Effects And Mechanisms Of Mycobacterium Vaccae Nebulization On Asthmatic Model And Respiratory Syncytial Virus Infection In Mice

OBJECTIVE:To explore the effects and mechanisms of Mycobacterium vaccae(M.vaccae)nebulization on prevention and treatment of respiratory syncytial virus(RSV)infection and asthmatic model in mice.METHODS:To establish the asthmatic models with OVA sensitization and RSV(respiratory syncytial virus infection)infected models with RSV nasal drop.M.vaccae were nebulized or intraperitoneal injected.Mice in asthmatic prevention groups were nebulized or intraperitoneal injected with M.vaccae one month before asthmatic models were established,they were executed after asthmatic models were established;Mice in asthmatic treatment groups were nebulized with M.vaccae after asthmatic models were established,they were executed on treat days 3and 5 respectively;Mice in RSV prevention groups were investigated through time-effect relationship and dose-effect,M.vaccae were nebulized or intraperitoneal injected one month or one week before 106PFU RSV infected models in time-effect relationsip were established.22.5μg or 22.5μg*3 M.vaccae were nebulized one week before 106PFU or 105PFU RSV infected models in dose-effect relationship were established,the mice were executed 4 days after RSV infected;Mice in RSV treatment groups were nebulized with M.vaccae the day after RSV infected,they were executed 5 days after nebulization.The whole groups were controlled with normal and model groups.lung function were measured with non-invasive lung function machine.the lung tissue and bronchoalveolar lavage fluid(BALF)were harvested.In asthmatic groups,airway inflammation were observed by pathological HE staining and PAS staining,concentration of cytokine interlukin-9 and OVA-IgE in BALF were detected with ELISA,the percentage of lungγδTCR+CD3+,IL-9+CD3+,IL-10+CD3+,IL-9+γδTCR+,IL-10γ+γδTCR+lymphocytes were detected by flow cytometry,lung IL-9R mRNA level were evaluated by fluorescent quantitation polymerase chain reaction(PCR),In the asthmatic prevention groups,the expression of lung BDNF,NF09 and Acetylcholine were detected by immunohistochemisty and NGF mRNA were evaluated by fluorescent quantitation PCR either.Besides the above methods,in the RSV groups,the condition of RSV infecton in the lung were observed by electron microscope and lung immunofluorescence,the level of IL-9R,PU.1,PD-L2,JAK1,STAT1,STAT5A,RSV,IFN-y,TLR7 and TLR8 mRNA were detected by fluorescent quantitation PCR,the expression of EGFR,NF09 and Acetylcholine in the lung were detected by immunohistochemisty.RESULTS:Compared with asthmatic control group,airway inflammation relieved,the concentration of IL-9 in BALF and the percentage of lung IL-9+CD3+、IL-9+γδ8TCR+lymphocytes decreased,the percentage of lung IL-10+CD3+、IL-10+γδTCR+lymphocytes increased in asthmatic prevention and treatment groups.Simultaneously,the airway responsiveness were improved,the concentration of OVA-IgE,the level of IL-9R、NGF mRNA and the expression of BDNF,NF09,Acetylcholine decreased obviously in asthmatic prevention groups.Compared with RSV infection controls,the quantity of RSV decreased obviously in electron microscope,the immunofluorescence of lung anti-RSV attenuated,the expression of RSV mRNA,the level of IL-9 decreased and the level of IL-10,the expression of JAK1,STAT1,STAT 5 A mRNA in IL-9-JAK-STAT signal transduction pathway and the level of TLR7 and TLR8 mRNA increased notably,the level of PU.1mRNA decreased sharply,the expression of NF09,Acetylcholine and EGFR were decreased obviously in the RSV prevention and teatment groups.The group nebulized with low dose and short time of M.vaccae showed preferable effects.In RSV treatment group,the percentage of γδTCR+lymphocytes increased sharply while IL-9+γδTcells decreased obviously also.CONCLUSIONS:The experimental dose of M.vaccae nebulization is contributed to the prevention and treatment of asthma and RSV infection,the mechanisms include the regulation of Th9 cells,IL-9-JAK-STAT signal transduction pathway,neuromechanism,TLR7,TLR8,PU.1 and EGFR.Part I The effects and mechanisms of Mycobacterium vaccae nebulization on bronchial asthma in miceSection I The effects of Mycobacterium vaccae nebulization on the prevention of bronchial asthma and Th9 cells in miceOBJECTIVE:To investigate the effects of Mycobacterium vaccae(M.vaccae)nebulization on the prevention of asthmatic model and Th9 cells in mice.METHODS:Twenty-four Balb/c mice were randomized as normal control group(normal control),asthma control group(asthma control),nebulization prevention group(Neb.group)and intraperitoneal injection group(i.p.group),M.vaccae were nebulized in Neb.group and intraperitioneal injected in i.p.group once a day for 5 days one month before asthmatic models were established.Normal control and asthma control groups were nebulized with PBS.the asthmatic models were established with OVA sensitization.All mice were executed after airway responsiveness were measured by non-invasive lung function machine,the lung tissue and bronchoalveolar lavage fluid(BALF)were harvested.airway inflammation were measured with pathological HE staining and PBS staining.the concentration of IL-9 and OVA-IgE in BALF were detected by ELISA,the percentage of lung γδTCR+CD3+,IL-9+CD3+,IL-10+CD3+,IL-9+γδTCR+,IL-10+γδTCR+lymphocytes were detected by flow cytometry,lung IL-9R mRNA level were evaluated by fluorescent quantitation polymerase chain reaction(PCR).RESULTS:Compared with normal control group,asthma control group showed apparent airway inflammation,airway hyperreponsiveness,the increased concentration of IL-9 and OVA-IgE in BALF,the increased percentage ofγδTCR+CD3+,IL-9+CD3+,IL-9+γδTCR+lymphocytes,and the decreased IL-10+CD3+lymphocytes obviously.Compared with asthma control group,the airway responsiveness alleviated,the airway inflammation relieved,the concentration of IL-9 and OVA-IgE、the percentage ofγδTCR+CD3+,IL-9+CD3+,IL-9+γδTCR+lymphocytes and the level of IL-9R mRNA decreased obviously in asthmatic prevention group.CONCLUSIONS:Both nebulizaiton and intraperitoneal injection of M.vaccae are contributed to the prevention of asthma.the mechanisms is likely to increase the secretion of cytokine IL-10 and to decrease the production of cytokine IL-9 by γδT cells.which presented a IL-10 dominance and maybe a member of Th9 cells family.Section II The neuromechanism of Mycobacterium vaccae nebulization on the prevention of bronchial asthma in miceOBJECTIVE:To explore the neuromechanism of Mycobacterium vaccae(M.vaccae)nebulization on the prevention of bronchial asthma in mice.METHODS:Twenty-four Balb/c mice were randomized as normal control group(normal control),asthma control group(asthma control),nebulization prevention group(Neb.group)and intraperitoneal injection group(i.p.group),M.vaccae were nebulized in Neb.group and intraperitioneal injected in i.p.group once a day for 5 days one month before asthmatic models were established.Normal control and asthma control groups were nebulized with PBS.the asthmatic models were established with OVA sensitization.after that,the expression of BDNF、NF09、Acetylcholine were detected with immunohistochemisty,the level of NGF mRNA were evaluated by fluorescent quantitation PCR.RESULTS:Compared with normal control group,the expression of BDNF、NF09 and Acetylcholine in the lung were apparent,the level of NGF mRNA were increased in asthma control group.Compared with asthma control group,the expression of BDNF、NF09、Acetylcholine in the lung and the level of NGF mRNA were decreased in Neb.group,while the expression of BDNF and Acetylcholine decreased in i.p.group.CONCLUSIONS:Mycobacterium vaccae nebulization can protect against asthma by downregulation the expression of BDNF、NF09、Acetylcholine and NGF,It attributed to the regulation of neuromechanism and anti-cholinergic procedure.Section Ⅲ The effects of Mycobacterium vaccae nebulization on the treatment of bronchial asthma and Th9 cells in miceOBJECTIVE:To observed the effects of Mycobacterium vaccae(M.vaccae)nebulization on the treatment of asthmatic model and Th9 cells in mice.METHODS:Thirty-six Balb/c mice were randomized as normal control group(Normal,A),before treatment group(Before treatment,BO),M.vaccae treat group(treat 3 days,treat 5 days,(B3,B5))and treat control group(treat 3 days control,treat 5 days control,(C3,C5)).Mice were sensitised and challenged with OVA to establish a murine asthmatic model.Group B3,B5 were nebulized with M.vaccae for 3 days and 5 days respectively.Group C3,C5 were nebulized with PBS for 3 days and 5 days respectively,All mice were executed after airway responsiveness were measured by non-invasive lung function machine,the lung tissue and bronchoalveolar lavage fluid(BALF)were harvested.airway inflammation were measured with pathological HE staining and PBS staining.the concentration of IL-9 and OVA-IgE in BALF were detected by ELISA,the percentage of lung γδTCR+CD3+,IL-9+CD3+,IL-10+CD3+,IL-9+γδTCR+,IL-10+γδTCR+lymphocytes were detected by flow cytometry,lung IL-9R mRNA level were evaluated by fluorescent quantitation polymerase chain reaction(PCR).RESULTS:Compared with group A,group BO showed apparent airway inflammation,airway hyperreponsiveness,the increased concentration of IL-9 and OVA-IgE,the increased percentage of γδTCR+CD3+、IL-9+CD3+、IL-9+y8TCR+lymphocytes,the decreased percentage of IL-10+CD3+lymphocytes,and the increased level of IL-9R mRNA.The percentage of IL-9+CD3+、IL-9+γδ8TCR+lymphocytes decreased,and the percentage of IL-10+CD3+lymphocytes increased in group B3 and group B5 compared with group B0,The airway inflammation were relieved in group B3 but not in group B5 compared with group BO.The above index were almost the same in group C3 and C5 compared with group BO.The level of IL-9R mRNA in group B3 and B5 were deceased obviously compared with group C3 and C5 respectively.CONCLUSIONS:Mycobacterium vaccae nebulization can alleviate airway inflammation in asthmatic model,both Th9 cells and y8T cells participated into the procedure.The mechanism is likely to induce the secretion of cytokine IL-10 and to restrain the production of cytokine IL-9 by γδT cells.which presented a IL-10 dominance and maybe a member of Th9 cells’ family.but the airway inflammation may aggravate by nebulization of M.vaccae repeatedly,The M.vaccae may act as the antigen in the procedure.Part II The effects of Mycobacterium vaccae nebulization on the prevention and treatment of respiratory syncytial viruse infection and Th9 cells in miceSection I The effects of Mycobacterium vaccae nebulization on the prevention of respiratory syncytial viruse infection and Th9 cells in miceOBJECTIVE:To investigate the effects of Mycobacterium vaccae(M.vaccae)nebulization on the prevention of respiratory syncytial viruse(RSV)infection and Th9 cells in mice.METHODS:Seventy-two Balb/c mice were randomized as time-effect relationship group and dose-effect relationship group.Mice in time-effect relationship group were randomized as normal control(N),RSV infection group(C6)and M.vaccae intervention group(Wneb.,Wi.p·,Mneb.,Mi.p·),Mice in dose-effect relationship group were randomized as normal control(N),RSV infection group(C6,C5)and M.vaccae intervention group(Wneb16,Wneb36,Wneb15,Wneb35).Mice in time-effect relationship group were infected with 106PFU RSV once a day for consecutive 5 days.Group Wnebwere nebulized while group Wi.p were intraperitoneal injected with M.vaccae one week before RSV infection,Group Mnebwere nebulized while group Mi.p were intraperitoneal injected with M.vaccae one month before RSVinfection.Lung funciton were measured by non-invasive lung function machine and the mice in time-effect relationship research were executed 4 days after RSV infection.The BALF and lung tissue were harvested.Group C6,Wneb16 and Wneb36 were infected with 106 PFU RSV while group C5,Wneb15 and Wneb35 were infected with 105PFU RSV once a day for consecutive 3 days.Group Wneb16 and Wneb15 were nebulized with 22.5μg M.vaccae while group Wneb36 and Wneb35 were nebulized with 22.5μg*3 M.vaccae once a day for consecutive 5 days after the last time of RSV infection.Lung funciton were measured by non-invasive lung function machine and the mice in dose-effect relationship research were executed after the last time of M.vaccae nebulization.The BALF and lung tissue were harvested.RSV infection condiiton were observed with electron microscope,airway inflammation were detected with pathological HE staining and PAS staining,the expression of lung anti-RSV were detected with immunofluorescence,the concentration of IL-9 in BALF were measured with ELIS A,the expression of EGFR、NF09、Acetycholine were detected with immunohistochemisty,the level of IL-9R、PU.l、PD-L2、JAK1、STAT1、STAT5A、RSV、IFN-γ、TLR7、TLR8 mRNA were detected with fluorescent quantitation PCR,the percentage of IL-9+CD3+、IL-10+CD3+lymphocytes were detected by flow cytometry.RESULTS:①Airway responsiveness were raised up in group C6 compared with normal group,decreased in group Mi.p and Wi.p compared with group C6,M.vaccae nebulization had no effect on the airway responsiveness.②Airway inflammationwere apparent in group C6 compared with C5,M.vaccae nebulization can lead to airway inflammation,which were correlated with nebulization dose of M.vaccae,airway inflammation can be alleviated by M.vaccae intraperitoneal injection,which had no time-effect relationship.③The concentration of IL-9 and OVA-IgE were increased obviously in both group C6 and C5,and decreased in both group Mneb、Mi.p and Wneb16 compared with group C6.④The IOD results of lung anti-RSV immunofluorescence were increased sharply in both group C6 and C5 compared with normal group,decreased in all M.vaccae intervention group compared with C6 and C5 respectively.group Wneb had better effect in the procedure.⑤Amounts of RSV were found in alveolar interstitial in group C6,no RSV in group Wneb16,and small amounts of RSV in group C5 and the other M.vaccae intervention group.⑥The expression of ENFR、NF09、Acetylcholine increased in both group C6 and C5 compared with normal group.The expression of EGFR in group Wi.p and Wneb16 decreased obviously compared with group C6.The expression of NF09 decreased in Wneb16 and Wneb36 compared with group C6.The expression of Acetylcholine decreased in Wneb compared with C6,Both the expression of NF09 and Acetylcholine decreased in Wneb15 and Wneb35 compared with group C5.⑦The level of IL-9R mRNA had no differences among normal group、C6、C5 and the other M.vaccae intervention group;PU.1 mRNA increased in group C6 and C5 compared with normal but decreased in group Wneb、Wneb36 and Wi.p compared with group C6;PD-L2 mRNA decreased in group C6 comapared with normal;JAKl mRNA decreased in group C6 compared with normal but increased in group Wneb36、Wneb15、Wneb35 compared with their infection control;STAT1 mRNA increased in group Wneb15 and Wneb35 compared with group C5,but had no differences among group C6、C5 and normal.STAT5A mRNA decreased in group C6 and C5 compared with normal,but increased in group Wneb16、Wneb36、Wneb15、Wneb35 compared with their infection controls;RSV mRNA increased sharply in group C6 and C5compared with normal and decreased in all M.vaccae intervention group compared with their infection controls expect Wneb35 group;IFN-y mRNA had no differences among all groups;TLR7 mRNA increased in group C6 compared with normal,also increased in group Wi.p、Mneb and Mi.p but decreased in Wneb16 and Wneb36 compared with group C6;TLR8 mRNA decreased in group C6 compared with normal,increased in group Wneb、Wi.p、Mneb、Mi.p、Wneb16 and Mneb36 compared with group C6;The percentage of IL-9+CD3+lymopghocytes increased in group C6 and had no difference in group C5 compared with normal,decreased in group Wi.p and Mi.p but increased in group Mneb、Wneb36、Wneb15、Wneb35 compared with their infection control groups,the percentage of IL-10+CD3+lymphocytes had no difference in both group C6 and C5 compared with normal,but increased in group Wi.p、Mi.p、Wneb15 compared with their infection controls.CONCLUSIONS:Experimental dose of M.vaccae nebulization can protect against RSV infection,Th9 cells play a important role in the procedure,the mechanisms include:①Regulation of Th9 cells through controlling PU.1 and PD-L2,reducing the level of cytokine IL-9 and increasing the level of cytokine IL-10,regulation the expression of JAK1、STAT1、STAT5A and the downstream genes.②Acting as the TLR7 and TLR8 agonist,playing the antivirus effect by activating TLR7 and TLR8;③Downregulation the expression of NF09 and Acetylcholine;④Downregulation the expression of EGFR.Section Ⅱ The effects of Mycobacterium vaccae nebulization on the treatment of respiratory syncytial viruse infection and Th9 cells in miceOBJECTIVE:To investigate the effects of Mycobacterium vaccae(M.vaccae)nebulization on the treatment of respiratory syncytial viruse(RSV)infection and Th9 cells in mice.METHODS:Eighteen Balb/c mice were randomized as normal group,RSVinfection group(RSV group)and M.vaccae treat group(treat group).Both the RSV group and treat group were infected with 106PFU RSV through nasal drip once a day for consecutive 3 days.the treat group were nebulized with M.vaccae while the RSV group with PBS once a day for 5 days after the last time of RSV infection.All animals were executed after last nebulization.Airway reponsivenee were measured by non-invasive lung funciton machine before execution,BALF and lung tissue were harvested.RSV infection condition were investigated by electron microscope,airway inflammation were detected by pathological HE staining and PAS staining,the expression of anti-RSV in the lung were observed by immunofluorescence,the concentration of IL-9 were detected by ELISA,the content of EGFR、NF09、Acetylcholine were detected by immunohistochemistry,the level of IL9R、PU.1、PD-L2、JAK1、STAT1、STAT5A、RSV、IFN-γ、TLR7、TLR8 mRNA were detected by fluorescent quantitation PCR,flow cytometry were performed to detect the percentage of IL-9+CD3+、IL-10+CD3+、γδTCR+CD3+、IL-9+γδTCR+、IL-10+γδTCR+lymphocytes.RESULTS:M.vaccae nebulization can alleviate the airway responsiness and airway inflammation markedly,the anti-RSV immunofluorescence attenuated sharply in treat group.Compared with RSV group,no RSV were found by electron microscope.the expression of EGFR、NF09 and Acetylcholine were decreased in treat group.The fluorescent quantitation PCR showed that the level of RSV、IL-9R、PU.1、IFN-y mRNA decreased while JAK1、STAT1、STAT5A、TLR7、TLR8 mRNA increased obviously in treat group compared with RSV group.Flow ctyometry results showed that the percentage of γδTCR+CD3+lymphocytes decreased compared with normal while increased in treat group compared with RSV group.the percentage of IL-9+CD3+lymphocytes decreased and IL obviously in treat group compared with RSV group.CONCLUSIONS:Experimental dose of M.vaccae nebulization can treat RSV infection,Th9 cells play a important role in the procedure,the mechanisms include:①Regulation of Th9 cells through controlling PU.1 and PD-L2,reducing the level of cytokine IL-9 and increasing the level of cytokine IL-10,regulation the expression of JAK1、STAT1、STAT5A and the downstream genes.②Acting as the TLR7 and TLR8 agonist,playing the antivirus effect by activating TLR7 and TLR8;③Downregulation the expression of NF09 and Acetylcholine;④Downregulation the expression of EGFR;⑤Recruitment of y8T cells to the lung to participate into antivirus procedure.Part III Non-invasive lung function measurements in normal Balb/c miceOBJECTIVE:To measure normal murine lung function,to provide references for researchers.METHODS:Total 300 normal Balb/c mice were divided into 6 groups according to sex and weight:group AF(n=50,female,16-20g),group AM(n=50,male,16-20g),group BF(n=50,female,20-24g),group BM(n=50,male,20-24g),group CF(n=50,female,24-28g),group CM(n=50,male,24-28g),Lung function measurements were performed with TBL4500 FinePointe NAM(Non Invasive Airway Mechanics)system,correlation between lung function parameters were analyzed.RESULTS:No significant differences among groups were seen in f,sRaw,sGaw,Raw,Ti,Te and Dt.The parameters including TV,MV,Frc,R2,PIF,PEF,Rpef,EF50 and Rinx showed obviously different among groups.correlation analys showed that sex is correlated with W,Frc,R2,Rpef,W is positively correlated with TV,MV,sRaw,Frc,PIF,PEF,Rpef,EF50 and Rinx;f is positively correlated with TV,MV,R2,PIF,PEF,Rpef,EF50 and Rinx while negatively correlated with sRaw,sGaw,Raw,Ti,Te,Dt.CONCLUSIONS:Lung function parameters in different groups showed significant differences.our experiment provided references for researchers with appropriate choice of animals.