Effects Of Dai Medicine Wubao Capsule On Airway Inflammation And Mucus Hypersecretion In Asthmatic Mice

Objective:As a folk prescription of Dai people in Xi Shuang Ban Na,Yun Nan province,Wubao Capsule(Ya-jiao Ha-dun San)is included in the pharmacopoeia of the people's Republic of China and widely used in clinic.It has the functions of clearing heat,detoxifying,stopping bleeding and stopping pain.In this study,ovalbumin(OVA)was used as sensitizer to induce BALB/c mice to establish asthma model,in order to explore the mechanism of Wubao Capsule in inhibiting airway inflammation and mucus hypersecretion in asthmatic mice.Method:1.experiment methods:(1)Animal models: OVA sensitization and OVA solution atomization were used to establish asthma model.OVA sensitizer was injected into mice abdominal cavity three times on the 1st,8th and 15 th day.OVA solution was atomized on the 21st-27 th day.Normal control mice were sensitized and atomized with normal saline.(2)Drug administration method: This experiment is a therapeutic drug,which is administered by intragastric administration.The time of administration is one hour before the atomization excitation period(21-27 days).Normal and model control mice were given normal saline,positive drug group mice were given dexamethasone solution,drug control mice were given Wubao Capsule high and low dose solution respectively.(3)Animal material: Serum,alveolar lavage fluid(BALF)and lung tissue of mice were obtained on the 28 th day of the experiment(24 hours after the last administration).2.Indicator detection:(1)Observe the whole condition of the animals and record the latent period of asthma induction on the 25th-27 th day of the experiment.(2)Fast Rayleigh-Jimsa staining was used to observe the changes of white blood cells in each group(3)HE staining and PAS staining were used to observe the pathological changes of lung tissue and goblet cell proliferation in mice.(4)The serum levels of IgE,OVA-IgE and IFN-?,IL-25,IL-33,TSLP,IL-4,IL-5,IL-13,IL-6,IL-9,IL-10,IL-8,IL-17 A,MCP-1,Eotaxin and MUC5 AC in BALF were determined by ELISA.(5)Immunohistochemical method(IHC)was used to detect the expression of EGFR,p-EGFR,IL-13,FOXA2 and MUC5 AC in lung tissue of mice.Result:1.Changes of animal behavior and latent period of asthma induction: Except for the normal group,mice in each group showed asthma-like symptoms such as head licking,shortness of breath,itching on the head and face,quietness and inactivity,retardation of movement,restlessness,erection of hair,back and abdomen convulsions in varying degrees after aerosol inhalation of OVA;during aerosol inhalation,mice lost weight and fed less.After treatment with dexamethasone and Wubao capsule,the above symptoms of mice in drug control group were significantly improved,and the asthmatic latency of mice in dexamethasone group and Wubao capsule group was significantly longer than that of model group(P < 0.01).2.Changes in the number of white blood cells: The total number of white blood cells,monocytes,lymphocytes,neutrophils and eosinophils in BALF of mice in model group were higher than those in normal group(P < 0.01);after treatment with dexamethasone and Wubao capsule,all kinds of white blood cells in asthmatic mice decreased significantly(P < 0.05;P < 0.01).3.Pathological changes of lung tissue in mice: HE staining showed that the alveolar periphery of model group was loose and infiltrated with inflammatory cells,a large number of inflammatory cells infiltrated around trachea and pulmonary interstitium,basement membrane was thickened,airway mucosal epithelium was incomplete,and exfoliation occurred.The pathological inflammation score of lung tissue in model group was higher than that in normal group.After treatment with dexamethasone and Wubao capsule,lung tissue was infiltrated.PAS staining showed that goblet cell proliferation and mucus secretion increased significantly in the model group.After treatment with dexamethasone and Wubao capsule,goblet cell proliferation and mucus hypersecretion were improved,and the positive area statistics showed that goblet cell proliferation and mucus hypersecretion in the drug group were significantly lower than those in the model group(P < 0.01).4.Changes of inflammatory cytokines: The levels of serum IgE,OVA-IgE and IL-4,OVA-IgE and IL-4,IL-5,IL-5,IL-6,IL-8,IL-8,IL-9,IL-13,IL-17 A,IL-25,IL-33,IL-33,TSLP,TSLP,MCP-1,Eotaxin,Eotaxin,MUC5 AC in the model group were higher than those in the normal group(P < 0.01).The levels of serserum IgE,OVA-IgE and MUC5 AC MUC5AC and MUC5 AC in the model group mice were higher than those in the normal group(P < 0.01).The levels of IgE,OVA-IgE,OVA-IgE and ILThe results of this study are as follows:1.-The levels of IL-33,TSLP,MCP-1,Eotaxin and MUC5 AC in BALF of model group were lower than those of model group(P < 0.05;P < 0.01);the contents of IFN-? and IL-10 in BALF of model group were lower than those of normal group(P < 0.01);the contents of IFN-? and IL-10 in BALF of dexamethasone group and Wubao capsule group were higher than those of model group(P < 0.05;P < 0.01).5.Changes of mucus hypersecretion-related proteins: The expression of IL-13,EGFR,P-EGFR and MUC5 AC in lung tissue of model group mice was higher than that of normal group,and the positive area of IL-13,EGFR,P-EGFR and MUC5 AC in lung tissue of mice in dexamethasone group and Wubao capsule group was lower than that of model group,and the positive area of IL-13,EGFR,P-EGFR and MUC5 AC in lung tissue of model group mice had significant difference(P<0.05;P<0.01).(P < 0.05;P < 0.01),positive regulation of mucin production;FOXA2 expression in lung tissue of model group was lower than that of normal group,and the positive area of FOXA2 was significantly different(P < 0.05;P < 0.01);the expression of FOXA2 in lung tissue of mice in dexamethasone group and Wubao capsule group was higher than that of model group,and the positive area of FOXA2 was significantly different(P < 0.05;P < 0.01),negatively regulating mucin(P < 0.05;P < 0.01).Conclusion:1.Through the observation and analysis of animal behavior,latency of asthma induction,HE staining and PAS staining,the asthma model of this experiment was established successfully.2.According to the analysis of various white blood cell counts,Wubao Capsule may alleviate the inflammatory reaction of asthmatic mice by reducing the number of monocytes,lymphocytes and granulocytes.3.The analysis of cytokines in serum and BALF by ELISA showed that Wubao Capsule could improve the inflammatory response and mucus hypersecretion of asthmatic mice by regulating immune cells such as mast cells,granulocytes,monocytes,Th1,Th2,Th17,Treg,ILC2 s and their secreted cytokines.Its role might be related to the inhibition of IgE production,Th1/Th2 balance,Treg/Th17 balance.The formation of ILC2 s is related.4.The expression of mucin-related proteins in lung tissues of asthmatic mice was detected by immunohistochemical method,suggesting that Wubao Capsule may inhibit the production of mucin MUC5 AC through IL-13 signaling pathway and EGFR signaling pathway,thereby reducing mucus hypersecretion in asthmatic mice.