The Pharmacodynamic Material Basis And Molecular Mechanism Of Yingdu Shufei Recipe In The Treatment Of New Coronary Pneumonia

Objective:To study the protective effect of Yi-Du-Bi-Fei-Fang on lipopolysaccharide(LPS)-induced acute lung injury in mice.A UHPLC-Q-TOF/MS method was applied to identify the absorbed prototype chemical compounds of Yi-Du-Bi-Fei-Fang in rat serum after its oral administration,which was recognized as the active compounds.Then a UHPLC-MS/MS method was established to assay the main active compounds in Yi-Du-Bi-Fei-Fang.Meanwhile,network pharmacology was used to explore the action targets of the identified active compounds in Yi-Du-Bi-Fei-Fang,as well as the gene ontology analysis and pathway enrichment,etc.,in an attempt to systematically predict the molecular mechanism of this decoction in the treatment of COVID-19.Methods:1.Sixty healthy C57BL/6 male mice were randomly divided into blank control group(NS),model group,positive group(dexamethasone,1 mg·kg^-1),and Yi-Du-Bi-Fei-Fang low-dose and Yi-Du-Bi-Fei-Fang medium-dose groups(406 and 812 mg·kg^-1),with 12 mice in each group.The low-dose,medium-dose and positive groups were given 20 m L·kg^-1as standard for 7 days,respectively.The control group and model group were also given normal saline orally at the same dose;On the 8th day,model group,positive group and the low-dose and medium-dose of Yi-Du-Bi-Fei-Fang groups were given LPS(5 mg·kg^-1)intraperitoneally,while the control group was given the same dose of normal saline in the same way.Mice in each group were killed immediately 6 hours after receiving the drug and their eyeballs were removed for blood collection.Serum levels of interleukin-6 and interleukin-1?were detected by ELISA kit,and lung tissues of mice were analyzed by HE staining.2.Ultra-high-performance liquid chromatography-triple/time-of-flight mass spectrometry technology was used to analyze and identify the components absorbed into the blood after the oral administration of Yi-Du-Bi-Fei-Fang,and the original medicinal materials of each component were classified.3.On this basis,the prototype component into blood was taken as the pharmacodynamic substance to further target its quality control index components.UHPLC-MS/MS method was used to verify the linear range,precision,stability,reproducibility and recovery rate,and to determine the content of the index components in three batches of Yi-Du-Bi-Fei-Fang.4.By screening corresponding targets for the identified prototype components into the blood from both TCMSP and Swiss Target Prediction database,and the target genes of COVID-19 from Gene Cards.After screening the common targets,we constructed the associated network diagram by Cytoscape 3.6.1.The STRING platform was used to construct the target protein interaction network,and the key targets were extracted.The GO gene function and pathway of the key targets were analyzed,so as to predict the molecular mechanism of Yi-Du-Bi-Fei-Fang in the treatment of COVID-19.Results:1.Yi-Du-Bi-Fei-Fang can effectively improve the lung tissue injury caused by LPS,improve the infiltration of inflammatory cells,alveolar wall edema and lung interstitial thickening;moreover,the expression of interleukin-6 and interleukin-1?in serum was significantly inhibited.2.A total of 18 prototype components were identified through comparative analysis of typical chromatogram of Yi-Du-Bi-Fei-Fang sample,mixed standard added serum and normal rat serum containing drug.3.Taking 18 prototype components as reference objects,10 measurable components were selected for quantitative analysis.It was found that 10 components of the Yi-Du-Bi-Fei-Fang had good linear relationship in the concentration range of 0.05?4.5ug·m L^-1(r²(29)0.99),and the average recovery rate was 93.07%?112.01%.3.The 18transfusion components above acted on 22 key targets,most of which were immune,inflammatory factors or related apoptotic proteins,such as TNF,IL-6,IL-10,CCL2,IL-2,CASP3,TP53,MAPK1,RELA,etc.The key targets enrichment pathways mainly include TNF signaling pathway,HIF-1 signaling pathway,Toll-like receptor signaling pathway,Fox O signaling pathway,PI3K-Akt signaling pathway and other inflammatory regulation pathways;T cell receptor pathway,B cell receptor pathway and other immune regulation;influenza A infection,Epstein-Barr virus infection,herpes simplex virus infection and other regulatory virus infection.Conclusion:In this study,under the guidance of mouse ALI model,the pharmacodynamics analysis was carried out on the treatment of ALI by Yi-Du-Bi-Fei-Fang.Moreover,UHPLC-Q-TOF/MS and UHPLC-MS/MS were used to carry out qualitative identification and quantitative analysis of Yi-Du-Bi-Fei-Fang,so as to provide reference for preliminary screening of pharmacodynamic substances and research on improving quality standards.At the same time,network pharmacological methods and techniques were used to predict the molecular mechanism of the treatment of COVID-19by the prototype component of Yi-Du-Bi-Fei-Fang.