芽孢杆菌影响Caco-2、RAW264.7细胞及小鼠免疫功能的研究
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摘要
益生菌主要通过免疫调节途径(增强肠道免疫、活化全身免疫系统及减轻炎症反应)和非免疫调节途径(改善肠道微生态平衡和肠道形态结构)改善动物健康,从而促进动物生长。目前,关于益生菌调节机体免疫功能的研究主要集中于乳酸菌,而针对目前广泛应用的饲用芽孢杆菌的研究较少。因此,有必要系统、深入地研究饲用芽孢杆菌的作用机理,特别是其提高动物免疫功能的机理。因此本实验分别以Caco-2细胞和RAW264.7细胞为体外肠上皮细胞和巨噬细胞模型,探讨芽孢杆菌B1、B4和B10的营养体和芽孢分别对肠上皮细胞和巨噬细胞免疫功能调节作用及其机理；并且通过动物实验,研究三株芽孢杆菌对BALB/c正常小鼠免疫功能调节作用以及芽孢杆菌B10对免疫抑制小鼠免疫功能的调节作用。
主要研究结果如下：
(1)芽孢杆菌对Caco-2细胞粘附、活性、细胞因子分泌及免疫相关基因表达的影响。三株芽孢杆菌对Caco-2细胞的粘附率均低于大肠杆菌K88和沙门氏菌,且均不能通过竞争、排斥和置换作用明显抑制大肠杆菌K88和沙门氏菌对Caco-2细胞的粘附(P>0.05)。三株芽孢杆菌对Caco-2细胞的存活率和膜完整性无显著影响,而大肠杆菌K88和沙门氏菌对Caco-2细胞造成严重损伤,表明了三株芽孢杆菌作为益生菌的安全性。三株芽孢杆菌、大肠杆菌和沙门氏菌显著促进Caco-2细胞分泌促炎细胞因子-增殖诱导配体(APRIL)(P<0.01),而对白介素(IL)-6和IL-8的分泌无显著影响(P>0.05)；芽孢杆菌B4、大肠杆菌K88和沙门氏菌显著降低Caco-2细胞分泌抗炎细胞因子IL-10(P<0.01),而芽孢杆菌B1和B10对其无显著影响(P>0.05)。PCR array分析结果显示,三株芽孢杆菌均可双向性调节Caco-2细胞先天免疫和获得性免疫应答相关基因的表达,可通过Toll样受体(TLR)介导的一系列信号通路影响某些基因的表达。芽孢杆菌B1、B4、B10显著上调的基因分别有27、10和19个,显著下调的基因分别有3、9和5个
(2)芽孢杆菌芽孢对RAW264.7细胞活性和免疫功能的影响。三株芽孢杆菌·芽孢均不会影响RAW264.7细胞活性,而大肠杆菌K88和沙门氏菌会对RAW264.7细胞造成严重损伤。三株芽孢杆菌芽孢均显著提高FRAW264.7细胞胞外一氧化氮(NO)含量和超氧化物岐化酶(SOD)活性、胞内酸性磷酸酶(ACP)、乳酸脱氢酶(LDH)和诱导型一氧化氮合酶(iNOS)活性(P<0.01)。大肠杆菌和沙门氏菌显著提高胞外NO含量、SOD活性和胞内iNOS活性(P<0.01),而显著降低胞内ACP和LDH活性(P<0.01)。芽孢杆菌B1芽孢显著提高胞内SOD活性(P<0.01),而芽孢杆菌B4和B10芽孢对胞内SOD活性无显著影响(P>0.05)。芽孢杆菌芽孢、大肠杆菌和沙门氏菌均显著提高巨噬细胞分泌的促炎细胞因子肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ、IL-1β、IL-6和IL-12含量,其中芽孢杆菌B10作用较强且与病原菌接近,B4最弱,而B1介于二者之间。芽孢杆菌B1和B4芽孢、大肠杆菌和沙门氏菌均显著提高IL-10含量(P<0.01),而B10芽孢显著降低IL-10含量(P<0.01)。以上结果表明,芽孢杆菌芽孢对RAW264.7细胞无毒害作用,同时能调节RAW264.7细胞免疫功能相关酶活性、NO和细胞因子分泌量。
(3)饲料中添加芽孢杆菌对BALB/c小鼠肠道细菌酶活、肠道、体液和细胞免疫功能的影响。芽孢杆菌可通过改变小鼠肠道内容物的细菌酶活性改善肠道环境。芽孢杆菌显著提高小鼠小肠细胞因子、抗菌肽β-防御素(DF)、髓过氧化物酶(MPO)和分泌型磷脂酶A2(sPLA2)活性(P<0.01),显著降低iNOS活性(P<0.01),而对分泌型免疫球蛋白A(sIgA)和二胺氧化物酶(DAO)活性无显著影响(P>0.05)；表明芽孢杆菌可增强小鼠肠道免疫功能。芽孢杆菌B1和B10显著提高小鼠的血清抗体滴度和溶血空斑数(P<0.01),芽孢杆菌B4对小鼠血清抗体滴度和溶血空斑数无显著影响(P>0.05)；表明芽孢杆菌B1和B10可增强小鼠体液免疫功能。芽孢杆菌显著提高小鼠血液吞噬细胞呼吸爆发活性、单核巨噬系统吞噬功能、NK细胞杀伤活性、脾脏Thl和Tc1细胞数(P<0.01)和显著提高CD3+、CD4+细胞数(P<0.05),而对T、B细胞增殖活性无显著影响(P>0.05)；表明芽孢杆菌可增强小鼠细胞免疫功能。以上结果表明芽孢杆菌可改善小鼠肠道环境,增强小鼠肠道、体液和细胞免疫功能。
(4)通过连续3d腹腔注射环磷酰胺(CP)建立BALB/c小鼠免疫抑制模型,研究饲料中添加芽孢杆菌B10对免疫抑制小鼠肠道细菌酶活、肠道、体液和细胞免疫功能的影响。免疫抑制剂(CP)组小鼠肠道内容物中的细菌酶活性远远低于对照组,肠道环境恶化。免疫抑制剂会显著降低小鼠肠道、体液和细胞免疫功能。芽孢杆菌B10对免疫抑制剂降低的盲肠内容物中α-葡萄糖苷酶活性、血液吞噬细胞呼吸爆发活性和单核巨噬系统吞噬功能有一定的恢复作用,而对肠道、体液和其他细胞免疫功能无显著影响(P>0.05)。
综上所述,三株芽孢杆菌对Caco-2细胞和(?)RAW264.7细胞无毒害作用,同时能调节Caco-2和RAW264.7细胞和小鼠的免疫功能。
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.
主要研究结果如下：
(1)芽孢杆菌对Caco-2细胞粘附、活性、细胞因子分泌及免疫相关基因表达的影响。三株芽孢杆菌对Caco-2细胞的粘附率均低于大肠杆菌K88和沙门氏菌,且均不能通过竞争、排斥和置换作用明显抑制大肠杆菌K88和沙门氏菌对Caco-2细胞的粘附(P>0.05)。三株芽孢杆菌对Caco-2细胞的存活率和膜完整性无显著影响,而大肠杆菌K88和沙门氏菌对Caco-2细胞造成严重损伤,表明了三株芽孢杆菌作为益生菌的安全性。三株芽孢杆菌、大肠杆菌和沙门氏菌显著促进Caco-2细胞分泌促炎细胞因子-增殖诱导配体(APRIL)(P<0.01),而对白介素(IL)-6和IL-8的分泌无显著影响(P>0.05)；芽孢杆菌B4、大肠杆菌K88和沙门氏菌显著降低Caco-2细胞分泌抗炎细胞因子IL-10(P<0.01),而芽孢杆菌B1和B10对其无显著影响(P>0.05)。PCR array分析结果显示,三株芽孢杆菌均可双向性调节Caco-2细胞先天免疫和获得性免疫应答相关基因的表达,可通过Toll样受体(TLR)介导的一系列信号通路影响某些基因的表达。芽孢杆菌B1、B4、B10显著上调的基因分别有27、10和19个,显著下调的基因分别有3、9和5个
(2)芽孢杆菌芽孢对RAW264.7细胞活性和免疫功能的影响。三株芽孢杆菌·芽孢均不会影响RAW264.7细胞活性,而大肠杆菌K88和沙门氏菌会对RAW264.7细胞造成严重损伤。三株芽孢杆菌芽孢均显著提高FRAW264.7细胞胞外一氧化氮(NO)含量和超氧化物岐化酶(SOD)活性、胞内酸性磷酸酶(ACP)、乳酸脱氢酶(LDH)和诱导型一氧化氮合酶(iNOS)活性(P<0.01)。大肠杆菌和沙门氏菌显著提高胞外NO含量、SOD活性和胞内iNOS活性(P<0.01),而显著降低胞内ACP和LDH活性(P<0.01)。芽孢杆菌B1芽孢显著提高胞内SOD活性(P<0.01),而芽孢杆菌B4和B10芽孢对胞内SOD活性无显著影响(P>0.05)。芽孢杆菌芽孢、大肠杆菌和沙门氏菌均显著提高巨噬细胞分泌的促炎细胞因子肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ、IL-1β、IL-6和IL-12含量,其中芽孢杆菌B10作用较强且与病原菌接近,B4最弱,而B1介于二者之间。芽孢杆菌B1和B4芽孢、大肠杆菌和沙门氏菌均显著提高IL-10含量(P<0.01),而B10芽孢显著降低IL-10含量(P<0.01)。以上结果表明,芽孢杆菌芽孢对RAW264.7细胞无毒害作用,同时能调节RAW264.7细胞免疫功能相关酶活性、NO和细胞因子分泌量。
(3)饲料中添加芽孢杆菌对BALB/c小鼠肠道细菌酶活、肠道、体液和细胞免疫功能的影响。芽孢杆菌可通过改变小鼠肠道内容物的细菌酶活性改善肠道环境。芽孢杆菌显著提高小鼠小肠细胞因子、抗菌肽β-防御素(DF)、髓过氧化物酶(MPO)和分泌型磷脂酶A2(sPLA2)活性(P<0.01),显著降低iNOS活性(P<0.01),而对分泌型免疫球蛋白A(sIgA)和二胺氧化物酶(DAO)活性无显著影响(P>0.05)；表明芽孢杆菌可增强小鼠肠道免疫功能。芽孢杆菌B1和B10显著提高小鼠的血清抗体滴度和溶血空斑数(P<0.01),芽孢杆菌B4对小鼠血清抗体滴度和溶血空斑数无显著影响(P>0.05)；表明芽孢杆菌B1和B10可增强小鼠体液免疫功能。芽孢杆菌显著提高小鼠血液吞噬细胞呼吸爆发活性、单核巨噬系统吞噬功能、NK细胞杀伤活性、脾脏Thl和Tc1细胞数(P<0.01)和显著提高CD3+、CD4+细胞数(P<0.05),而对T、B细胞增殖活性无显著影响(P>0.05)；表明芽孢杆菌可增强小鼠细胞免疫功能。以上结果表明芽孢杆菌可改善小鼠肠道环境,增强小鼠肠道、体液和细胞免疫功能。
(4)通过连续3d腹腔注射环磷酰胺(CP)建立BALB/c小鼠免疫抑制模型,研究饲料中添加芽孢杆菌B10对免疫抑制小鼠肠道细菌酶活、肠道、体液和细胞免疫功能的影响。免疫抑制剂(CP)组小鼠肠道内容物中的细菌酶活性远远低于对照组,肠道环境恶化。免疫抑制剂会显著降低小鼠肠道、体液和细胞免疫功能。芽孢杆菌B10对免疫抑制剂降低的盲肠内容物中α-葡萄糖苷酶活性、血液吞噬细胞呼吸爆发活性和单核巨噬系统吞噬功能有一定的恢复作用,而对肠道、体液和其他细胞免疫功能无显著影响(P>0.05)。
综上所述,三株芽孢杆菌对Caco-2细胞和(?)RAW264.7细胞无毒害作用,同时能调节Caco-2和RAW264.7细胞和小鼠的免疫功能。
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.
引文
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