Study Of Evaluation Of Clinical Efficacy And Molecular Mechanism Of Wenfei Shufeng Fang In The Treatment Of Cough Variant Asthma

Cough variant asthma(CVA)is one of the leading causes of chronic cough with high incidence,and it is prone to recurrence.This seriously affects the quality of life of patients.The guidelines recommend inhalation of glucocorticoids/beta-receptor agonists(ICS/LABA)as the preferred treatment for CVA.However,due to long treatment period and resistance to hormone,most patients have poor compliance with hormone therapy.Studies have confirmed that even though CVA symptoms can be effectively controlled after standard inhalation of ICS/LABA,cough symptom and airway inflammation can aggravate again after a period of drug withdrawal,and lung function can also deteriorate again.So stopping or discontinuing medication is considered an unwise choice.Therefore,how to reduce the use of inhalation drugs and reduce the aggravation of cough symptom,lung function and airway inflammation after withdrawal while the cough symptom can be effectively controlled during the treatment is a more important point for research.The treatment of cough with Traditonal Chiese Medicine has a long history.Doctors believe it should belong to the category of“wind cough”and the treatment of CVA should be to disperse the wind and diffuse the lung.In the clinical practice of CVA,we find CVA not only have obvious characteristics of wind”,but also have characteristics of“deficiency”and“cold”.It suggest the treatment of CVA is not only to disperse wind and diffuse the lung,but also to warm the lung.Based on early clinical observation,we found that ICS/LABA combined with Wenfei Shufeng Fang could improve the cough symptom for a long time and reduce the recurrence frequency,thus reducing the use of ICS/LABA.Therefore,we believe that the advantages of Wenfei Shufeng Fang combined with ICS/LABA in the treatment of CVA may be in reducing cough recurrence,improving cough symptom,lung function and airway inflammation.In this study,a randomized,double-blind,placebo-controlled trial will be conducted to evaluate the clinical efficacy of Wenfei Shufeng Fang.The possible pathways of Wenfei Shufeng Fang in the treatment of CVA will be screened by means of network pharmacology,and experimental studies will be carried out to explore the molecular mechanism of Wenfei Shufeng Fang in the treatment of CVA.Objective1.To objectively evaluate the safety and efficacy of Wenfei Shufeng Fang in the treatment of CVA through a randomized,double-blind and placebo-controlled clinical trial.Clarify the advantages of Wenfei Shufeng Fang in the treatment of CVA.2.Through the method of network pharmacology,the possible pathways of Wenfei Shufeng Fang in the treatment of CVA were screened.And it provided theoretical basis for the study of its mechanism.3.The pathways screened by network pharmacological were used as a breakthrough point to explore the mechanism of Wenfei Shufeng Fang in the treatment of CVA.Methods1.A randomized,double-blind,placebo-controlled trial was conducted.The subjects were patients diagnosed with CVA by doctors of respiratory outpatient department and Lingnan Zhenshi Miscellaneous Diseases School of of Guangdong Province Hospital of Chinese Medicine.Patients in the experimental group took granules of Wenfei Shufeng Fang,while patients in the control group were given the matched placebo.An administration of salmeterol/fluticasone propionate combination for 12 weeks was the basic therapy for the two groups.The cough visual analogue score,Leicester quality of life score and lung function were observed and compared between the experimental group and the control group at 4,8 and 12 weeks after treatment,12 and 24 weeks after drug withdrawal.The cumulative recurrence rate of cough symptom and the anti-inflammatory treatment rate of recurrent patients at 12 and 24 weeks after drug withdrawal were also compared,and the safety of Wenfei Shufeng Fang was evaluated.2.The effective chemical constituents and corresponding targets of Wenfei Shufeng Fang were inquired through TCMSP database,and the related targets of asthma were inquired through Disgenet,OMIM,Drugbank and KEGG database.Then the targets of Wenfei Shufeng Fang were mapped to the targets of asthma,and the possible targets of Wenfei Shufeng Fang on asthma were obtained.Using String platform to construct the in vivo response network of Wenfei Shufeng Fang on asthma.According to the protein-protein interaction score,the interaction relationships with high score were screened.The key targets of Wenfei Shufeng Fang on asthma were obtained by network topology analysis with Cytoscape software.The DAVID database was used to enrich and analyze KEGG signaling pathways of mapped targets,and the possible pathways of Wenfei Shufeng Fang on asthma were obtained.The key targets and enriched pathways of Wenfei Shufeng Fang on asthma were comprehensively analyzed,and choose one pathway that was closely related to asthma for animal experiment.3.The asthma model of mouse was established by intraperitoneal injection of ovalbumin(OVA)and aerosol stimulation.Experimental mice were randomly divided into normal group,model group,dexamethasone group and Wenfei Shufeng Fang group,with 8 in each group.The model group,dexamethasone group and Wenfei Shufeng Fang group were sensitized by intraperitoneal injection of OVA and aluminium hydroxide mixture on the 0th,6th and 12th day of the experiment,while the normal group was sensitized by intraperitoneal injection of phosphate buffer(PBS).The model group,dexamethasone group and Wenfei Shufeng Fang group were stimulated for 40 minutes by nebulization of the OVA solution,while the normal group were stimulated for 40 minutes by nebulization of the PBS.The mice of the Wenfei Shufeng Fang group were intragastric administrated with Wenfei Shufeng Granule at dose of 2.55g/kg 1 hour before each stimulation.The mice of the dexamethasone group were injected with dexamethasone intraperitoneally at dose of 2.5mg/kg 1 hour before each stimulation.The mice of the normal group and model group were intragastric administrated with 0.5 ml saline 1 hour before each stimulation.Observe the performance of mice in each group during aerosol stimulation.Invasive pulmonary function was performed to detect airway resistance within 48 hours after treatment.The bronchoalveolar lavage of mice was collected and the liquid supernatant was used for ELISA detection after centrifugation.The cell precipitation was suspended for smears to count and classify nucleated cells.HE staining was used to observe the pathological changes of lung tissue,and PAS staining was used to observe the proliferation of goblet cells in airway epithelium.ELISA was used to detect the contents of inflammatory factors IL-6,TNF-a and IL-1? in bronchoalveolar lavage fluid.Real-time PCR was used to detect the mRNA expression level of key targets TLR4?NF-? Bp65?p38MAPK?Erk1/2 and JNK in the TLR4-NF? B/MAPK signaling pathway.Western Blot was used to detect the protein expression level of TLR4?p-NF-? Bp65?p-p38MAPK?p-Erk1/2 and p-JNK.Results1.The clinical study included 80 cases.Four subjects were excluded.Intention-to-treat analysis(ITT)included 76 cases and per-protocol analysis(PPS)included 57 cases.The results of the two analyses are consistent.(1)Analysis of cough visual analog scales:There were no significant differences in cough visual analogue scores between the two groups at weeks 4,8,12,12 and 24 weeks after withdrawal(P>0.05).Compared with before treatment,the cough visual analogue scores of the two groups decreased significantly at each time point after treatment(P<0.05).(2)Analysis of cumulative relapse rate of cough symptoms:There were no significant differences in the cumulative relapse rate of cough symptoms between the experimental group and the control group at weeks 12 and 24 after drug withdrawal(P>0.05).(3)Analysis of the re-anti-inflammatory treatment rate of recurrent patients:There were no significant differences between the two groups in the rate of re-anti-inflammatory treatment for relapsed cough symptoms at weeks 12 and 24 after drug withdrawal(P<0.05).(4)Analysis of lung function:There were no significant differences in FVC?FEV1?MEF75?MEF50 and MEF25 between the two groups at weeks 12,12 and 24 weeks after withdrawal(P>0.05).There was significant difference in MEF75 of the experimental group after treatment and before treatment(P<0.05).(5)Analysis of total score of Leicester Cough Quality of Life Questionnaire:There were no significant differences in total scores of Leicester Cough Quality of Life Questionnaire between the two groups at weeks 4,8,12,12 and 24 weeks after withdrawal(P>0.05).Compared with before treatment,the total scores of Leicester Cough Quality of Life Questionnaire of the two groups decreased significantly at each time point after treatment(P<0.05).(6)Analysis of combined drug use:Both groups used drugs that had influences on observation of the efficacy,but there was no significant difference in the utilization rate(P>0.05).(7)Analysis of safety:There was no significant difference in abnormal changes of laboratory indicators between the two groups after treatment(P>0.05),and there was no significant difference in the incidence of suspicious adverse reactions between the two groups(P>0.05).2.A total of 239 human target proteins and 1413 human genes related to asthma were obtained by TCMSP database.110 targets were obtained from the overlaps of the targets of Wenfei Shufeng Fang and asthma.17 key targets including JUN,STAT3,QBHIJ4,TNF,EDN1 and MAPK et al were obtained through topological analysis.11 Kegg pathways of Wenfei Shufeng Fang were obviously enriched.Highly enriched pathways include TNF signaling pathway,Toll-like receptor signaling pathway,MAPK signaling pathway,cAMP signaling pathway,NOD-like receptor signaling pathway and so on.The key targets and enriched pathways of Wenfei Shufeng Fang on asthma were comprehensively analyzed.On the basis of literature review,TLR4-NF-K B/MAPK signal pathway,which was highly related to asthma,was selected to study.3.The results of animal experiments:(1)Compared with the model group,The airway resistance of mice in Wenfei Shufeng Formula group decreased significantly.The difference is signifcant(P<0.05).(2)Compared with the model group,The proportion of eosinophils in bronchoalveolar lavage fluid of mice in Wenfei Shufeng Formula group decreased significantly.The difference is signifcant(P<0.05).(3)Compared with the model group,The infiltration of inflammatory cells in lung tissue and the proliferation of goblet cells in airway epithelium were reduced in Wenfei Shufeng Fang group.(4)Compared with the model group,The expression levels of IL-6,TNF-? and IL-1? in alveolar lavage fluid in the mice of Wenfei Shufeng Fang group decreased significantly.The difference is signifcant(P<0.05).(5)Compared with the model group,the mRNA expression levels of TLR4?NF-? Bp65?p38MAPK?Erkl/2 and JNK in lung tissue of Wenfei Shufeng Fang group decreased significantly.The difference is signifcant(P<0.05).(6)Compared with the model group,the protein expression levels of TLR4?p-NF-K Bp65?p-p38MAPK?p-Erkl/2 and p-JNK in lung tissue of Wenfei Shufeng Fang group decreased significantly.The difference is signifcant(P<0.05).Conclusion1.Compared with inhalation of salmeterol/ticasone powder,this study did not find the advantages of Wenfei Shufeng Fang combined with inhalation of salmeterol/ticasone powder in improving cough symptoms and quality of life of CVA patients during the treatment and within 24 weeks after withdrawal.2.Compared with inhalation of salmeterol/ticasone powder,Wenfei Shufeng Fang combined with inhalation of salmeterol/ticasone powder has a tendency to reduce the recurrence rate of cough symptoms and it can reduce the rate of re-anti-inflammatory treatment in relapsed patients.3.Compared with before treatment,Wenfei Shufeng Fang combined with inhalation of salmeterol/ticasone powder can improve small airway function(MEF75)in CVA patients.4.Wenfei Shufeng Fang may play an anti-inflammatory role in CVA by inhibiting TLR4-NF-K B/MAPK signaling pathway.