Research On The Mechanism Of Action Of Phlegm-resolving And Collateral-dredging Method In Treating Cough Variant Asthma In Children Based On Complex Networ

Objective:To summarize the clinical experience of Professor Wu Liqun’s treatment of children with cough variant asthma(CVA)with resolving phlegm and dredging collaterals method based on the data mining techniques.The target and signal pathway of Huatan Tongluo compound in the treatment of CVA were investigated by network pharmacology.The results of network pharmacology were verified through animal experiments,and the mechanism of the treatment of CVA guinea pigs with resolving phlegm and dredging collaterals method was preliminarily revealed,so as to provide reference and guidance for the clinical treatment of CVA in children and provide experimental basis for the clinical application of phlegm-removing and collateralremoving method.Research methods:1 Data mining section:The medical records of children with CVA diagnosed and treated by Professor Wu Liqun from January 2016 to December 2022 were collected.This thesis adopted the methods of following the principle of "integration of human and machine,focusing on experts",using online analysis technology and complex network methods to analyze the rules of syndrome and treatment and academic experience of Professor Wu Liqun’s treatment of children with cough variant asthma with resolving phlegm and dredging collaterals method.2 Network Pharmacology section:Huatan Tongluo Compound was analyzed by network pharmacology.TCMSP was used to obtain the active ingredients and targets of traditional Chinese medicine,and Genecards,OMIM,Disgenet,TTD and DrugBank were used to obtain the CVA targets respectively.Cytoscape and STRING platforms were used to construct proteinprotein interaction networks,and Metascape was used for gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis,and column and bubble graphs were drawn by microbiograph.3 Animal experiment section:40 healthy 4-week-old male clean Hartley guinea pigs were randomly divided into normal group,model group,Montelukast sodium group and Huatan Tongluo group,with 10 rats in each group.The ovalalbumin and aluminum hydroxide was used for modeling.After 14 days of intervention,the samples were taken after the coughing frequency was measured.The appearance and behavior of guinea pigs were observed.Hematoxylin-eosin staining and Masson staining,immunohistochemistry staining and immunofluorescence staining were used to observe the pathological changes of airway and blood vessels in lung tissue.The concentrations of IL-17,TGF-β1,VEGF and PDGF in serum and bronchoalveolar lavage fluid were detected by enzyme-linked immunoassay.The protein and gene expressions of p-ERK1/2,p-p38MAPK,Bcl-2,Bax,HIF-1α,VEGF,DLL4 and Notch4 in lung tissues were detected by Western blotting and real-time quantitative PCR.Research results:1 Data mining section1.1 General information:149 children with CVA who meet the nclusion and exclusion criteria were included in this study,384 treatment times.The male to female ratio was 96 to 53.The oldest age of first diagnosis was 18 years old,the youngest was 2 years old,and the average age was 5.90 years old.The age of onset was preschool age and school age.The longest course of disease was 5 years,the shortest was 1 month,and the average course of disease was 6.47 months.The past history of allergic rhinitis is more common.14.09%of the children have a clear history of allergy,9.40%have a clear family history of allergy,and most of them were first-degree relatives.1.2 Clinical characteristics and core prescriptions of CVA main syndromes:The syndrome of wind phlegm invading lung was most common,followed by the spleen deficiency phlegmdampness syndrome,spleen deficiency phlegm-heat syndrome,deficiency of lung and spleen,syndrome of spleen deficiency phlegm stasis,yin deficiency of lung heat and syndrome of wind heat invading lung.(1)The clinical characteristics of the syndrome of wind-phlegm invading lung include coughing,coughing after encountering cold air,coughing after exercise,less sputum,nasal congestion,runny nose,reddish tongue,thin white tongue coating,thin and smooth pulse,etc.The core prescriptions are stir-fried Raphani seeds,licorice,silver bupleurum,honey ephedra,Perilla seed,etc.(2)The clinical characteristics of spleen deficiency phlegmdampness syndrome include morning cough,night cough,white sputum cough,fatigue,poor appetite,pale complexion,reddish tongue,white and greasy tongue coating,thin and smooth pulse,etc.The core prescriptions were stir-fried seeds of Raphani,poria cocos,pinellia sinensis,honey aster,stir-fried bitter almond,etc.(3)The clinical characteristics of spleen deficiency phlegm-heat syndrome include cough,yellow sputum,cough after exercise,fatigue,dry stool,pharyngeal congestion,red tongue,thick and greasy tongue coating,pulse slip,etc.The core prescriptions are stir-fried seeds of Raphani,radix pseudostellariae,trichosanthes,earthdragon,stir-fried bitter almonds,etc.(4)The clinical characteristics of deficiency syndrome of lung and spleen include morning cough,night cough,cough after exercise,excessive sweating,fatigue,poor appetite,pale yellow complexion,pale red tongue,white tongue coating,thin or smooth pulse,etc.The core prescriptions are stir-fried white fungus with bran,stir-fried semen raphani,parsnips,astragalus,licorice,etc.(5)The common clinical features of yin deficiency lung heat syndrome include nocturnal cough,cough phlegm,dry throat,night sweat,red tongue,white or thin yellow tongue coating,thin pulse,etc.The core prescriptions were stir-fried Semen Raphani,licorice,Radix japonicus sinensis,wine and yellow essence,ophiopogon japonicus,etc.(6)The common clinical features of wind-heat lung syndrome include cough,nasal congestion,runny nose,fever,sore throat,dry stool,reddish or reddish tongue,thin yellow tongue coating,floating pulse,etc.The core prescriptions are honeysuckle,forsythia,fried burdock seed,fried bitter almond,fried Raphani seed,etc.1.3 CVA treatment prescriptions:Core prescriptions include stir-fried seeds of Raphani,licorice,trichosanthes,poria,stir-fried bitter almond,silver bupleurum,etc.The most common prescription is Modified Liu’an Decoction.The prescriptions with high frequency of coordination include Sanao Decoction and Sanzi Yangqin Decoction,Sanzi Yangqin Decoction and anaphylactic decoction,Sanao Decoction and anaphylactic Decoction,Yupingfeng Powder and anaphylactic Decoction,anaphylactic Decoction and Xanthium Powder,etc.The main types of drugs were heat-clearing drugs,phlegm-reducing cough and antiasthmatic drugs,tonifying deficiency drugs and relieving surface drugs.Commonly used drugs to promote blood circulation and collaterals are dilong,peach kernel,loofah,red peony root and so on.The properties of common traditional Chinese medicines are mainly warm,cold,slightly cold and flat,and the medicinal tastes are mainly sweet,bitter and occinative.The main channels of regulating meridian are lung meridian,stomach meridian,spleen meridian followed by and liver meridian,large intestine meridian and so on.2 Network Pharmacology sectionThere were 211 active components of Huatan Tongluo Compound prescription for CVA treatment,corresponding to 416 TCM targets,2349 CVA disease targets,215 intersections of the two targets,including AKT1,TNF,CASP3,VEGFA,PTGS2,BCL2L,BAX and other key targets.The main signaling pathways are MAPK,IL-17,TNF,VEGF,NF-κB,JAK-STAT,etc.3 Animal experiment section3.1 Effect of resolving phlegm and dredging collaterals method on body weight and airway sensitivity of CVA guinea pigs:(1)Body weight:The model group and drug intervention group showed an increasing trend compared with the normal group,but there was no significant difference between the drug intervention group and the model group.(2)Airway sensitivity:Cough frequency increased in the model group and decreased in the drug intervention group.3.2 Effect of resolving phlegm and dredging collaterals method on lung histopathology of CVA guinea pigs:Macroscopic observation:(1)The model group had dark red lung tissue,hyperemia and edema,petechiae and ecchymosis;(2)HE staining:In the model group,the bronchial basement membrane was thickened,lumen became smaller,mucus plug was visible,inflammatory cell infiltration,epithelial cell arrangement was disordered,and alveolar wall was destroyed under the microscope.In the drug intervention group,the lung tissue color became lighter,the bronchial basement membrane became thinner,the mucus thrombus was reduced,the lumen was relatively intact and unobstructed,the inflammatory cell infiltration was reduced,and the alveolar cavity was clear.(3)Masson staining:There were thicker collagen deposits around the airway in the model group,and thinner collagen deposits in the drug intervention group.(4)Airway morphological parameters:Wall area of bronchial tube/perimeter of basement membrane,wall area of bronchial smooth muscle/perimeter of basement membrane and collagen volume fraction in the model group were increased compared with the normal group,while the drug intervention group was decreased compared with the model group.(5)Cytokines:In serum and bronchoalveolar lavage fluid,the levels of IL-17,TGF-β1,VEGF and PDGF were increased in the model group,but decreased in the drug intervention group.3.3 Effect of resolving phlegm and dredging collaterals method on airway smooth muscle of CVA guinea pig:(1)Immunohistochemistry:Microscopically,the proliferation of airway smooth muscle cells was obvious in the model group,and decreased in the drug intervention group.(2)Immunofluorescence:Under microscope,the density of type I collagen,type III collagen and α-smooth muscle actin in lung tissue of model group was higher and concentrated,while the density of drug intervention group was decreased.(3)Average optical density value:Airway smooth muscle cells,type I collagen,type III collagen and α-smooth muscle actin increased in the model group,while decreased in the phlegm-clearing and collagal-clearing group.(4)Relative protein expression:p-ERK1/2,p-p38MAPK and Bcl-2 increased in model group,decreased in Huatan Tongluo group,Bax decreased in model group,and increased in huatan Tongluo group.(5)mRNA relative expression:p-ERK1/2,p-p38MAPK and Bcl-2 increased in the model group,decreased in the drug intervention group,Bax decreased in the model group,and increased in the drug intervention group.3.4 Effect of resolving phlegm and dredging collaterals method on blood vessels of CVA guinea pig lung tissue:(1)HE staining:In the model group,the vascular wall beside the air duct was thickened,the smooth muscle in the media layer was hyperplasia,and a large number of blood cells in the lumen;in the drug intervention group,the vascular wall was thinner,the smooth muscle in the media layer was thinner,and the lumen was unobstructed.(2)Pulmonary vascular morphological parameters:Lumen area/total vessel area decreased in model group,but increased in drug intervention group;Vascular smooth muscle layer area/total vascular area,vascular smooth muscle thickness/vascular outer diameter:Increased in model group,decreased in drug intervention group.(3)Relative protein expression:HIF-1α,VEGF and DLL4 increased in model group,decreased in Huatan Tongluo group;Notch4 decreased in model group,and increased in huatan Tongluo group.(4)mRNA relative expression level:HIF-1α,VEGF and DLL4 increased in model group,decreased in Huatan Tongluo group;Notch4 decreased in model group,and increased in huatan Tongluo group.Research conclusion:1 Professor Wu Liqun believes that the pathogenesis of CVA in children is deficiency of the original body,phlegm stasis in the lungs,and wind induces.The treatment should be slow and urgent and treated in stages.In the acute stage,it is necessary to attack evil,distinguish the outside and inside and treat it,fostering righteousness and dispelling evil,resolving phlegm and dredging collaterals,promoting spleen and stomach,fostering righteousness in the recovery period,nourishing qi and nourishing Yin,and dredging collaterals.Healthy spleen and stomach,resolving phlegm and dredging collaterals throughout the treatment.The combination of hard drugs,cold and temperature regulation,mainly lung meridian,stomach meridian,followed by spleen meridian,suit the pathogenesis of CVA.2 CVA is commonly used in Huatan Tongluo Compound prescription,which contains various active ingredients.By regulating multiple key targets and regulating multiple signaling pathways,CVA interferes with cell proliferation,differentiation and apoptosis,inhibits airway smooth muscle cell proliferation,extracellular matrix deposition,α-smooth muscle actin expression and vascular remodeling in lung tissue,thus reducing airway remodeling.Reduce airway hyperresponsiveness and thus play a role in the treatment of CVA.3 Resolving phlegm and dredging collaterals method may regulate the levels of IL-17,TGF-β1,VEGF and PDGF and the ERK/p38MAPK and VEGF/Notch signaling pathways and then interfere with the proliferation of airway smooth muscle and vascular smooth muscle,extracellular matrix deposition and α-smooth muscle actin expression in lung tissue,thereby improving airway remodeling,reducing airway hyperresponsiveness and relieve cough symptoms.