The Ameliorative Effect And Mechanism Study Of Huatan Huoxue Formula On Airway Inflammation In Obese Asthma Mice

Objective:Obesity asthma,as a new phenotype of asthma,has significant neutrophil inflammation and type 17 immune response.In this study,a mouse model of obesity-type asthma was established by feeding high-fat feed to induce obesity and House dust mite(HDM)induced asthma,and investigated the effect of Huatan Huoxue Formula(HHF)on airway inflammation in obese asthmatic mice and explored its mechanism.Method:1.The content of main components in HHF was determined by HPLC.The HPLC dual wavelength switching method with methanol-0.5%phosphoric acid aqueous solution as the mobile phase for gradient elution was used to identify seven indicator components of mustard base thiocyanate,paeoniflorin,rosmarinic acid,bitter amygdalin,hydroxy saffron yellow pigment A,digitonin D and ferulic acid of nine herbs in HHF.2.Network pharmacology analysis of potential targets and pathways of HHF for the treatment of obesity-type asthmaWe used TCMSP to screen the active ingredients in HHF fractions and retrieve all the targets that meet the screening criteria;use Gene Cards database and OMIM database to retrieve Obesity Asthma disease-related targets;construct "ingredient-disease-target"network to obtain common targets for HHF treatment of obesity asthma;and construct common targets.Construct "Component-Disease-Target" network to obtain common targets of HHF for the treatment of obesity asthma;Construct PPI network of common targets,and use Metascape database to perform key target searches.GO enrichment analysis and KEGG pathway enrichment analysis of key targets using Metascape database.3.Observation on the efficacy of HHF in obese asthmatic miceC57BL/6J mice(male)were randomly divided into normal group,asthma group,obesity group,obesity asthma group,dexamethasone group,HHF-low-dose group and HHF-high-dose group.Obesity was induced by high fat diet and asthma was induced by HDM.Weekly observation and recording of the effect of HHF on body weight and general condition of mice.AHR was measured on day 85 in mice.Blood was obtained and mice were executed,BALF was obtained,and lung tissue was routinely sectioned with HE and PAS fixation,and serum lipid quadruple was measured by ELISA.Immunofluorescence assay was performed to detect neutrophil production in lung tissues.4.Study on the mechanism of action of HHF against obesity-type asthma in miceELISA was performed to detect the cytokines IL-1β,IL-17A,IL-6,IL-18,TNF-α and GM-CSF in mouse lung tissue BALF.The expression of M1 macrophage NLRP3 inflammatory vesicle-associated proteins in mouse lung tissues was detected by protein immunoblotting,and the expression of IL-1β,IL-6,IL-17A and TNF-α proteins in mouse lung tissues was detected by immunohistochemistry.NLRP3 in M1 macrophages in lung tissues by immunofluorescence staining,and ILC3s and Th17 in lung tissues by flow cytometry generation.Results:1.The content of main components in HHF was determined by HPLC.The content of rehmannitin D,paeoniflorin,ferulic acid,rosinic acid,hydroxysafflower yellow pigment A,sinapine thiocyanate and amygdalin in HHF was 193.13μg/mL,489.27 μg/mL,22.93 μg/mL,86.71 μg/mL,250.30 μg/mL,95.06 μg/mL,595.67 μg/mL.2.Network pharmacology analysis of potential targets and pathways of HHF for the treatment of obesity-type asthmaThere are 94 common targets between HHF and obese asthma.PPI network analysis,IL6,TNF,AKT1,IL-1β,PTGS2,CXCL8 can be considered as the key targets of HHF for treating obese asthma.GO enrichment analysis showed that it mainly involves the response of lipopolysaccharide,response to molecules of bacterial origin,membrane rafts,membrane microdomains,cytokine receptor binding,cytokine KEGG enrichment analysis showed that HHF treatment of obese asthma was associated with lipid and atherosclerosis,TNF signaling pathway and IL-17 signaling pathway.3.Observation on the efficacy of HHF in obese asthmatic miceThe effect of HHF on body weight of mice was not significant;both HHF-L and HHF-H were effective in improving asthma attacks in mice;HHF-H significantly improved AHR in mice(P<0.05);serum lipid assay showed that all dose groups significantly reduced LDL-C,T-CHO and TG in mice.Both HHF-L and HHF-H significantly improved the inflammatory cell infiltration around the trachea and alveoli of mice,and reduced the thickening of tracheal wall to different degrees.HHF could improve the mucus secretion and cupped cell proliferation around the trachea of mice;HHF could reduce the production of neutrophils in the lung tissue of mice.4.Study on the mechanism of action of HHF against obesity-type asthma in miceHHF significantly reduced the contents of IL-1β,IL-17A,IL-6,IL-18,TNF-α,GMCSF in mouse BALF;it significantly reduced the expression of NLRP3 inflammatory vesicle-related protein NLRP3,pro-IL-1β,pro-Caspase1,IL-1β,Caspasel,in mouse lung tissue,ASC;the results of immunohistochemical assay showed that HHF could reduce the expression of IL-1β,IL-6,IL-17A,TNF-α proteins in mouse lung tissue;the results of immunofluorescence staining assay showed that HHF could reduce the content of NLRP3 proteins in M1 macrophages in mouse lung tissue;the results of flow cytometry assay showed that HHF could reduce the expression of ILC3s and Th17 cell production in lung tissues.Conclusions:HHF alleviated obese asthma attacks,improved lung histopathology,inhibited airway mucus hypersecretion and improved airway inflammation in mice.HHF significantly attenuated type 17 immune response and neutrophil inflammation in mice,the latter possibly associated with downregulation of NLRP3 inflammatory vesicle activation product IL-1β in M1 macrophages and reduced neutrophil production.