The Effect Of Dachengqi Decoction On The Polarization State Of Helper T Cells And MAPK Pathway In Mice With Allergic Asthma

Objective:To explore the effect and molecular mechanism of Dachengqi decoction(DCQD)on murine allergic asthma model by detecting the polarization of helper T cell and MAPK in order to provide the biological evidences of the method "Treatment of Lung and intestine".Methods:1 Establishment of muirine allergic asthma model and exploring the appropriate dosage of tranditional Chinese prescription.20 female C57BL/6 mice were randomly divided into 4 groups,including normal group,model group,DCQD group and dexamethasone positive control group.Each group had 5 mice.The murine model of allergic asthma was established by OVA sensitization and challenge.On the 0,14th days,mice from model and treatment groups were injected i.p with the mixed solution 0.2ml of OVA and aluminum hydroxide gel.On the 21st day,these mice were placed into a plexiglass box and exposed to the 1%OVA aerosol for 30min by using medical nebulizer.This procedure was applied once every day and persisted 5 days.Normal group mice were modeled with sterile PBS by intraperitoneal injection and inhalation,continuing for 5 days.DCQD group,dexamethasone(DXM)positive control group animals were treated by intragastric administration every day an hour before challenging.Each volume of intragastric administration was 0.2ml.Normal group and the model group mice were gavaged by equivalent sterile PBS,continuing for 5 days.2 Effects of traditional Chinese medicine on lung index,spleen index,lung and intestinal pathology of allergic asthmatic mice;On 26th day,all the mice were scraficed by cervical dislocation.At the time of sacrifice,removed the lung and spleen and recorded the weights of these tissues.Then,the middle lobe of right lung and intestines were fixed with 10%formaldehyde solution for 4 hrs.It was then dehydrated in ethylic alcohol,embedded with paraffi sectioned in 6μm,and stained with haematoxylin and eosin(HE).Tissue slices were evaluated through light microscope(Nikon)by trained technician in a blind.3 Effects of traditional Chinese medicine on the levels of cytokines and the classification of inflammatory cells in bronchoalveolar lavage fluid of allergic asthmatic mice;Mouse lungs were flushed via the trachea cannula with 0.8 ml sterile PBS solution with 1%BSA3 times following the final OVA challenge.BALF was centrifuged at 200 g for 5 mins.Supernatants were stored at-20℃for subsequent cytokine measurements.The total cell suspension was resuspended in 1 ml PBS.Cells were counted using a hemocytometer.Differential cell counts were carried out after Diff-Quick.staining.After blood was withdrawn from the orbital venous plexus,serum was separated from the blood clot by centrifugation at 2,500 rpm for 20 mins at 4℃.The levels of OVA-specific IgE,total IgE,IFN-gamma,IL-4,IL-5,IL-17A in BALF and serum,were measured using enzyme-linked immunosorbent assay.4 Eflfects of traditional Chinese medicine on MAPK signaling pathway in lung tissue of allergic asthmatic mice;At the time of sacrifice,80mg lung tissues were clipped and stored at-80℃ for subsequent protein detection.MAPK protein expression was detected by Western blotting techniques.Results:1 After sensitization and challenge of OVA,mice from model group appeared wheezing,shortness of breath,sneezing,scratching nose,etc.What’s more,their hair lacked of luster and shit becomed harder.However,these symptoms in DCQD treatment group and DXM treatment group were significantly reduced.2 The results of total and OVA-specific IgE level,spleen index and lung index,lung and intestinal phothology:compared with normal group,total and OVA-specific IgE level,spleen index and lung index of model group significantly increased(P<0.01),lung and intestinal phothology of model group appeared evident pathological changes:infiltration of abundant inflammatory cells;compared with model group,total and OVA-specific IgE level,lung index of DCQD treatment group and DXM treatment group significantly reduced(P<0.05),spleen index of DCQD treatment group increased(P<0.05),lung and intestinal inflammation of DCQD treatment group and DXM treatment group improved obviously.3 The results of IFN-γ、IL-4、IL-5 and IL-17A level and classified cell count in BALF:compared with normal group,IFN-γ in BALF of model group significantly reduced(P<0.0 1),IL-4、IL-5 and IL-17A level in BALF of model group significantly increased(P<0.01);compared with model group,IFN-y in BALF of DCQD treatment group and DXM treatment group significantly increased(P<0.01),IL-4 and IL-17A level in BALF of DCQD treatment group and DXM treatment group reduced(P<0.01),IL-5 level in BALF of DCQD treatment group and DXM treatment group reduced but had no obvious changes.4 The results of differential cell counts in BALF:compared with normal group,the numbers of total cells in BALF of model group significantly reduced(P<0.01),the numbers of eosinophils neutrophils lymphocytes and monocytes in BALF of model group significantly increased(P<0.01);compared with model group,the numbers of eosinophils neutrophils lymphocytes in BALF of DCQD treatment group and DXM treatment group significantly reduced(P<0.05),the numbers of monocytes in BALF of DCQD treatment group and DXM treatment group had no obvious changes.5 The results of p38、ERK1/2、P-p38 and P-ERK1/2 in lung tissues:p38 and ERK1/2 in lung tissues of groups had no significant changes;compared with normal group,P-p38and P-ERK1/2 level in lung tissues of model group significantly increased(P<0.01);compared with model group,P-p38 and P-ERK1/2 level in lung tissues of DCQD treatment group and DXM treatment group significantly reduced(P<0.01).Conclusion:Dachengqi decoction might affect the polarization state of Thelper cells from allergic asthmatic mice through MAPK pathway,which contributed to alleviate the pulmonary inflammation and exerted its theraptic effect on allergica asthma.