Experimental Study On The Effect Of Flavored Anaphylactic Decoction On CD4~+T Cell Subsets And Cytokines In Asthmatic Mice

Objective:To establish a mouse of allergic asthma model by OVA,and to observe the effect of flavored anaphylactic Decoction on the CD4⁺T cell subsets and its cytokines in asthmatic mice,in order to clarify the immune molecular mechanism and target of antiasthmatic of flavored anaphylactic Decoction,fill the study on the gene level of allergic asthma in the treatment of flavored anaphylactic Decoction.In order to provide an experimental basis for the development of flavored anaphylactic decoction of a new,safe and effective drug to antagonize asthma,it provides a scientific and reliable basis for the clinical use of this prescription for the treatment of allergic diseases.Methods:60 BALB/c female mice of 5 weeks old were randomly divided into 6 groups（blank group,model group,positive control group with dexamethasone acetate,low,medium and high dose group of flavored anaphylactic decoction）,10 mice in each group.There are two stages that is sensitization and stimulation,sensitization-the day of the experiment of sensitization was sensitized on the day of first,eighth,fifteenth experiments,and 3 times,in addition to the blank group,all the mice in the other groups were intraperitoneally injected with OVA sensitizer（containing 20μg OVA+150μL aluminum hydroxide adjuvant）,0.2m L/only.In the blank group,the phosphate buffer solution（PBS）was used instead of the OVA sensitizing solution.The method was excited by the other groups;stimulation-the 25-29day of the experiment of stimulation.In addition to the blank group,other mice were placed in the self-made closed glass container（25cm x 30cm x40cm）,and the atomization particles were inhaled by 10g/L atomization（the average diameter of the atomized particles was 3.7μm）,20min/times,1times/d,to stimulate asthma,during this period,we observed the behavioral performance of mice when asthma was activated.The blank group was treated with PBS solution instead of OVA,The method was excited by the other groups.Drug intervention:Each group was intragastrical on the 16-29 day of the experiment.The dosage of low,medium and high dose group was 13.6,27.2,54.4g·kg^-1·d^-1of flavored anaphylactic decoction respectively.The dosage of dexamethasone group was 1.25mg·kg^-1·d^-1.The volume of gavage was 20m L/kg,1 times/day,continuous 14d.The blank group and the model group were given equal volume of normal saline.After the last stimulation of24h,serum was taken and the content of Ig E,IFN-γ,IL-4,IL-17 and IL-10 in the serum of mice were detected by ELISA,splenic tissue was taken and the proportion of the subsets of Th1/Th2/Th17/Treg and other cells was detected by flow cytometry,and the lung tissue was detected by RT-PCR technique to detect the m RNA expression level of type Th1 cytokine IFN-γ,m RNA expression level of type Th2 cytokine IL-4,m RNA expression level of Th17type cytokine IL-17,and m RNA expression level of Treg type cytokine IL-10in the lung tissue.Results:1.The study on the efficacy of flavored anaphylactic decoction in the treatment of allergic asthma:Compared with the blank group,the behavioral score of the model group was significantly higher（P<0.01）.Compared with the model group,the behavioral manifestations of the asthmatic mice in the group of flavored anaphylactic decoction,such as sneezing,choking cough,shortness of breath,and abdominal muscle contraction,were significantly reduced（P<0.01）.Compared with the blank group,the concentration of OVA-s Ig E in the serum of model mice increased significantly（P<0.01）.Compared with the model group,the content of OVA-s Ig E in the serum of low dose group of flavored anaphylactic decoction had a decreasing trend,and the concentration of OVA-s Ig E in the middle and high dose groups could obviously decrease the concentration of OVA-s Ig E in the serum of mice（P<0.05～0.01）.2.The experimental study on the effect of flavored anaphylactic decoction on CD4⁺T cell subsets and its cytokines in asthmatic mice:The results of ELISA experiment showed that the expression level of cytokines IL-4 and IL-17 in serum of asthmatic mice could be significantly reduced（P<0.01）,and the expression level of cytokine IFN-γand IL-10 was significantly improved（P<0.01）.The results of flow cytometry showed that flavored anaphylactic decoction can significantly reduce the proportions of Th2 and Th17 cells in the spleen tissue of OVA induced asthma mice（P<0.05～0.01）,and the ratio of Th1 and Treg cells could be increased,and the ratio of Th1/Th2 was significantly increased and the ratio of Th17/Treg was significantly reduced.The results of RT-PCR experiment showed that flavored anaphylactic decoction could significantly reduce the expression level of Th2 cytokine IL-4 and Th17 type cytokine IL-17 in lung tissues of asthmatic mice,and the expression level of IFN-γ（P<0.01）of Th1 type cytokine was significantly increased,and the m RNA expression water of Treg cytokine IL-10 could be obviously improved.Conclusion:1.Flavored anaphylactic decoction has the effect of treating allergic asthma.2.Flavored anaphylactic decoction in the treatment of mechanism of allergic asthma may be an regulation of the differentiation of Th1/Th2 cells:Up regulation of the expression level of Th1 cells and their cytokine IFN-γ,down regulation of the expression level of Th2 cells and their cytokine IL-4;or an regulation of the differentiation of Treg/Th17 cells:Up regulation of Treg cells and their cytokine IL-10 expression and down regulation of Th17 cell and its cytokine IL-17 expression level.