Study On The Theoretical Basis And Effect Mechanism Of Qufeng Xuanbi Formula On Asthma Airway Inflammation

Objectives:Qufeng Xuanbi therapy was proposed by Professor Shi Suofang for asthma with Fengtanzufei and Xiongyangbizu syndrome.On this basis,Qufeng Xuanbi formula was invented,which had a significant curative effect in the treatment of asthma.In this study,the asthma mouse model was induced by ovalbumin(OVA),and the TLR9 high expression RAW264.7 model was constructed with agonists to further study the anti-asthmatic effect and mechanism of Qufeng Xuanbi formula.Combined with Chinese medicine literature research,clinical application research of Qufeng Xuanbi therapy,network pharmacology research and lipid metabolomics research,this paper discussed the theoretical basis and effect mechanism of Qufeng Xuanbi formula in reducing asthma airway inflammation,so as to provide basis for the treatment of asthma.Methods:1.Collect asthma related literature and systematically sort out the etiology,pathogenesis and treatment methods of asthma.2.To summarize Professor Shi Suofang experience in treating asthma with Fengtanzufei and Xiongyangbizu syndrome.3.Explore the potential mechanism of Qufeng Xuanbi formula in the treatment of asthma by using the method of network pharmacology.4.In vivo experimentThe asthma mouse model was induced by ovalbumin(OVA),and the following tests were carried out with high and low doses of Qufeng Xuanbi formula respectively:(1)HE staining,Masson staining and PAS staining were used to observe the effects of Qufeng Xuanbi formula on airway inflammatory infiltration,collagen deposition and mucin secretion in asthmatic mice.(2)The secretion levels of IgE and Th2 cytokines IL-4,IL-5,IL-13 and proinflammatory cytokines IL-6,IL-1β and IL-18 in BALF were detected by ELISA.(3)The cells in BALF were classified and counted by whole blood cell counter.(4)The expression levels of TLR9,MyD88,NF-κB P65,NF-κB P-P65,IL-6,NLRP3,ASC,caspase-1,IL-18 and IL-1β protein were detected by Western blot(WB).(5)The protein expression levels of TLR9,NLRP3 and ASC in lung tissue were detected by immunofluorescence(IF),and the phosphorylation level of NF-κB P65 was observed.5.In vitro experimentTLR9 overexpression RAW264.7 model was constructed with TLR9 agonist.Qufeng Xuanbi formula was used for intervention,and the following tests were carried out:(1)MTT was used to detect the effects of different concentrations of Qufeng Xuanbi formula on raw264.7 viability,and the safe concentration was selected for follow-up experiment.(2)WB was used to detect the expression of TLR9,MyD88,NF-κB P65,NF-κB P-P65,IL-6,NLRP3,ASC,caspase-1,IL-18 and IL-1β in RAW264.7 after the intervention of agonist and Qufeng Xuanbi formula.(3)IF was used to detect the expression of TLR9,NLRP3 and ASC after the intervention of agonist and Qufeng Xuanbi formula,and the nuclear translocation of p65 was observed.(4)ELISA was used to detect the proinflammatory factors IL-6,IL-18 and IL-1β in the supernatant of cell culture after the intervention of agonist and Qufeng Xuanbi formula.6.Asthma mouse model was induced by ovalbumin(OVA),and Qufeng Xuanbi formula was used to intervene.The lung tissue of mice was selected for lipid metabolomics detection,the callback of lipid metabolites was observed,and the lipid metabolism mechanism of Qufeng Xuanbi formula in the treatment of asthma was explored.Results:1.Ancient Chinese medicine doctors believed that the inducements of asthma included Qi,wind cold,phlegm,diet and so on;The treatment criterion of asthma is dispersing cold and resolving phlegm during attack;Supplement when there is no attack.Modern doctors’understanding of the pathogenesis of asthma includes that all five internal organs can cause asthma,and wind,phlegm and dampness can cause asthma;In terms of treatment,it puts forward the treatment principles of five zang organs and wind phlegm.2.Professor Shi Suofang summarized the pathogenesis of Fengtanzufei and Xiongyangbizu in the attack period of asthma,and proposed the Qufeng Xuanbi therapy.Professor Shi Suofang took Qufengtan and Xuanbi as the key points of treatment.If there was a fixed time for asthma attack,Professor Shi believed that it can be treated based on the time point of asthma attack.3.Network pharmacology research showed that the effective components such as quercetin,luteolin and sitosterol in Qufeng Xuanbi formula can improve the symptoms of asthma,and Qufeng Xuanbi formula can act on TNF-α、IL-1β,VEGFA and other targets affect asthma symptoms.Qufeng Xuanbi formula can regulate asthma by regulating Toll like receptor signal pathway,Nod like receptor signal pathway,MAPK,NF-κB,VEGF and TNF signal pathway.4.Experiment(1)Qufeng Xuanbi formula can reduce airway inflammatory cell infiltration,collagen deposition and mucin secretion in ova asthmatic mice;Qufeng Xuanbi formula can reduce the secretion levels of IgE and Th2 cytokines IL-4,IL-5,IL-13 and proinflammatory cytokines IL-6,IL-18 and IL-1β in BALF;Qufeng Xuanbi recipe can reduce the production of airway inflammatory cells such as eosinophils,neutrophils,basophils and monocytes in ova asthmatic mice.(2)Qufeng Xuanbi formula can regulate TLR9/NF-κB/NLRP3 signaling pathway reduces airway inflammation in asthma.(3)Qufeng Xuanbi formula intervened in OVA asthmatic mice,resulting in the production of fatty acids,phosphatidylethanolamine,phosphatidylglycerol,phosphatidylinositol,n-acylethanolamine,phosphatidylcholine,sphingomyelin and other lipid metabolites.Conclusions:Wind and phlegm are the key pathogenic factors leading to the attack of asthma.Phlegm is glued inside and induced by external pathogens such as wind and cold.Professor Shi Suofang summarized that asthma had the mechanism of Fengtanzufei and Xiongyangbizu in the attack period,and proposed to use the Qufeng Xuanbi therapy to treat asthma,and based on this,he proposed the formula of Qufeng Xuanbi.Qufeng Xuanbi recipe can significantly reduce airway inflammation,collagen deposition and airway mucus secretion in mice.The improvement of airway inflammation is mainly reflected in the reduction of IgE secretion,Th2 cytokines and related pro-inflammatory factors,and the reduction of airway inflammatory cell infiltration.TLR9 mediates inflammation by regulating NF-B/NLRP3.After the intervention of Qufeng Xuanbi formula,the expression of TLR9,NF-κB,NLRP3 and related proteins decreased,and the secretion of proinflammatory cytokines decreased.It is confirmed that Qufeng Xuanbi formula can reduce asthma airway inflammation by regulating TLR9/NF-κB/NLRP3 pathway.The results of lipid metabolomics confirmed that Qufeng Xuanbi formula can interfere with lipid metabolites and play an anti-inflammatory and anti-asthmatic role.