Study On The Mechanism Of Huanglong Decoction On Airway Hyperresponsiveness In Children With Cough Variant Asthma By Regulating IL-4/P-STAT6 And CaSR

Objective:To explore the effect of Huanglong Decoction on airway hyperresponsiveness in children with cough variant asthma(CVA)from the spasmolytic effect on airway smooth muscle.Moreover,the mechanism of multi-target and multi-effect of in the treatment of children with CVA was further clarified,providing a more solid theoretical basis for the development of new drugs of traditional Chinese medicine for specific diseases.Methods:(1)Establishment of a young mouse model of cough variant asthma(CVA):Seventy young(4-week-old)BALB/c mice were randomly divided into 7 groups:blank control group,model group,dexamethasone group,montelukast sodium group,and low,medium,and high dose groups of Huanglong Decoction,with 10 mice in each group.The model was established by intraperitoneal injection of ovalbumin(OVA)mixed with aluminum hydroxide for sensitization and atomization inhalation of ovalbumin for challenge.Each treatment group was intragastrically administered with dexamethasone(2mg/kg),montelukast sodium(1.5mg/kg),and Huanglong Decoction at low,medium,and high doses(8.4g/kg,16.8g/kg and33.6g/kg).After the completion of modeling,samples were collected to observe tissue inflammation and airway structural changes.(2)Extraction of rat primary airway smooth muscle cells and establishment of cell model:After the primary rat smooth muscle cells extracted were passaged to the F4 generation,the cell model was established by using IL-4 as a modeling agent.Day 1:Airway smooth muscle cells were seeded into 8 medium dishes.Day 2:4 dishes were pre-incubated with high glucose DMEM containing 1%fetal bovine serum to prepare Huanglong Decoction solution(containing crude drug 1 mg/ml)(more than 12h).The other 4 dishes were replaced with culture medium(1%fetal bovine serum in high-glucose DMEM)to continue the culture.Day 3:At this time,the cells were divided into Huanglong and non-Huanglong group according to the presence or absence of pre-incubation with Huanglong Decoction,and stimulated with IL-4 at different concentrations of 0,1,2,and 5 ng/ml for 30 min in these two groups of cells(4 dishes each).On the basis of the cell model,immunofluorescence assay was further performed to detect P-STAT6,calcium probe Fluo-4 assay was used to observe calcium concentration changes,and Western blot was used to detect STAT6/P-STAT6 and CaSR protein expression levels in airway smooth muscle cells.Results:(1)Effect of Huanglong Decoctionon on OVA-induced cough variant asthma in young mice model:After the completion of modeling,the mice in the model group showed symptoms including cough,sneezing,rubbing nose,itching,tail clenching,anxiety and irritability.The symptoms of mice in each group treated were improved,and the mice in the blank control group showed no significant abnormality.(2)Observation results of HE staining of trachea and lung tissues:The airway structure of mice in the blank control group was relatively intact,and there was no abnormality of lung structure.In the model group,the sections showed extensive infiltration of inflammatory cells into the trachea and around the blood vessels,bronchial smooth muscle thickening,reduced ventilation range,and goblet cell proliferation in the inner wall of the trachea.Airway inflammatory infiltration and airway structural changes in mice of each treatment group were milder than those in the model group.Results of F4/80 staining:Macrophage accumulation was observed around the airways in the model group,but not in the blank control group and the medium dose group of Huanglong Decoction.(3)Effect of Huanglong Decoction on intracellular calcium concentration of airway smooth muscle cells stimulated by IL-4:The intracellular calcium level of airway smooth muscle cells in IL-4 group increased,and the calcium concentration in Huanglong group was lower than that in IL-4 group(P<0.05).(4)Effect of Huanglong Decoction on the expression of α-sma,STAT6,P-STAT6 and CaSR protein in airway smooth muscle cells stimulated by IL-4:There was no significant difference inα-sma and STAT6 protein expression among the groups(P>0.05),P-STAT6 protein expression increased with increasing stimulation concentration,and P-STAT6 expression was lower in the Huanglong Decoction group than in the IL-4 group(P<0.05),CaSR expression was lower in the Huanglong Decoction group than in the IL-4 group(P<0.05).Conclusions:1.Huanglong Decoction can significantly improve the symptoms of OVA-induced CVA young mouse model,reduce airway and lung pathological changes,so as to play a role in the treatment of cough variant asthma.2.Huanglong Decoction can inhibit the airway hyperresponsiveness,airway inflammation and immune response of CVA by inhibiting the IL-4/P-STAT6 pathway expression level.At the same time,Huanglong Decoction can inhibit the protein expression level of CaSR,regulate the imbalance of CaSR-mediated calcium homeostasis,and improve the abnormal contraction of airway smooth muscle stimulated by inflammatory factors,thereby reducing airway hyperreactivity.3.Huanglong Decoction may reduce airway hyperresponsiveness through multi-target effects related to airway smooth muscle contraction and has the potential to be developed as a new drug for pediatric CVA specific diseases.