| Part1 Effects of Mycobacterium vaccae inhalation on Mouse Asthma prevention and its relationship with VDRObjective:to investigate the Intervention effects of mouse asthma model formation by inhalation Mycobacterium vaccae and its effects on Vitamin D receptor(VDR).Methods:24 male Balb/c mices were randomLy divided into tree groups:normal control group (A), asthma model group (B), the prevention group(C).Group B and group C was sensitisated and challenged by ovalbumin to establish Asthmatic model,but group C were treated with Mycobacterium vaccae for 3 days(inhaled 22.5?g Mycobacterium vaccae dissolved in lOmL saline for once a day) before sensitisated, group A were sensitisated and treated by PBS instead. After model established,mearsured the deviation from PBS of Specific airway resistance(sRaw) by Finepointe NAM system with PBS ? 6.25mg/mL?12.5mg/mL? 25mg/mL of Mch. The murines were sacrificed to extract lung tissue and bronchoal veolar lavage fluid (BALF). Used the HE and AB-PAS staining to measure the lung inflammation and Bronchus mucus production.Counted the inflammatory cells in the BALF.Detected the concentration of IFN-y,IL-4, IL-13 in BALF with ELISA. The protein and mRNA of VDR in lung tissue were detected by immunohistochemisty and real-time fluorescence quantitative PCR.Results:The deviation from PBS of sRaw of group B[(2.49±2.54)cmH2-O×s,(3.66±0.83)cmH2O×s,(4.80±1.66)cmH2O×s]were significantly higher than group A[(0.37±0.54)cmH20×s,(1.41±0.94)cmH20×s,(2.04±0.91)cmH2O×s](P<0.05), There is no statistically significant difference in the group C[(1.07±0.80)cmH2O×s,(1.89±0.25)cmH2O×s,(2.34±0.15)cmH2O×s] and the normal group A(P>0.05). can be seen from HE staining,inflammatory infIL-tration ,mucus secretion of group B were more severe than the group A,also EosinophIL-s counts of group B(12.38±4.62) is significantly increased compared with group A(2.12×1.73) (P<0.05),but EosinophIL-s counts of group C(6.62±2.98) had been reduced compared with group B(P<0.05),inflammatory infIL-tration and mucus secretion of group C were less than group B. As the VDR immunohistochemisty, 12.5% showed positive staining for group A, 87.5%for group B,25% for group C,group B was significantly increased compared with the group A(P<0.05),whIL-e group C was decreased,when compared with group B(P<0.05).The level of VDR mRNA was increased in group B compared with group A(P<0.05),and the group C was decreased Compared with group B.In addition,as the group B compared with group A ,the concentration of IFN-y reduced and IL-4, IL-13 increased in the BALF(P<0.05); compared with the group B,the level of IFN-y in the BALF of the group C were increased(P<0.001),whIL-e IL-4, IL-13 were decreased (P<0.05).Conclusion: Inhalation of Mycobacterium vaccae may prevent the formation of OVA-sensitized mouse asthma model, it Relate to the down regulated expression of VDR, VDR involve in the pathogenesis of asthma.Part 2 Effects of inhaled inactivated-Mycobacterium phlei on CCL25,CCL20,CCR6+IL-17+??T cell, CCR9+y8T cell in a murine asthmatic modelObjective:to investigate the effects of inhaled inactivated-Mycobacterium phlei on airway hyprresponsiveness,inflamation and CCL25, CCL20,CCR6+IL-17+??T cell, CCR9+??T cell in a Murine Model.Methods:male Balb/c mices were randomLy divided into four groups:PBS-sensitized/challenged group (A), OVA-sensitized/challenged group (B),Preventive inhaled inactivated-Mycobacterium phlei group (C), Inhale inactivated-Mycobacterium phlei therapy group(D).Group A was treated with vehicle, i.e. mice with OVA-induced model of allergic asthma,group C was treated with inhaled Mycobacterium vaccae for 5 days before sensitisated, group D was treated with inhaled Mycobacterium vaccae for 5 days after sensitisated.After model established,mearsured the deviation from PBS of Specific airway resistance(sRaw) by Finepointe NAM system with PBS.6.25mg/mL?12.5mg/mL? 25mg/mL of Mch before the murine were sacrificed to extract lung tissue and bronchoalveolar lavage fluid (BALF). Measure the Histopathological of lung by HE and AB-PAS staining. Classified and count the inflammatory cells in the BALF.measured the concentration of CCL25 in BALF with ELISA.CCR9,CCR6,IL-17 were Detected dy immunohistochemisty staining. mearsured the mRNA levels of CCL25,CCL20,CCR9,CCR6,IL-17 in lung by real-time fluorescence quantitative PCR. Flow cytometry analysis was for measured the percentage of CCR9+CD3+? CCR6+CD3+? CCR9+??TCR+?CCR6+??TCR+and IL-17+CCR6+??TCR+ lymphocytes in lung.Results:The airway resistance of group B were significantly increased at 12.5mg/mL,25mg/mL concentrations of methacholine (P<0.05), There is no difference in all groups at 6.25mg/mL of methacholine (P>0.05). Group C mice show a dreased of sRaw at 12.5mg/mL,25mg/mL of methacholine compared to group B.Mucus secretion, and eosinophIL-s ratio of group Cand group D were less than group B(P<0.05). CCL25, CCL20, CCR9, CCR6, IL- 17 mRNA expression in lung tissue of Group B were significantly higher than the A group,the CCL25, CCL20, CCR9, CCR6, IL-17 expression of group C were decreased compared with group B. CCR9, IL-17 expression of group D were reduced compared with group B (p <0.05), but CCL25, CCL20 and CCR6 have no difference in group B. Immunohistochemistry: CCR6, CCR9, IL-17 expression of group B was significantly higher than that in group A, CCR6, CCR9, IL-17 expression of group C, IL-17 expression of group D was reduced compared with group B. Flow cytometry:percentage of CCR9+CD3+, CCR6+CD3+,CCR9+??TCR +, CCR6+??TCR+ and CCR6+IL-17+??TCR+lymphocytes was significantly increased in group B (P <0.05) when compared with group A, the percentage of CCR6+CD3+, CCR9+??TCR+, CCR6+??TCR+ and CCR6+IL-17+??TCR+T lymphocytes in group C ware decreased while compared with group B. the percentage of CCR6+IL-17+??TCR+T lymphocytes in group D was lower than group B.Conclusion:CCL25, CCL20 and CCR6+IL-17+??T cell, CCR9+??T cells involved in the formation of OVA sensitized mice asthma model, inhaled inactivated-Mycobacterium phlei reduce asthma airway hyperresponsiveness and airway inflammation through reduced CCL25, CCL20 and CCR6+IL-17+??T cell, CCR9+??T cells. |
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