Influence Of Bifidobacterium On Gut Barrier Function And Th1/th2 Cytokine In Food Allergic Mice

BACKGROUNDThe treatment of infectious disease has achieved a great success along with the development of new type of antibiotic and vaccine.However,in the past a few decades,the prevalence of allergic diseases has been significantly increased worldwide.The clinical symptoms of food allergy(FA),one of the common allergic diseases,vary from mild discomfort such as vomiting,abdominal pain and diarrhea to life-threatening allergic shock.The initiation of sensitization in human intestinal remains unclear.Probably the concurrent exposure of intestine to infectious agents and food antigens produces a synergistic action,so that the resting T-cells differentiate into antigen-specific Th2 cells;intestinal immune tolerance is disturbed and lead to food allergy.Fuller reported that intake of probiotics such as Lactobacillus and Bifidobacterium can be a prevention remedy of allergic diseases.Recently,Herz et al. reported that probiotics had been demonstrated the role of anti-allergic in human and mice models.Attention has therefore been focused on probiotics therapeutics. Probiotics may be involved in regulating immune imbalance of the intestine;its mechanism still needed to be understood in further study.Staphylococcal enterotoxin B(SEB) which is commonly found in the human intestine has been proved to be related with allergic diseases.It is reported that SEB has adjuvant effect and to be used in food allergy animal model development with food antigens such as ovalbumin(OVA).In this study,we sensitized mice to OVA with SEB as an adjuvant,and examined the impact of Bifidobacterium on improving gut barrier function,intestinal flora and immune function in food allergic mice. Smecta,a common used protectant of intestina mucosa,was employed together with Bifidobacteria in the present study to investigate whether there was synergistic effect on gut barrier function of the twos.AIMSStaphylococcal enterotoxin B(SEB) and ovalbumin(OVA) were used together on BALB/c to construct a new mice model of food allergy to investigate the gut barrier function of ovalbumin-induced allergic mice and analyze whether there was bacteria translocation or not.The numbers of CD4~+CD25~+ regulatory T cells in the spleens of FA mice were tested in order to find the relationship between CD4~+CD25~+ regulatory T cells and food allergy.And Bifidobacterium was employed in the study to treat the FA mice,so that the impact of Bifidobacterium on improving gut barrier function,intestinal flora and immune function in food allergic mice could be examined.METHODSForty BALB/c mice fed with ovalbumin(OVA)-free diet were randomly divided into four groups(10 mice per group):A group of mice were sensitized to OVA mixed with Staphylococcal enterotoxin B(SEB) as an adjuvant;other three groups of mice were treated with normal saline(NS),or SEB or OVA.All the mice were challenged by intragastric gavage with OVA on the 7th and 14th days.Then the experiment control group,intervention by Bifidobacterium group,intervention by smcta group and intervention by both Bifidobacterium and smecta group were set on the base of SEB+OVA treatment group.Diarrhea,the signal of food allergy,was also noted by direct observation of the colon and cecum;the liquid stool observed following OVA challenge-induced diarrhea in the sensitized mice contrasts with the solid pellets seen in the distal colon of other mice in control groups.Intestinal microbial flora in the feces was detected by culturing the feces.1.The level of total IgE in blood serum was analyzed by ELISA.2.The intestinal microbial flora was detected by culturing the feces.3.The levels of IL-4,IL-5,IL-10,IL-13 and INF-γwere tested by ELISA.4.The level of diamine oxidase in blood serum was measured by spectrophotometer.5.The mesenteric lymph nodes,homogenated tissues of the lives,lungs and kidneys were cultured to determine bacterial translocation.6.The numbers of CD4~+CD25~+ regulatory T cells(CD4~+CD25~+ Treg cells) in spleens were tested by flow cytometry. Data were expressed as the means±SD.All statistical analysis was performed using SPSS statistical version 13.0.Differences between groups were determined with one-way ANOVA;The rates of bacterial translocation were determined with Chi-square test,a P value of less than 0.05 was considered to be statistically significant.RESULTS1.Mice treated with both SEB and OVA showed higher titers of IgE(0.634±0.220) in the sera compared with naive control group(P=0.044).The high level of IgE indicates successful establishment of murine food allergy model.After the treatment of Bifidobacterium,serum total IgE(0.440±0.126) in SEB+OVA treatment group decreased significantly(P=0.035).2.The serum DAO level of SEB and OVA treatment group was significantly higher than that of naive control group(0.385±0.186 vs 0.145±0.094,P=0.000).The high level of DAO in the sera indicated the high permeability of allergic mice.After treatment with Bifidobacterium,serum DAO level(0.157±0.071) decreased significantly(P=0.035).There was no significant difference between Bifidobacteria+ smecta group and Bifidobacteria group of the serum DAO level.3.CD4~+CD25~+ Treg cells in suspension of spleen mononuclear cell of mice were accounted and presented as percentage of mononuclear cells.The number of CD4~+CD25~+Treg cells was much less in SEB+OVA treatment group than that in naive control group(4.216%±0.439%vs 5.969%±0.469%,P=0.000).After treatment by Bifidobacteria,the CD4~+CD25~+ Treg cell number in SEB+OVA treatment group increased significantly(5.778%±0.773%vs 4.216%±0.439%, P=0.000).There was no significant difference between Bifidobacteria+ smecta group and Bifidobacteria group of the number of CD4~+CD25~+Treg cells.4.Th1/Th2The level of IL-4,a typical Th2 cytokine,was slightly higher in mice exposed to SEB or OVA alone but increased significantly in mice exposed to both SEB and OVA compared with that of naive control group(69.980±9.103 vs 45.877±2.804, P=0.000).After treatment by Bifidobacterium,the IL-4 level(51.314±3.785) decreased significantly in allergic mice(P=0.000).In contrast,the typical Th1 cytokine,IFN-γ,decreased most dramatically in mice treated with both SEB and OVA(204.018±32.031 vs 133.875±33.822,P=0.000),while it decreased slightly in mice treated with SEB or OVA alone.After treatment by Bifidobacterium,the IFN-γlevel(194.281±12.144) increased significantly(P=0.000).The ratio of IL-4/INF-γ, being representative of Th2/Th1 cytokine,was higher in allergic mice than naive control group.After treatment by Bifidobacterium,the ratio of IL-4/INF-γdecreased.The levels of IL-5,IL13,the other two Th2 cytokines,were also higher in allergic mice and decreased after treatment by Bifidobacterium.However the level of IL-10 increased after treatment by Bifidobacterium.It confirms that Th2 polarization has been induced,and eventually induces food allergy.However Bifidobacterium can suppress the skewed Th2 reaction.5.The numbers of conditioned pathogen Escherichia coli(P=0.000) and Bacteroides (P=0.000) were significantly higher in SEB+OVA treatment group than those in naive control group,but the numbers of probiotics Bifidobacterium(P=0.000) and lactobacilli(P=0.000) were significantly lower in SEB+ OVA treatment group. After the treatment with Bifidobacteria,the numbers of Escherichia coli(P=0.000) and Bacteroides(P=0.000) decreased in SEB+OVA treatment group.In contrast,the numbers of Bifidobacterium(P=0.000) and lactobacilli(P=0.000) increased significantly.The numbers of conditioned pathogen Escherichia coli and Bacteroides were higher than those of the naive control group,in contrast the numbers of probiotics Bifidobacteria and lactobacilli were lower.After treatment with Bifidobacteria,the Escherichia coli and Bacteroides decreased in food allergic mice,but Bifidobacteria and lactobacilli increased.Probiotics can suppress the growth of conditional pathogenic bacteria and modulate the gut flora.6.Bacteria translocation was more frequently noted in SEB+OVA treatment group than in naive groups(37.5%vs 7.5%,P=0.001).After treatment with smecta or Bifidobacterium,the rate of bacterial translocation in SEB+OVA treatment group decreased(P=0.001);There was no significant difference between Bifidobacterium+ smecta group and Bifidobacterium group.The present study confirms that there is bacteria translocation in allergic mice.And Bifidobacterium can cure it.CONCLUSIONS1.SEB and OVA were used together to construct a food allergic mice model successfully.2.There was intestinal dysbacteria in allergic mice.The numbers of conditional pathogenic bacterias(Escherichia coli and Bacteroides) were significantly higher while the numbers of probiotics(Lactobacillus and Bifidobacterium) were significantly lower in FA mice.The high level of DAO in blood serum indicated high intestinal permeability in FA animals.These results manifested the gut barrier dysfunction of FA mice.The phenomenon of bacteria translocation was certified in FA mice.3.CD4~+CD25~+ Treg cells may play an important role in food allergy.4.Bifidobacterium could coordinate the immunologic function,cure the alteration of intestinal flora and protect the intestinal tract mucosa barrier function.