Study On Rules Of Syndrome Of Prescription And Mechanism In The Intervention Of Jiawei Shengjiang Powder On Bronchial Asthma

Objective:As a common heterogeneous disease of the respiratory system,the prevalence of bronchial asthma(asthma)is increasing year by year in the world,reaching 4.2%in Chinese adults.Asthma control for most patients is hard to achieve.Western medicine uses inhaled glucocorticoids,systemic hormones,β2 receptor agonists,leukotriene modulators,and other methods to control and alleviate the disease,while Chinese medicine applies methods such as promoting lung and eliminating phlegm,relieving wind and spasmolysis to treat asthma with unique advantages.Composed of Bombyx Batryticatus,Cicadae Periostracum,Rhei Radix Et Rhizoma,Curcumae Longae Rhizoma,Fritillariae Thunbergii Bulbus,Uncariae Ramulus Cum Uncis,and Mori Cortex,Jiawei Shengjiang Powder is an effective TCM formula for prevention and treatment of asthma based on the method purging the viscera and regulating the lung,dispelling wind and resolving phlegm.The preliminary clinical trial of our research group found that the Jiawei Shengjiang Powder was effective in the treatment of asthma,significantly alleviating major clinical manifestations such as shortness of breath and wheezing in the acute exacerbation stage,improving lung function and reducing eosinophil level.On the basis of early clinical study,this series of studies carried out follow-up studies such as clinical data-mining and basic experiments.According to the treatment literature of modified Shengjiang Powder,we clarified the research direction.Relying on data mining,we explored the four diagnostic information and the distribution law of prescriptions of Jiawei Shengjiang Powder in the treatment of asthma.Network pharmacology and molecular docking was to explore the effective components and potential mechanism of Jiawei Shengjiang Powder in intervening asthma through the "gut-lung axis".And through animal experiments,we studied the effect of Jiawei Shengjiang Powder on airway inflammation,airway hyperresponsiveness and airway remodeling caused by asthma,based on the short chain fatty acid-GPCR-ERK signaling pathway of "gut-lung axis" to illustrate the mechanism of Jiawei Shengjiang Powder in the intervention of asthma.Methods:This manuscript was divided into five chapters.Chapter 1 included the discussion and visual analysis of the clinical efficacy of modified Shengjiang Powder in the treatment of asthma.Chapter 2 showed the data mining research of diagnostic information and prescription rules of Jiawei Shengjiang Powder in the treatment of asthma.The basic research involved the network pharmacology and molecular docking research of Jiawei Shengjiang Powder intervening asthma through "gut-lung axis"(Chapter 3),observation of the effect of Jiawei Shengjiang Powder on airway inflammation,remodeling and responsiveness of asthma through animal experiments of pharmacodynamics(Chapter 4),and explanation of the mechanism of Jiawei Shengjiang Powder’s intervention on asthma through short chain fatty acid GPCR-ERK signaling pathway of "gut-lung axis"(Chapter 5).(1)Researchers applied CiteSpace 5.7.R2 and VOSviewer 1.6.15 to count and analyze the author,organization,key words and their changes by searching for the literatures about Shengjiang Powder in treating asthma in CNKI database.(2)The symptoms,tongue and pulse information of patients with asthma and corresponding prescriptions containing Jiawei Shengjiang Powder was collected.Researchers established an information database and explored the information of TCM diagnosis and the distribution of prescriptions and medicines by frequency statistics,correlation analysis,cluster analysis and other statistical methods based on SPSS 26.0,SPSS modeler 18.0 and traditional Chinese Medicine Inheritance auxiliary platform.(3)Through TCMSP,Batman-TCM and other databases,combined with literature,the main effective components and corresponding prediction targets of Jiawei Shengjiang Powder were searched and integrated,and the actual drug components were identified by UHPLC-MS/MS.The intersection targets of asthma and "gut-lung axis" were screened by Genecards,CTD,etc.The traditional Chinese medicine component and component-target network of Jiawei Shengjiang Powder were constructed by using Cytoscape 3.7.2 software.Then,researchers sought for the common targets of the compounds-asthma-gut-lung axis,and constructed PPI network by STRING database.The key targets were screened by topology operation,cytohubba,and MCODE of Metascape.David database was used for GO and KEGG analysis of core targets to explore biological processes,cell components,molecular functions,and related signaling pathways.Molecular docking was simulated based on CB-DOCK.(4)SD rats were randomly divided into normal group,model group,budesonide group and Jiawei Shengjiang Powder group.After ovalbumin sensitization and stimulation,the asthma model was successfully established.The budesonide group was given Budesonide inhalation.And the Jiawei Shengjiang Powder group was given Jiawei Shengjiang Powder by gavage.The model group was given control intervention,while the normal group served as blank control.Researchers observed behavioral changes such as shortness of breath,coughing and sneezing.FEV0.3,FVC,FEV0.3/FVC,PEF and Ri,Re,Cdyn airway provocation test were detected.According to the predicted targets of network pharmacology and Th2 response,the contents of IL-5,IL-6,TNF-α and ERK2 in serum and BALF of rats were determined by ELISA.Lung and colon tissues were stained with hematoxylin-eosin(HE),Masson and PAS.(5)The contents of acetic acid,propionic acid,butyric acid,isobutyric acid,valeric acid,iso-valeric acid,hexanoic acid and isohexanoic acid in feces of rats in each group were determined based on gas chromatography coupled ion mobility spectrometry and mass spectrometry(GC-MS).The protein expressions of GPR41,GPR43,ERK1/2,p-ERK1/2,H3,AC-H3 in lung tissue and GPR41,GPR43,ERK1/2,p-ERK1/2 in colon tissue were detected by Western blot and Immunohistochemistry(IHC).RT-qPCR was applied to detect the mRNA expression levels of GPR41,GPR43,ERK1 and ERK2 in lung tissues and colon tissues of rats in each group.Results:(1)A total of 92 literatures were included,involving 20 major institutions.The top 20 key words implied that there were multi-dimensional and multi-level research types involving clinical trials,basic experiments,experience of famous TCM physicians.The relative studies were mainly based on the theory of latent phlegm,Nine geographical palaces and eight directional winds,gut-lung correlation.The prescriptions of Shengjiang powder and Jiawei Shengjiang powder were aimed at the treatment of allergic asthma,cough-variant asthma and other phenotypes of asthma following the principle of lung and intestine combined treatment and other treatment principles and methods.(2)The core syndromes in the treatment of asthma with Jiawei Shengjiang Powder were gasp for breath,wheezing in the throat,cough,nasal itching,wheezing with odor,chest tightness,sticky and white phlegm,runny nose,shortness of breath,soft stool/dry stool,red tongue with white or white and greasy coating,and slippery pulse.Wheezing had a high correlation with other information.Cluster analysis showed five types of syndrome types:wind-phlegm obstruction of lung,phlegm-heat obstruction of lung,Qi deficiency of lung and kidney,Qi stagnation in lung,wind-phlegm obstruction of lung and sthenia-viscera syndrome.And the main TCM syndromes were wind-phlegm obstruction of lung and wind-phlegm obstruction of lung and sthenia-viscera syndrome.Herbs in Traditional Chinese medicine prescriptions combined with Jiawei Shengjiang Powder for the treatment of asthma are mainly with cold Qi and bitter,sweet,pungent flavours,which attributed to the lung,liver,stomach,spleen,large intestine meridian.We get the combined TCM prescriptions of Jiawei Shengjiang Powder,which included Sangmei Zhike decoction,Qianjin Weijing decoction and so on.Medicine combinations include Armeniacae Semen Amarum,Eriobotryae Folium,and Glycyrrhizae Radix Et Rhizoma(cluster 1),Magnoliae Flos,Xanthii Fructus,Menthae Haplocalycis Herba(cluster 2).(3)In the network pharmacology research,94 kinds of active ingredients and 152 key prediction targets were obtained,and the drug-component and component-target network of Jiawei Shengjiang Powder was constructed.There were 5973 asthma targets,2241 gut-lung axis targets and 661 intersection targets.Then 60 common targets of Jiawei Shengjiang Powder and asthma-gut-lung axis were selected,and PPI network was deduced based on STRING database.Through topological operation,30 targets were selected,and the top targets such as TNF、IL6、MAPK8、MAPK1 were obtained based on cytohubba.Based on the MCODE of Metascape,two main functional modules are obtained.Go analysis of David database showed that there were biological processes involved in G-protein-coupled acetylcholine receptor signaling pathway,cell components including postsynaptic membrane,molecular functions including G-protein coupled acetylcholine receptor activity and MAPK activity.KEGG analysis showed that there were signal pathways including TNF,HIF-1,PI3K-Akt,VEGF,NOD like receptor signal pathway,FoxO,Toll like receptor,T cell receptor,calcium,MAPK and cAMP.The top three small molecules were linked with the top five MCC targets IL6,PTGS2,TNF,VEGFA and Jun of cytohubba respectively,and the simulation results were obtained.The top three small molecules of quercetin,kaempferol β-sitosterol according to topology were docked with the top five targets of cytohubba,and the simulation results were obtained.(4)Through animal experiments of pharmacodynamics,results were as follows:①Construction of model:Model of asthma was constructed successfully.②Behavior:Compared with the normal group,the rats in the model group showed obvious wheezing,dyspnea and abdominal contraction.Compared with model group,rats in budesonide group and Jiawei Shengjiang Powder group had less wheezing,dyspnea and abdominal contraction.③Pulmonary function:In terms of pulmonary ventilation function,the levels of FEV0.3,FVC,PEF and FEV0.3/FVC in the model group were significantly lower than those in the normal group.The levels of FEV0.3 and FEV0.3/FVC in budesonide group and Jiawei Shengjiang Powder group were higher than those in model group.Airway provocation test showed that the inspiratory phase resistance of the model group was significantly higher than that of the normal group.Compared with the model group,budesonide group and Jiawei Shengjiang Powder group showed that intervene could inhibit the continuous increase of biphasic resistance and the decrease of Cdyn at different concentrations.④Cell count in blood and BALF:The EO count in the model group was significantly increased while the EO count in the budesonide group and Jiawei Shengjiang Powder group was lower than that in the model group.The EO count in BALF of model group was significantly higher than that of normal group,while the EO percentage of budesonide group and Jiawei Shengjiang Powder group decreased,and the EO of Jiawei Shengjiang Powder decreased with statistical difference.⑤ELISA:We detected ERK2,IL-6,IL-5 and TNF in serum and BALF.The levels of serum ERK2,IL-6,IL-5 and TNF in the model group were significantly higher than those in the normal group.The levels of serum ERK2,IL-6,IL-5 and TNF in budesonide group and Jiawei Shengjiang Powder group were lower than those in model group.The levels of ERK2,IL-6,IL-5 and TNF in BALF of model group were significantly higher than those of normal group.The contents of ERK2,IL-6,IL-5 and TNF in BALF of budesonide group and Jiawei Shengjiang Powder group were significantly lower than those of model group.⑥Histopathological staining of HE,Masson,PAS:Lung histopathology in model group showed airway mucosal epithelial structure disorder,inflammatory cell infiltration,increase of the collagen deposition in airway wall,and dense proliferation of goblet cells.Colonic histopathology showed infiltration of many inflammatory cells in the mucosa,destruction of mucosal structure,atrophy and reduction of goblet cells.The above pathological changes were mild in budesonide group and Jiawei Shengjiang Powder group.(5)Determination of short chain fatty acid content,proteins and genes are as follows:① Determination of short chain fatty acid content based on GC-MS:The content of propionic acid,butyric acid and valeric acid in model group was significantly lower.Besides,the content of propionic acid and butyric acid in budesonide group was significantly higher than that in model group,and the content of acetic acid,propionic acid,isobutyric acid and butyric acid in Jiawei Shengjiang Powder group was significantly higher than that in model group.②Western blot and IHC showed that the protein expression levels of GPR41 and GPR43 in the model group were lower than those in the normal group,and the protein expression levels of ERK1/2,pERK1/2,H3 and AC-H3 were higher.The protein expression levels of GPR41 and GPR43 in budesonide group and Jiawei Shengjiang Powder group were significantly higher than those in model group,while the protein expression levels of ERK1/2,p-ERK1/2,H3 and AC-H3 in budesonide group and Jiawei Shengjiang Powder group were lower than those in model group.The expression of GPR41 and GPR43 in colon tissue of model group was lower than that of normal group,and the expression of ERK1/2 and p-ERK1/2 was higher than that of normal group.The protein expression levels of GPR41 and GPR43 in budesonide group and Jiawei Shengjiang Powder group were significantly higher than those in model group,while the protein expression levels of ERK1/2 and p-ERK1/2 were significantly lower.③RT-qPCR:In lung and colon tissues,mRNA expression levels of GPR41 and GPR43 in the model group were not significantly different from those in the normal group,while mRNA expression levels of ERK1 and ERK2 were higher.After intervention with budesonide and Jiawei Shengjiang Powder,the mRNA expression levels of GPR41 and GPR43 were higher than those in the model group,while the mRNA expression levels of ERK1 and ERK2 were lower.Conclusion:Through clinical research literature,it is preliminarily confirmed that modified Shengjiang Powder is effective in the treatment of asthma which based on the traditional Chinese medicine theory of " the lung and the large intestine are exterior and interior ".The core syndrome of TCM diagnostic information in the treatment of asthma with Jiawei Shengjiang Powder is wind-phlegm obstruction of lung.The rules of combined TCM prescription and modified medicine of Jiawei Shengjiang Powder proved that Sangmei Zhike decoction and Qianjin Weijing decoction were frequently-used formulas purging the viscera and regulating the lung,and dispelling wind and resolving phlegm.The process is to regulate the Qi movement of liver and lung,and harmonize the exterior and interior relationship of lung and intestine.The network pharmacology research preliminarily suggests that Jiawei Shengjiang Powder may regulate G protein coupled acetylcholine receptor and other biological processes,affect MAPK activity and other molecular functions,regulate TNF,MAPK and other signal pathways.Those key targets are MAPK1(ERK2),IL6,TNF,etc.Through the intervention of Jiawei Shengjiang Powder,the behavior,lung function index,lung and intestinal histopathology of asthma model rats were improved in varying degrees.The contents of pro-inflammatory cytokines such as IL-6 and TNF in serum and BALF decreased,and the level of eosinophils decreased.The experiment suggests that Jiawei Shengjiang Powder has influence of overall adjustment and comprehensive intervention on airway inflammation,airway hyperresponsiveness and airway remodeling.The internal mechanism of Jiawei Shengjiang Powder in the intervention of model rats is related to shortchain fatty acid-GPCR-ERK signaling pathway through the "gut-lung axis".We conclude that this formula can effectively increase the content of acetic acid,propionic acid,isobutyric acid,butyric acid and other short chain fatty acids,and act on the corresponding short chain fatty acid receptors.It can up regulate the protein and gene expression of protein GPR41 and GPR43,down regulate the protein and gene expression of ERK1/2,and inhibit the protein phosphorylation process.Thus the mechanism of Jiawei Shengjiang Powder in the treatment of asthma is preliminarily explained at the micro level.