| Background: Asthma is one of the chronic airway diseases affecting public health worldwide seriously. In 2014, the Global Initiative for Asthma(GINA) committee updated the definition of asthma as: “asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation”[1]. The morbidity and mortality of asthma are increased year by year, which seriously affects human health, however, there are no radical cure for asthma at present. Therefore, to seek a safe and effective drug for prevention and treatment of asthma is urgently needed. Change in microbiota contributes to the development of allergies and asthma[2, 3]. Therefore, oral probiotics were tested on mouse model of asthma to restore the normal function of gut flora and reduce the incidence of asthma. The United Nations Food and Agriculture Organization/World Health Organization defined probiotics as: “live microorganisms which confer a health benefit on the host when administered in adequate amounts[4]”. Probiotics protect against allergic diseases by modifying theimmune function of host[5]. Some probiotic strains including lactobacilli and bifidobacteria have been intensively investigated as novel alternative options for the management of allergic diseases[5, 6], but the research about the benefit of Clostridium butyricum CGMCC313-1(C.butyricum) on allergy is rarely reported.Objective: We administered C.butyricum to mouse model of asthma, and determined lung function by Flexi Vent system, counted leukocyte and its subgroup populations in the bronchoalveolar lavage fluid(BALF) by optical microscope, examined the levels of mouse mast cell protease 1(m MCP-1), cytokines, OVA specific immunoglobulins and matrix metalloproteinases 9(MMP-9) by enzyme linked immunosorbent assay(ELISA), observed histopathologic changes of lung tissue by histological analysis. This study aimed to explore whether C.butyricum has preventive and therapeutic effect on airway inflammation and airway remodeling in asthmatic mice and provide theoretical basis for the prevention and treatment of asthma.Methods:(1)Mouse model of asthma: The mice received 10μg ovalbumin(OVA) plus 1.5mg alum or only equal alum intraperitoneally on days 7 and 14. Mice were then subjected to airway allergen challenges by exposure to 1%OVA aerosols or NS for 30 min from day 21 to day 28 with an INQUA NEB plus.(2)Oral C.butyricum:The animals in the preventive and treated group received 200μl C.butyricum feeding from day 1 to day 14 or from day 15 to day 28 respectively.(3)Anesthetized and tracheostomized mice were nebulized with increasing doses of acetyl-?-methylcholine chloride(Mch) in a range of 0~100 mg/ml to assess airway hyperresponsiveness(AHR).(4)Whole blood of mice was collected and serum was separated on day 29. Levels of m MCP-1, cytokines, OVA specific immunogloblins and MMP-9 in the serum were examined.(5)Bronchoalveolar lavage was performed after collection of whole blood on day 29. Leukocyte and its subgroup populations in the bronchoalveolar lavage fluid(BALF)were counted by optical microscope, and levels of m MCP-1, cytokines, and MMP-9 in the BALF were detected by ELISA.(6)Lungs of mice were isolated and pathological changes of the lungs were evaluated by histological analysis.(7)Statistical analysis.Results:(1)Mouse model of asthma was successfully established.(2)Airway resistance: mice in the positive control group showed a significant increase in the total lung resistance(basal, 1.5, 3, 12, 25, 50, 100mg/ml Mch) compared with that in the normal control group. Preventive usage of C.butyricum resulted in a significant decrease in the total lung resistance(25,50mg/ml Mch) as compared to the positive control group, C.butyricum treatment also significantly decreased the total lung resistance(12, 25, 50, 100mg/ml Mch).(3)Inflammatory cells infiltration of lungs: the mice in the positive control group showed a significant increase in the number of total inflammatory cell(P<0.05), which was due to a relative increase in the number of eosinophils, neutrophils, and macrophage as compared to the normal control group. C.butyricum significantly decreased the total inflammatory cell number(P<0.01) and relative number of eosinophils and neutrophils both in the preventive and treated mice.(4)Mast cell degranulation: the expression of m MCP-1 was significantly increased in the mice of positive control group as compared to the control group in both BALF(P<0.001) and serum(P<0.01). C.butyricum was effective in suppressing mucosal mast cell degranulation in mice of preventive(P<0.001) and treated group(P<0.01) in serum, however, the suppressive effect was not significant in BALF(P >0.05).(5)The expression of cytokines: Th2 cytokines measured includes IL-4, IL-5, IL-13 and IL-17. Th1 cytokines includes INF-γ. Compared with the normal control group, the levels of Th2 cytokines in the BALF(IL-4, IL-5, IL-13 and IL-17) and serum(IL-5, IL-17) were significantly increased in the positive control group, whiledecreased after oral C.butyricum, however, IL-13 in BALF of the preventive group was not significant different from the positive control group(P>0.05). Both INF-γ and IL-10 in the positive control group were decreased compared with the normal control group, and increased after oral C.butyricum. However, IL-10 in BALF was not significantly changed in the treated group compared with the positive control group(P>0.05).(6)The levels of OVA specific Ig E/G1: compared with the normal control group, OVA-specific Ig E/G1 significantly elevated in the OVA/OVAsensitized/challenged group(P<0.001), it decreased dramatically in the groups that received C.butyricum compared with that of the positive control group, especially for the level of Ig G1 in the treated group.(7)The expression of MMP-9: the mice of the positive control group had stronger expression of MMP-9 in serum(P<0.05) and BALF(P<0.01) compared with the normal control group. MMP-9 decreased in BALF(P<0.01) and serum(P<0.05) in the groups received C.butyricum orally compared with the positive control group.(8)Histological analysis: in the lung sections of OVA/OVA-sensitized/challenged mice, increased inflammation and collagenous connective tissue fibers, and the expression of α-smooth muscle actin(α-SMA), and Ki67 were observed as compared to the normal control group. Oral administration of C.butyricum reduced inflammation, the number of collagenous connective tissue fibers, and the expression of α-SMA and Ki67 in the airway epithelia as compared to the positive control group.Conclusion: C.butyricum significantly reduced lung resistance in the asthmatic mice. Pulmonary airway inflammation, mast cell degranulation, OVA specific immunoglobulins and the expression of MMP-9 were suppressed by oral C.butyricum. C.butyricum can also reversed the Th1/Th2 imbalances and reduce the expression of α-SMA and Ki67 in the airway epithelia. Therefore, C.butyricum has preventive andtherapeutic effect on airway inflammation and airway remodeling in asthmatic mice... |
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