Based On The Activation Of Alveolar Macrophages To Investigate The Mechanism Of Combined Treatment Of Lung And Intestine On Mouse Bronchial Asthma

Objective:In vitro experiments were conducted to investigate whether vasoactive intestinal peptide(VIP)regulates the activation of alveolar macrophages in mice through the p38 MAPK signaling pathway.In vivo experiments were conducted to investigate whether p38 MAPK signaling pathway could be inhibited by increasing endogenous VIP,regulate the activation of alveolar macrophages in mice,and treat bronchial asthma in mice,so as to provide experimental basis for the clinical application of pg-intestinal therapy.Methods:In vitro experiment,primary mouse alveolar macrophages cultured in vitro were used as the research object.Mouse alveolar macrophages were divided into control group,activation group and VIP treatment group.By LPS+IFN-γ induced alveolar macrophage activation,VIP treatment group in addition to exogenous VIP stimulation.Use ELISA to detect the content of TNF-ɑ,IL-6 in each group;Use Western Blot to detect the protein content of TNF-ɑ,IL-6,p38MAPK、p-p38 MAPK in each group;Use Q-PCR to detect the expression level of TNF-ɑ,IL-6,p38 MAPK mRNA in each group.In vivo experiments,KM mice were randomly divided into blank group,model group,western medicine treatment group,lung treatment group with traditional Chinese medicine,and combined treatment of lung and intestine group.The mouse bronchial asthma model was sensitized by egg albumin(OVA),the state of each group was observed,and lung histopathological sections were prepared.HE staining,ab-pas staining and MASSON staining were used to observe the inflammatory infiltration of lung tissue,mucus secretion and airway fibrosis,and semi-quantitative analysis was conducted.The serum VIP content of mice was detected by ELISA.Use Western Blot to detect the protein content of TNF-ɑ,IL-6,p38 MAPK,p-p38 MAPK in lung tissue;Use Q-PCR to detect the expression level of TNF-ɑ,IL-6,p38 MAPK mRNA in lung tissue.Results:In vitro experiment: 1,the ELISA results showed that groups compared with control group,the activation cell cultures of TNF-ɑ,IL-6 levels increased significantly(P<0.01);Compared with activation group,VIP treatment group of TNF-ɑ,IL-6 levels in cell cultures decreased significantly(P<0.01).2,Western Blot results showed that groups compared with control group,the activation cell TNF-ɑ,IL-6,p-p38 MAPK protein content increased significantly(P<0.01);Compared with activation group,VIP treatment group of intracellular TNF-ɑ,IL-6,p-p38 MAPK protein content decreased significantly(P<0.01).3,Q-PCR results show that compared with the control group,the activation cell groups TNF-ɑ,IL-6,p38 MAPK mRNA expression level increased significantly(P<0.01);Compared with activation group,VIP treatment group cells TNF-ɑ,IL-6,p38 MAPK mRNA expression level decreased significantly(P<0.05).In vivo experiments: 1.Behavior changes: During modeling,mice in the normal group show glossy fur,strong appetite,bright eyes and quick actions.Mice in the model group show dim hair color,decreased appetite,restlessness or listlessness,increased and accelerated breathing,a purple color around the lips,frequent scratching of the mouth,nose and limbs,nod-like breathing,arched back,forelimb constriction,nasal fans,convulsions,obvious abdominal muscle contraction and other symptoms when stimulated by atomization.With the increase of stimulation times,the symptoms of mice in the model group were gradually aggravated,and the symptoms of each intervention group were alleviated to different degrees compared with the model group.2.Pathological changes: HE staining: in the blank group,no obvious inflammatory cell infiltration was observed around the airway,and no exfoliated cells were observed in the airway.In the model group,a large number of inflammatory cells were infiltrated around the airway and blood vessels,and a large number of exfoliated cells were found in the airway.The infiltration of inflammatory cells around the airway was alleviated in each intervention group.Ab-pas staining: no obvious goblet cell metaplasia was observed in the airway epithelium of the blank group,and there was no mucus in the airway.In the model group,goblet cell metaplasia increased and mucus secretion was high.The changes of airway goblet cell metaplasia and mucous secretion in each intervention group were significantly reduced compared with the model group.MASSON staining: no obvious collagen deposition was observed in the airway of the blank group.Collagen deposition was obvious in the model group and around the airway,and the airway smooth muscle thickened in the large airway.Collagen deposition was also observed in various intervention groups.3.ELISA results: compared with the blank group,the serum VIP content of mice in the model group was significantly reduced(P<0.05).Compared with the model group,the serum VIP content of mice in the lung treatment group with traditional Chinese medicine and the combined treatment of lung and intestine group increased to different degrees(P<0.05),but the growth in western medicine treatment group is significant(P>0.05).4,Western Blot results: compared with blank group,model group’s protein content of TNF-ɑ,IL-6,p-p38 MAPK increased significantly(P<0.05);Compared with model group,protein levels of TNF-ɑ,IL-6,p-p38 MAPK in the lung treatment group with traditional Chinese medicine and the combined treatment of lung and intestine group decreased to different degrees(P<0.05).What’s more,the decrease of protein levels of TNF-α、IL-6 in the combined treatment group was more significant than that in the lung treatment group with traditional Chinese medicine(P<0.05),while no significant difference was found in the protein content of p-p38 MAPK in the western medicine treatment group(P>0.05).5,Q-PCR test results: compared with blank group,the expression of TNF-ɑ,IL-6,p38 MAPK mRNA in model group are increased(P<0.05).Compared with model group,the rest of the three groups of TNF-ɑ,IL-6,p38 MAPK mRNA expression levels are in different degrees of lower(P<0.05),in which,the decrease of TNF-ɑ,IL-6 mRNA expression level in the combined treatment group are more significant than that in the lung treatment group with traditional Chinese medicine(P<0.05).Conclusions:1,exogenous VIP can inhibits alveolar macrophage activation by p38 MAPK signaling pathway,thus reducing the release of TNF-ɑ,IL-6 inflammatory cytokines;2.The combined treatment of lung and intestine can inhibit the activation of alveolar macrophages,reduce pulmonary inflammation and treat bronchial asthma by increasing the generation of endogenous VIP.