no influence on lymphocyte proliferation induced by T cell mitogen and B cell mitogen. These results suggested that dietary Bacillus could improve intestinal health through affecting bacterial enzyme activities of intestinal digesta, and enhance intestinal, humoral and cellular immune functions.(4) The study was designed to determine whether dietary Bacillus B10could affect bacterial enzyme activities of intestinal digesta, intestinal, humoral and cellular immune functions of cyclophosphamide (CP)-induced immunosuppressive mice. A total of120female BALB/c mice (4weeks old) with similar weight, were randomly assigned to group A, group B, group E and group F, with6replicates per group,5mice per replicate. To develop immunosuppressive mice, group E (CP) and group F (CP+B10) were intraperitoneally (i.p.) with a dose of CP (80mg/kg body weight) for3consecutive days. All groups were fed control diet (basal diet) during the three days. After injection of CP, group A and group E (CP) were given the control diet, whereas group B and group F (CP+B10) were fed the treatment diet (basal diet containing Bacillus B10) for60days.The results demonstrated that bacterial enzyme activities of caecal and small intestinal digesta in group E (CP) were far less than group A (the control group)(P<0.01). In addition, CP impaired intestinal, humoral and cellular immune function of BALB/c mice. Finally, dietary Bacillus B10did not improve intestinal, humoral and cellular immune function of CP-induced immunosuppressive BALB/c mice, except for a-glucosidase activity, respiratory burst activity of blood phagocytes, and phagocytic function of the MPS. These results demonstrated that dietary Bacillus B10has no obvious effects on bacterial enzyme activities of intestinal digesta, intestinal, humoral and cellular immune functions of CP-induced immunosuppressive BALB/c mice.In conclusion, Bacillus strains, showed no cytoxicity towards Caco-2and RAW264.7cells, and these strains could modulate the immune function of Caco-2, RAW264.7cells and BALB/c mice. |
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