The Mechanism Of The Flavonoids Of Qingfei Oral Liquid Regulating AM Energy Metabolism In ALI Mice

Objective:To systematically sort out the literature on prevention and treatment of pneumonia in children with traditional Chinese medicine,explore the rules of modern physicians for pneumonia in children,and provide a basis for the treatment of flavonoids in the treatment of pulmonary inflammation;based on the method of network pharmacology to study the main activities of flavonoids in the lung cleansing Ingredients improve the potential target of acute lung injury;to study the effect and mechanism of the flavonoid component of Qingfei Oral Liquid in improving the effect and mechanism of lipopolysaccharide(LPS)-induced acute lung injury in mice,from the Qingfei flavonoid group Adjust the energy metabolism angle of mouse RAW264.7 macrophages and adjust the angle of mitochondrial function-mediated pulmonary inflammation in different doses to evaluate the low dose of Qingfei flavonoids on LPS-stimulated RAW264.7 The effect of macrophage energy metabolism,the impact of high dose of Qingfei flavonoids on energy metabolism of RAW 264.7 macrophages,and the relationship between IFN-β induced by high dose of Qingfei flavonoids and inflammation and mitochondria.Provide a reliable theoretical basis for optimizing Chinese herbal compound and treating ALI.Methods:Retrieving the relevant literature on the treatment of pediatric pneumonia by Chinese medicine from CNKI and Wanfang Medical from the establishment of the database to April 2020 as the research source.According to the search strategy that has been developed,a unified search was performed at the same time as a joint manual search.Manually screen Chinese medicine-related treatment references and extract treatment prescriptions from the original text for analysis.The qualitative and qualitative detection method of Qingfei Oral Liquid and flavonoid components:Based on the analysis results of the flavonoid components of Qingfei extracted by gradient ethanol,the same mixed standard product and the hospital preparation Qingfei Oral Liquid were compared,and the mixed standard product was passed Check and qualitatively analyze the flavonoids of Qingfei Oral Liquid,using Aquity H-Clas ultra-high performance liquid chromatograph combined with LTQ Orbitrap XL ETD liquid chromatography-mass spectrometer,using AQUITY UPLC HSS T3 chromatographic column,positive and negative ion mode switching Scan mode acquisition,using electrospray ionization source(HESI)to qualitatively identify target compounds.Pulmonary flavonoid component treatment ALI network pharmacology analysis:Use PubChem to obtain the SDF format file of the compound and its corresponding SMILES structure.Import the SDF file and SMILES structure of the flavonoids identified in the earlier stage into the Swiss Target Prediction database and the SEA database,and screen and merge to obtain the predicted target.Standardize and unify the obtained target information through UniProt database.Then use Genecards,DisGeNET,TTD databases to obtain ALI disease targets,STRING platform and Cytoscape software to construct a drug-component-target-disease interaction network,and finally DAVID 6.8 database for GO analysis and KEGG pathway enrichment research analysis.In vivo experimental study:A mouse ALI model was induced by LPS airway instillation.1 hour before modeling,the lung-clearing flavonoid low and high-dose groups were used for intragastric administration,while dexamethasone was used as a positive control,and the infection was 6 hours After that,the mice were tested for lung pathology and the dry/wet weight ratio was evaluated.The levels of IL-1β,TNF-α,IL-6,SOD,MDA,and MPO were detected by ELISA,and the cells in the alveolar lavage fluid were classified using a cell counter,and the low and high doses of lung-clearing flavonoids were evaluated for ALI.Inflammation in the lungs of rats.Through the combined analysis of non-target proteomics and non-target metabonomics,the mechanism of different doses of Qingfei flavonoids on the ALI mouse model was explored,and qPCR was used to detect the effects of low and high doses of Qingfei flavonoids on lung IFN-β.And IFN-α,IFN-β mRNA levels.In vitro experimental study:low-dose Qingfei flavonoids acted on LPS-stimulatedRAW 264.7 macrophages for 12 h.Through target metabolomics technology,the levels of glucose metabolism and TCA-related metabolites in RAW 264.7 macrophages were detected by qPCR analysis Cytoinflammatory factors,oxidative stress-related factors,and cellular mitochondrial-related proteins.The high dose of Qingfei flavonoids and the high dose of Qingfei flavonoids accompanied by IFN-βAb directly acted on RAW 264.7 macrophages for 24 hours.Through target metabolomics technology,the RAW 264.7 macrophages after the high dose of Qingfei flavonoids were detected.Carbohydrate metabolism and TCA-related metabolite levels of phages.Results:1.Carry out frequency analysis on 265 prescriptions,and a total of 30 traditional Chinese medicines used more than 15 times.The top 10 most frequently used medicines are skullcap,honeysuckle,forsythia,licorice,almond,roasted ephedra,goat horn,bear bile powder,raw gypsum,and fritillaria.Association rules can dig out the interrelationships hidden in the data.With a support degree of 0.23 and a confidence degree of 0.13,a total of 30 traditional Chinese medicine combinations for the treatment of bacterial pneumonia in children were obtained.The K-means clustering method was used to cluster analysis of all drugs involved in the 265 first prescriptions,and 7 groups of new core combination prescriptions for the treatment of bacterial pneumonia in children were obtained.2.In Qingfei Oral Liquid,6 compounds were identified in the negative ion mode,28 compounds in the positive ion mode,and a total of 34 compounds were identified.3.A total of 242 targets were mined for 82 types of flavonoids,and 73 common targets with acute lung injury were obtained.The top 5 targets were vascular endothelial growth factor(VEGFA),interleukin 6(IL-6),serine/threon Protein kinase(AKT1),epidermal growth factor receptor(EGFR),tumor necrosis factor alpha(TNF),etc.The target function enrichment results show that the mechanism of action of Qingfei flavonoids in the treatment of ALI is mainly related to the HIF-1 signaling pathway,PI3K-Akt signaling pathway,TNF signaling pathway and other pathways are related.4.The results of lung pathology showed that the lung tissue of ALI mice had severe alveolar edema accompanied by significant infiltration of inflammatory cells.ELISA test results show that low doses of flavonoids can effectively reduce IL-6,IL-1β and TNF-α,and improve the levels of SOD,MDA,and MPO.High doses of flavonoids for clearing lungs can effectively reduce IL-6,IL-1β and TNF in lung tissues.-α has no obvious improvement effect,and does not significantly improve the levels of SOD,MDA,and MPO.5.Non-target proteomics analysis of the mechanism of the low/high dose of Qingfei flavonoids on the ALI mouse model.Significant enrichment analysis found that the main activity of the protein is "granulocyte chemotaxis" and "neutrophil migration".However,the significant enrichment analysis of the high-dose Qingfei flavonoids found that the main activity of the active protein was "response to interferon beta".Analysis of non-target metabolic components analyzes the mechanism of Qingfei flavonoid low/high dose on ALI mouse model,and the related metabolic pathways are obtained through differential metabolite analysis.The metabolic pathways involved in low-dose Qingfei flavonoid intervention in ALI mice are The metabolism of alanine,aspartic acid and glutamate,the metabolism of D-glutamine and D-glutamate,the metabolism of glycine,serine and threonine,the metabolism of pyruvate,and the high-dose Qingfei flavonoids interfere with ALI.The metabolic pathways involved in rats are alanine,aspartic acid and glutamate metabolism,pyruvate metabolism,glycine,serine and threonine metabolism,glycolysis/gluconeogenesis,and citric acid cycle(TCA cycle).Compared with the Qingfei flavonoid low-dose group,the lung tissue of the Qingfei flavonoid high-dose group up-regulated IFN-α and IFN-β mRNA significantly.6.Target metabonomics technology test results show that the low dose of Qingfei flavonoids can improve cell energy metabolism,reduce the accumulation of succinic acid and citric acid,reduce HIF-la and reduce IL after 12 hours of LPS-stimulated RAW 264.7 macrophages.The release of-1β and other inflammatory factors reduces mitochondrial damage and increases the level of anti-inflammatory factors.Target metabonomics technology test results showed that the high dose of Qingfei flavonoids and the high dose of Qingfei flavonoids accompanied by IFN-β Ab directly acted on RAW 264.7 macrophages for 24 hours,and the high dose of Qingfei flavonoids group increased the energy metabolism of macrophages.The disorder reduces IDH and causes citric acid to accumulate.After IFN-β Ab inhibits IFN-β,it can regulate the energy metabolism disorder of macrophages caused by the high-dose lung-clearing flavonoid group,increase the level of IDH,and reduce the accumulation of citric acid.Conclusion:(1)As a whole,among the traditional Chinese medicines used by modern physicians to treat bacterial pneumonia in children,the commonly used(higher frequency)medicines are rich in flavonoids,and most of the medicines are bitter and cold in nature.(2)Using Aquity H-Clas combined with LTQ Orbitrap XL ETD technology,the use of standard product comparison can accurately and qualitatively analyze the 34 flavonoid-based compounds in Qingfei Oral Liquid.(3)Through network pharmacology research methods,the mechanism of action of the flavonoid component of Qingfei Oral Liquid in treating ALI is mainly related to the HIF-1 signaling pathway,PI3K-Akt signaling pathway,TNF signaling pathway,etc.,confirming the similarity of traditional Chinese medicines.The compound has the characteristics of multi-target and multi-path therapy.(4)Instillation of each C57BL/6 mouse through the airway can cause significant lung tissue inflammation and pathological damage;the low dose of Qingfei flavonoids has better effects on lung pathology,inflammation and oxidative stress.Qingfei flavonoid high-dose group.(5)The low dose of Qingfei flavonoids and the high dose of Qingfei flavonoids have different main activities of proteins that have an effect on the ALI mouse model.The low doses of flavonoids focus on"granulocyte chemotaxis" and "neutrophil migration".The high dose of flavonoids focuses on the "response to interferon beta".Similarly,the low dose of Qingfei flavonoids and the high dose of Qingfei flavonoids have different metabolic pathways in the ALI mouse model.The high doses of Qingfei flavonoids are different than the low doses.It focuses more on glycolysis/gluconeogenesis and citric acid cycle(TCA cycle).In addition,the high dose of Qingfei flavonoids can induce more INF-β in the lung tissues of mice.(6)The high dose of Qingfei flavonoids(0.128 mg/ml)has a long-term(24 h)effect on the production of RAW 264.7 macrophages,which is contrary to the initial cell activity reduction;the low dose of Qingfei flavonoids(0.032 mg/ml))The activity of RAW 264.7 macrophages can be maintained in a stable state(24 h);low-dose lung-clearing flavonoids can reduce the TCA cycle of succinic acid and citric acid,and can improve the energy metabolism of RAW 264.7 macrophages stimulated by LPS.Cell mitochondria function,and by reducing the accumulation of succinate,lowering the level of HIF-1α,down-regulating the inflammatory factors secreted by cells and alleviating oxidative stress.The high dose of Qingfei flavonoids induces excessive IFN-β to almost block the production of Isocitrate dehydrogenase,leading to the accumulation of citric acid,which is one of the reasons for the disturbance of energy metabolism in RAW 264.7 macrophages.