| Objective To explore the effect and mechanism of heat-killed Clostridium butyricum(HKCB)aerosol inhalation on airway inflammation in allergic asthmatic mice and neutrophilic asthmatic mice.Methods The asthmatic mouse model was established and was either given aerosol inhalation of HKCB,intragastric HKCB or intragastric Clostridium butyricum(CB)for intervention.Additionally,the neutrophilic asthmatic mouse model was established and was received HKCB aerosol inhalation for intervention.The total and differential inflammatory cells count in bronchoalveolar lavage fluid(BALF)were calculated.The airway inflammation,goblet cell hyperplasia and mucus secretion were observed by hematoxylin and eosin(HE)and periodic Acid-Schiff(PAS)staining.Enzyme linked immunosorbent assay(ELISA)was used to detecte the levels of interferon-γ(IFN-μ),interleukin(IL)-4,IL-17A and IL-10 in BALF.The percentage of T helper cell(Th)17 and regulatory T cell(Treg)in spleen were measured by flow cytometry.The expression of microtubule associated prtein 1 light chain 3β(LC3B),Beclinl,p62,Nuclear factor-kappa B(NF-κB)p65 and Nod-like receptor pyrin domain containing 3(NLRP3)in lung tissues were detected by immunohistochemistry staining.Western blotting assay was used to detect the expression difference of LC3B,Beclinl,p62,autophagy-related gene 5(AGT5),NF-κB p65,NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 in lung tissues.Results Compared with allergic asthma control group,the airway inflammation and mucus in aerosol inhalation of HKCB group,intragastric HKCB group and intragastric CB group were decreased,especially in aerosol inhalation of HKCB group.Compared with allergic asthma control group,the level of IFN-γ in BALF and the percentage of Treg cells in spleen of mice in aerosol inhalation of HKCB group were increased(P<0.05),while the level of IL-4,the percentage of Treg cells and the ratio of Th17/Treg were decreased(P<0.05).Compared with neutrophilic asthma control group,the mice in aerosol inhalation of HKCB group showed the the airway hyper responsivess,airway inflammation and mucus were decreased(P<0.05),the level of IL-17A in the BALF was decreased(P<0.05),on the contrary,the level of IL-10 was increased(P<0.05).Moreover,the expression of LC3B,LC3BII/LC3BI,Beclinl,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1βand IL-18 in lung tissues of the mice in aerosol inhalation of HKCB group were decreased compared with relevant asthma control group(P<0.05),the expression of p62 was increased(P<0.05).Conclusion HKCB aerosol inhalation can reduce the airway inflammation and mucus secretion in allergic asthmatic mice,and the mechanism may be related to regulation the balance of Th1/Th2 and Th17/Treg,autophagy and NF-κB/NLRP3 inflammasome signaling pathway;HKCB aerosol inhalation can alleviate airway hyperresponsiveness,airway inflammation and mucus secretion in neutrophilic asthmatic mice,which might be associated with the regulation of IL-10,autophagy and NF-κB/NLRP3 inflammasome signaling pathway.Part 1 The Effect of Heat-killed Clostridium Butyricum on Airway Inflammation in A Mouse Model of Allergic AsthmaObjective To explore whether aerosol inhalation of HKCB,intragastric HKCB or intragastric live CB can alleviate airway inflammation in a mouse model with ovalbumin(OVA)induced allergic asthma.Methods Sixty-four male Balb/c mice were randomly divided into 8 groups:normal control group(NC),asthma control group(OVA),aerosol inhalation of low dose HKCB group(OVA+a.i.HKCB-L),aerosol inhalation of high dose HKCB group(OVA+a.i.HKCB-H),intragastric low dose HKCB group(OVA+i.g.HKCB-L),intragastric high dose HKCB group(OVA+i.g.HKCB-H),intragastric low dose live CB group(OVA+i.g.CB-L)and intragastric high dose live CB group(OVA+i.g.CB-H),there were 8 in each group.The asthmatic mouse model was established by intraperitoneally injected with OVA to sensitize at Od,7d and 14d,and was challenged by aerosol inhalation with 2%OVA on 21st day,30 minutes daily for 7 consecutive days.Beginning on day 0,mice were either received aerosol inhalation of HKCB,intragastric HKCB or intragastric CB(1 X 107,1 X 108CFU/d)for intervention,once daily for 14 consecutive days.The behavioral changes of mice were observed during being challenged.The mice were sacrificed within 24 hours after the last challenge,and the BALF and lung tissues were collected.The total and differential inflammatory cells count in BALF were calculated.The airway inflammation and mucus production were observed by HE staining and PAS staining.Results The mice in OVA group displayed a series of typical symptoms of acute asthma during being challenged by OVA,goblet cells proliferation,mucus hypersecretion and massive inflammatory infiltration in lung tissues.Compared with NC group,the total inflammatory cells count and the percentage of eosinophils in the BALF of the mice in OVA group were increased(P<0.05),but the percentage of macrophages in BALF was decreased(P<0.05).The airway inflammation scores and PAS scores in lung tissues of the mice in OVA group were higher than those in NC group(P<0.05).Compared with OVA group,the mice in six intervention groups showed the symptoms of acute asthma were decreased,the total inflammatory cell count and the percentage of eosinophils in the BALF were decreased(P<0.05),the airway inflammation scores and PAS scores in lung tissues were decreased(P<0.05),the percentage of macrophages in BALF was increased(P<0.05),especially the OVA+a.i.HKCB-H group.Conclusion Aerosol inhalation of HKCB,intragastric HKCB and intragastric CB can attenuate airway inflammation and mucus secretion in allergic asthmatic mice,among which aerosol inhalation of high dose HKCB is the best.Part 2 Mechanism of Heat-killed Clostridium Butyricum Aerosol Inhalation on Alleviating Airway Inflammation in Allergic Asthmatic MiceObjective To study the effects of HKCB aerosol inhalation on the balance of Thl/Th2 and Th17/Treg,autophagy-associated protein and NF-κB/NLRP3 inflammasome signaling pathway in allergic asthmatic mice.Methods The BALF,spleen and lung tissues were collected from the mice of the NC group,OVA group,OVA+a.i.HKCB-L group and OVA+a.i.HKCB-H group in the first part.Cytokines IFN-γ and IL-4 levels in BALF were detected by ELISA.Flow cytometry analysis was used to measure the percentage of Th17 and Treg cells in spleen.Immunohistochemistry was used to detect the expression and localization of LC3B,Beclinl,p62,NF-κB p65 and NLRP3 in lung tissues.Western blotting assay was used to detect the expression difference of LC3B,Beclinl,p62,AGT5,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 in lung tissues.Results Compared with NC group,the level of IFN-γ in BLAF of the mice in OVA group was decreased(P<0.05),but the level of IL-4,the percentage of Th17 cells and the ratio of Th17/Treg in spleen were increased(P<0.05).Immunohistochemistry staining showed that the expression of NF-κB p65 and NLRP3 in airway inflammatory cells of the mice in OVA group were higher that that of NC group,the expression of LC3B and Beclinl in bronchial epithelial cells and airway inflammatory cells were increased,but the expression of p62 in these cells was decreased.Coincidentally,the results of western blot showed that the expression of LC3BⅡ/LC3BⅠ,Beclin1,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 in lung tissues of the mice in OVA group were higher than that of NC group(P<0.05),but the expression of p62 was decreased(P<0.05),there was no significant difference in the expression of AGT5(P>0.05).Compared with OVA group,the level of IFN-γ in BALF and the percentage of Treg cells in spleen of the mice in OVA+a.i.HKCB-H group were increased(P<0.05),while the level of IL-4,the percentage of Treg cells and the ratio of Th17/Treg were decreased(P<0.05).The expression of LC3BⅡ/LC3BⅠ,Beclinl,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 were decreased and p62 was increased in lung tissues of the mice in OVA+a.i.HKCB-H group,compared with OVA group(P<0.05).Conclusion The imbalance of Th1/Th2 and Th17/Treg,excessive autophagy of bronchial epithelial cells and airway inflammatory cells,and activation NF-κB/NLRP3 inflammasome signaling pathway of airway inflammatory cells are involved in the pathogenesis of allergic asthmatic mice.HKCB aerosol inhalation may regulate the balance of Th1/Th2 and Th17/Treg,autophagy and NF-κB/NLRP3 inflammasome signaling pathway to alleviate airway inflammation in allergic asthmatic mice.Part 3 The Effect and Mechanism of Heat-killed Clostridium Butyricum Aerosol Inhalation on Airway Inflammation in A Mouse Model of Neutrophilic AsthmaObjective To explore the effect of HKCB aerosol inhalation on the airway hyprresponsiveness,airway inflamation,autophagy-associated protein and NF-κB/NLRP3 inflammasome signaling pathway in neutrophilic asthmatic mice.Methods Thirty-two male Balb/c mice were randomly divided into 4 groups:normal control group(NC),neutrophilic asthma group(NA),dexamethasone group(DEX)and aerosol inhalation of HKCB group(HKCB),there were 8 in each group.The neutrophilic asthmatic mouse model was established by subcutaneous injected with OVA and fluorine complete adjuvant to sensitize at Od,and was challenged by aerosol inhalation with 1%OVA on 21st day,30 minutes daily for 3 consecutive days.Starting 3 days before sensitisated,the mice in HKCB group were received HKCB aerosol inhalation for intervention,once daily for 14 consecutive days.The mice in DEX group were intraperitoneally injected with DEX at 1 hour before each challenge.The behavioral changes of mice were observed during being challenged.After the last challenge,the airway resistance was measured.The BALF was collected for calculate the total and differential inflammatory cells count.The HE staining and PAS staining were used to observe the lung inflammation and airway mucus production.ELISA was used to detecte the levels of IL-17A and IL-10 in BALF.The expression and localization of LC3B,Beclinl,p62,NF-κB p65 and NLRP3 in lung tissues were detected by Immunohistochemistry.The expression difference of LC3B,Beclinl,p62,AGT5,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 in lung tissues were detected by Western blot.Results The mice in NA group showed typical symptoms of acute asthma during being challenged.Compared with NC group,the mice in NA group showed the airway hyper responsivess was increased(P<0.05),the total inflammatory cell count,the percentage of neutrophilics and eosinophils,the level of IL-17A in the BALF were increased(P<0.05),but the percentage of macrophages and the level of IL-10 in BALF were decreased(P<0.05).Compared with NC group,the inflammation scores and PAS scores in lung tissues of the mice in NA group were increased(P<0.05).Immunohistochemistry staining showed that the expression of LC3B,Beclinl,NF-κB p65 and NLRP3 in bronchial epithelial cells and airway inflammatory cells of the mice in NA group were higher that that of NC group,but the expression of p62 was decreased.Compared with NC group,the expression of LC3BII/LC3BI,Beclinl,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 in lung tissues of the mice in NA group were increased by Western blot assay(P<0.05),the expression of p62 was decreased(P<0.05),but there was no significant difference in the expression of AGT5(P>0.05).For index mentioned above,there were no significant difference between DEX group and NA group(P>0.05).Compared with NA group,the mice in HKCB group,the symptoms of acute asthma were decreased.Compared with NA group,the airway hyper responsivess,the total inflammatory cell count,the percentage of neutrophilics and eosinophils,the level of IL-17A in the BALF,the inflammation scores and PAS scores in lung tissues of the mice in HKCB group were decreased(P<0.05),while the percentage of macrophages and the level of IL-10 in BALF were increased(P<0.05).Compared with NA group,the expression of LC3B,LC3BII/LC3BI,Beclinl,NF-κB p65,NLRP3,ASC,Pro-caspasel,Caspase1 p20,Mature-IL-1β and IL-18 in lung tissues of the mice in HKCB group were decreased(P<0.05),the expression of p62 was increased(P<0.05).Conclusion Excessive autophagy and activation NF-κB/NLRP3 inflammasome signaling pathway of bronchial epithelial cells and airway inflammatory cells are involved in the pathogenesis of neutrophilic asthmatic mice;HKCB aerosol inhalation can alleviate airway hyperresponsiveness,airway inflammation and mucus secretion in neutrophilic asthmatic mice.Its mechanism may be related to the regulation of IL-10,autophagy and NF-κB/NLRP3 inflammasome signaling pathway. |
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