Research On The Network Regulation Characteristics And The Compatible Effect And Mechanism Of San’ao Decoction With Scorpio And Bombyx Batryticatus On Cough Variant Asthma

Objective:Cough variant asthma(CVA)is the most common cause of chronic cough,and it belongs to the category of wind induced cough in traditional Chinese medicine.This research is to sort out the features of wind induced cough in ancient and modern times according to tradition Chinese medicine,to explain the network regulation characteristics of the full prescription and the components of San’ao decoction with scorpio and bombyx batryticatus(SSB)in treating CVA,as well as to explore the effect and mechanism of SSB and its components in treating CVA through experiments in vivo and in vitro.Methods:1.The characteristics of the theory,pathogenesis and treatment of wind induced cough were systematically sorted out from the ancient medical records as well as modern published literature.2.Based on the researching strategy of network formulaology,the resources from public online database and published literature were used to search for the components and its potential targets of SSB in treating CVA.Analytical methods were used to explain and compare the network bioinformatic features and difference between SSB and its components in treating CVA,including gene ontology enrichment analysis,KEGG pathway enrichment analysis,protein-protein interactions as well as topological parameter analysis.3.Ovalbumin sensitization and challenge were used to establish the CVA mice model.The measurement indicators were:mice cough counts within 5min induced by capsaicine,eosinophils(EOS)counts in peripheral blood,inflammatory cells amout in brochoalveolar larage fluid(BALF),airway hyperresponsiveness(AHR)changes measured by whole body plethysmography(WBP),histopathological changes in lung tissues by HE staining,levels of IL-4,IL-13,NGF,CGRP and PGE2 in BALF by ELISA method,as well as the gene and protein expressions of TRPA1,TRPV1,and TRPV5 in both lung and medulla oblongata tissues by qPCR method and Western blotting method respectively.The difference in the effect and mechanism characteristics of SSB and its components in treating CVA were explored and analyzed.4.The inflammatory injury model of 16HBE cells was established induced by sulfur dioxide derivatives.The cytotoxicity of drugs and the effects of SSB lyophilized powder on cell survival rate were measured by MTT method.Cell reactive oxide species(ROS)level were detected by flow cytometry.The gene expressions of TRPV1 and TRPV5 in 16HBE cells were tested by qPCR method as well.Results:1.CVA belongs to wind-induced cough category.The meaning of wind induced cough in ancient times was rather generalized.The main etiology of wind induced cough included wind,coldness,heat,dryness and dampness,as well as stagnation phlegm and fire,deficiency of Qi and blood,emotions,etc.The pathogenesis included wind-coldness attacking lung,wind-heat attacking lung,stagnation of phlegm and fire in lung,endogenous wind in liver,and so on.The treatments were relieving exterior syndrome,diffusing lung and suppressing cough,reducing phlegm,tonifying spleen to nourish lung,etc.Modern doctors thought that wind induced cough manily showed as urgent,paroxysmal and spasmous cough.It was mainly induced or aggravated by cold air,allergen factors,etc.Diseases such as CVA,eosinophilic bronchitis,allergic cough belonged to the category of wind induced cough.The etiology and pathogenesis of wind induced cough in modern times included wind attacking lung,spasmous airway,the accumulation of internal wind and phlegm,liver wind agitation,the impairment of lung Qi,etc.The treatments included dispelling wind and ventilating lung,relieving spasmolysis,suppressing cough and relieving sore throat,eliminating phlegm and freeing channels,extinguishing wind and calming liver,and so on.Besides,patients were treated differently in different stages according to the characteristics of patients’ age,disease development,etc.Furthermore,it was recorded that ancient doctors used insect medicines in treating cough,while doctors in modern times also made good use of insect medicines to enhance the effect of dredging collaterals and relieving spasm during the treatment of wind induced cough.2.A total of 2082 targets related to CVA were collected.There were 530 compounds in SSB working on 1873 targets.(1)there were 108 potential targets of SSB in treating CVA,included SCN5A,MAPK,CYP3A4,TNF,TRPV1,EGFR,IL1β,IL-6,etc.Main compounds included quercitrin,kaempferol,tenellin,beauvericin,formononetin,etc.Targets of SSB in treating CVA mainly involved cellular response to stimulus,transcriptional regulation,translocation,antioxidation,and so on,as well as worked on the pathways of IL-17,MAPK,TNF,BDNF,NGF,regulation of TRP channels by inflammatory mediators,etc.(2)There were 84 potential targets of San’ ao decoction(SAD)in treating CVA,while 57 targets of scorpio and bombyx batryticatus(JQ)in treating CVA.Further analytic results showed that targets from SAD in treating CVA were mainly involved in the cellular response to chemical stimulus and oxygen-containing compounds,participating in cytokine receptor binding,cytokine activity and other functions,and mainly acted on signaling pathways such as IL-17,HIF-1,TNF,as well as Th17 cell differentiation and cytokine receptor interaction;targets from JQ in treating CVA were mainly involved in vascular regulation,regulation of enzyme activity,and mainly acted on calcium signaling pathway,stimulating ligand/receptor interaction in nerve tissue,vascular endothelial growth factor signaling pathway,etc.3.The experimental results showed that SSB could significantly reduce CVA model mice cough counts induced by capsaicin(P<0.05,P<0.01),decrease EOS numbers in peripheral blood(P<0.01)and white blood cell numbers in BALF(P<0.01),decrease AHR(P<0.05,P<0.01),improve histopathological changes in lung tissues,reduce the levels of IL-4,IL-13,NGF,CGRP and PGE2 in BALF(P<0.05,P<0.01),down regulate the gene and protein expressions of TRPA1,TRPV1,TRPV5 in both lung and medulla oblongata tissues(P<0.05,P<0.01),as well as increase cellular viability(P<0.05),reduce ROS level(P<0.05),and decrease TRPV1 and TRPV,mRNA expressions in 16HBE cells(P<0.05).Furthermore,in SSB group:cough counts were less than that in SAD group(P<0.05),EOS numbers and white blood cell numbers were lower than that in JQ group(P<0.05,P<0.01),AHR Penh value was lower than that in SAD group(P<0.05)and JQ group(P<0.01),IL-13 level was lower than that in JQ group(P<0.01),TRPA1 mRNA expressions were lower than that in JQ group(P<0.05),TRPV1 mRNA and protein expressions were lower than that in JQ group(P<0.05),TRPV5 mRNA and protein expressions were lower than that in SAD group(P<0.05,P<0.01).Besides,EOS numbers and white blood cell numbers in SAD groups were both lower than that in JQ group(P<0.05),and PGE2 level in BALF of JQ group was lower than that in SAD group(P<0.05).Conclusions:1.The theory of wind induced cough in ancient times were rather generalized and abundant.Modern doctors thought CVA may belong to wind induced cough.Its pathogenesis included wind attacking lung,spasmous airway,the accumulation of internal wind and phlegm,the impairment of lung Qi,etc.,and the treatments included dispelling wind and ventilating lung,relieving spasmolysis,suppressing cough and relieving sore throat,eliminating phlegm and freeing channels,and so on.2.The potential targets of SSB in treating CVA involved immunoinflammatory regulation,nervous system,oxidation reaction,TRP channels regulation,etc.There were differences of the characteristics between SAD and JQ in CVA treatment.SAD emphasized on the cellular respose to external stimulus and involved in immune regulation,inflammation reactions,and so on.JQ mainly took part in the intracellular changes,and worked on the regulation of metabolic enzymes,vascular regulation,and calcium signaling pathway,etc.3.SSB could reduce model mice cough counts,reduce EOS numbers in peripheral blood and white blood cells in BALF,decrease AHR,improve histopathological changes in lung tissues.The mechanism included the regulation of IL-4,IL-13,NGF,CGRP and PGE2 levels in BALF,regulation of the gene and protein expressions of TRPA1,TRPV1,TRPV5 in both lung and medulla oblongata tissues,as well as the protective effect on 16HBE cells.Generally,the effects of SSB were better than its components,and the effects of different components emphasized in different aspects.SAD mainly worked on the improvement of airway inflammation,regulation of TRPA1 and TRPV1 channels in lung and medulla oblongata tissues,while JQ mainly worked on reducing cough counts,improving neurogenic inflammation,and regulation of TRPV5 channel in lung and medulla oblongata tissues.