Changes Of Inflammatory Cytokines In Asthmatic Mice With Conditional Knockout Of Sema4D Gene In Airway Epithelium And The Intervention Effect Of Kechuannin

Objective: Observe the changes of inflammatory cytokines in airway epithelial brain signaling protein 4D(Sema4D)gene conditionally knocked out asthma mice,and study the role of Sema4 D in virus-induced neuroendocrine-immune(NEI)network disorder in asthma,revealing The mechanism of the interaction between the neuroendocrine and immune systems in asthma,and by observing Kechuanning intervention in respiratory syncytial virus(RSV)induced asthma mouse models,to explore the corresponding effect and mechanism of Kechuanning in the treatment of asthma.To clarify the pathogenesis of asthma provides a new theoretical basis.Methods: Use CRISPR/Cas9 gene editing technology to establish a Sema4 D gene conditional knockout mouse model of C57BL/6J mice.Randomly divide the Sema4 D gene conditional knockout mice into a Sema4 D knockout group and a Sema4 D knockout asthma group.Randomize C57BL/6J mice Divided into normal group,model group and Kechuanning group to construct RSV compound OVA asthma mouse model.Use animal calls Aspiration to measure airway reactivity of mice;HE staining method to observe the pathomorphological changes of lung tissue;collection of bronchoalveolar lavage fluid(BALF),protein detection and inflammatory cell classification of the lavage fluid;blood collection from mouse eyeballs,ELISA method to detect the levels of Ig E,IFN-γ and nerve growth factor(NGF)in peripheral blood.Results:1.F4 generation CKO knockout mice have reduced gene copy number,and the Sema4 D gene content of mice is lower than that of flox mice.The Sema4 D gene conditional knockout mouse model was successfully established.2.Airway responsiveness and lung histopathological results showed that RSV combined with OVA asthma model was successfully constructed.Compared with the normal group,the RL value of the model group and the Sema4D elimination group was significantly higher(P < 0.01);compared with the model group,the RL value of the Sema4 D elimination group was significantly lower(P<0.01).3.Comparison of Ig E content in serum and alveolar lavage fluid of each group of mice showed:(1)Compared with the model group,serum and alveolar lavage fluid of normal group,Sema4 D elimination group,Sema4 D elimination asthma group and Kechuanning group Ig E levels were significantly reduced(P<0.01);(2)Compared with the Sema4 D elimination group,the serum Ig E levels of the normal group and the alveolar lavage fluid and the Sema4 D elimination asthma group were similar,and there was no statistical significance(P> 0.05).Sema4 D eliminated the asthma group The level of Ig E in the alveolar lavage fluid was significantly higher(P <0.01);(3)Compared with the Kechuanning group,the serum Ig E level of the normal group was similar to that of the alveolar lavage fluid and the Sema4D-eliminated asthma group,and there was no statistical significance(P > 0.05),The level of Ig E in the alveolar lavage fluid of the Sema4 D exclusion asthma group was significantly increased(P<0.01).4.The comparison of IFN-γ content in serum and alveolar lavage fluid of each group of mice showed that:(1)Compared with the model group,serum and alveolar lavage fluid IFN-γ levels of normal group and Kechuanning group were significantly higher(P< 0.01),the serum IFN-γlevel in the Sema4 D elimination group and the Sema4 D elimination asthma group was significantly increased(P < 0.01),and the IFN-γ level in the alveolar lavage fluid was increased(P < 0.05);(2)Compared with the Sema4 D elimination group,the normal group,Sema4 D elimination asthma group serum and alveolar lavage fluid IFN-γ levels are similar,not statistically significant(P > 0.05);(3)Compared with Kechuanning group,normal group,Sema4 D elimination asthma group serum and alveolar lavage fluid The levels of IFN-γ were similar and not statistically significant(P>0.05).5.The comparison of NGF content in serum and alveolar lavage fluid of mice in each group showed that:(1)Compared with the model group,the NGF levels in serum and alveolar lavage fluid of normal group and Sema4 D knock-out group were significantly lower(P< 0.01),The serum NGF levels in the Sema4 D elimination group and the Kechuanning group were significantly reduced(P < 0.01),and the alveolar lavage fluid NGF level decreased(P<0.05);(2)Compared with the Sema4 D elimination group,the normal group and the Sema4 D elimination group The levels of NGF in serum and alveolar lavage fluid were similar and not statistically significant(P > 0.05);(3)Compared with Kechuanning group,serum and alveolar lavage fluid NGF levels of normal group and Sema4 D elimination group were similar,and there was no statistical significance.Significance(P >0.05).Conclusion: 1.Sema4 D is involved in the pathogenesis of asthma.It may aggravate asthma by stimulating the NEI network.Knockout of the Sema4 D gene in the lungs of asthmatic mice can reduce asthma airway responsiveness,improve lung histopathology,and reduce serum and alveolar lavage fluid The content of Ig E and NGF in the Chinese medicine can increase the level of IFN-γ and relieve asthma.2.Kechuanning may improve RSV-induced asthma airway inflammation by regulating Sema4 D gene.