The Mechanism Of Notch1 Signaling Regulates ??t17 Cells In Asthma And The Effect Of Inhaled Inactivated Mycobacterium Phlei

PART ? The expression of ??T17 cell in asthmatic mice and its significanceObjective To explore the effect of ??T17 cell in the asthmatic mice,and to further explore the possible mechanism.Methods Male Balb/c mice were used in the study.The mice were randomly divided into the control group and the asthmatic group.The mice in the asthmatic group were intraperitoneally injected with the mixture of ovalbumin(OVA)/ Al(OH)3 and then activated by exposure of the animals to OVA atomization.OVA-induced airway hyperresponsiveness(AHR)was determined by a non-invasive lung function machine,Hematoxylin and eosin(HE)and alcian blue-periodic acid Schiff(AB-PAS)were done for histopathological analysis,and the productions of IL-17 in bronchoalveolar lavage fluid(BALF)were detected by ELISA.The percentage of IL-17~+??T~+ CD3~+ cells was detected by flow cytometry,the transcription levels of ROR?t was determined by RT-PCR.Results Compared with the control mice,the severity of inflammation in lung tissue was evaluated by HE staining and AB-PAS staining,while AHR was assessed by lung function instrument in the asthmatic model mice which the s Raw was obvious increased at each dose of Mch(P <0.05).Furthermore,mice in the asthmatic group displayed significant increased in the presence of IL-17~+??T~+ CD3~+cells and in the level of IL-17 and the expression of ROR?t m RNA(P <0.05).Conclusion The effect of ??T17 cell in the asthmatic mice may contribute to the pathogenesis of OVA-induced asthma,which plays an important role in the pathogenesis of inflammation and provides a new way for prevention and treatment of asthma.PART ? The regulatory roles of ?-secretase inhibitor(DAPT)in the expression of ??T17 cells in the asthmatic miceObjective To study the roles of ??T17 cells in the asthmatic mice after ?-secretase inhibitor(DAPT)blocks Notch signaling.Methods Male Balb/c mice(6~8 weeks old)were randomly divided into three groups,the control group,the asthmatic group and the DAPT group with 6 mice in each group.The control group was treated with PBS instead of OVA at the sensitization and challenge stages.The asthmatic group and DAPT group were sensitized and challenged with OVA to establish the asthmatic model.A notch signal inhibitor,DAPT dissolved in 10% dimethyl sulfoxide(DMSO)was inhaled in the DAPT group 30 minutes before the OVA challenge.The control group and the asthmatic group were treated with the equal volumes of PBS.The pathological changes of lung tissue were analyzed by HE and AB-PAS staining,the lelve of IL-17 in BALF was detected by ELISA,the m RNA expression of ROR?t was measured by RT-PCR,the percentages of IL-17~+??T~+ CD3~+ cells were detected by flow cytometry.Furthermore,??T cells were isolated from spleens of the asthmatic mice by magneticbeads and the percentage of ??T17 cells were detected by flow cytometry.Results In vivo,compared with the asthmatic mice,the severity of airwy inflammation and mucus secretion in lung tissue were alleviated in the DAPT group.Furthermore,the level of IL-17 in BALF,the m RNA expression of ROR?t in lung tissue and the percentage of IL-17~+??T~+ CD3~+ cells were significantly decreased in the DAPT group(P <0.05).In addition,in vitro,the purity of ??T cells satistified the experiment requirement after isolation.The percentage of IL-17~+ ??T cells in the asthmatic group was significantly higher than those in the control group,those in the DAPT treated groups were lower than those in the asthmatic group in a dose-dependent manner(P <0.05).Conclusion ?-secretase inhibitor(DAPT)could block the Notch1 signaling and suppress the airway inflammation and the expression of IL-17,which provide a potential value for the target immunotherapy and prevention of asthma.PART ? Effects of inhaled inactivated mycobacterium phlei on Notch1 signaling pathways in asthmatic miceObjective To investigate the effects of early nebulized inhalation of inactivated mycobacterium phlei on Notch1 signaling in asthmatic mice,and to further explore the possible mechanism.Methods Male Balb/c mice were divided into the control group,the asthmatic group and the treatment group.Mice were made the asthmatic group with OVA.The treatment group inhaled the inactivated Mycobacterium phlei.HE staining and AB-PAS staining were done to measure lung inflammation and mucus production,Relative cytokinses in BALF were quantified by ELISA;The percentages of IL-17~+ among ??T~+ CD3~+cells in the spleen were analyzed by Flow Cytometry;Notch1 m RNA and protein expression levels were analyzed through RT-PCR and IHC,respectively.The protein expression of NICD was measured by Western Blot.Results After the treatment of Mycobacterium phlei,airway inflammation was attenuated.The levels of IL-17 and the percentages of IL-17~+??T~+ CD3~+cells were significantly decreased.In addition,the proteins and m RNA expression level of Notch1 was decreased.Conclusion The inhalation of Mycobacterium phlei before sensitization can regulate the expression of ??T17 cells and relative cytokines in asthmatic mice via Notch1 signaling pathway.