The Protective Effect And Mechanism Of Shuqing Granules On Mice Infected With H1N1 Influenza Virus Were Investigated Based On Network Pharmacology And Metabolomics

Influenza is an infection of the respiratory tract caused by influenza viruses that spread worldwide.Influenza-induced viral pneumonia is a major cause of morbidity and mortality in infected patients of all age groups worldwide,and the genetic characteristics of influenza viruses make vaccines lagging and prone to induce resistance to some antiviral drugs.Therefore,the development of new effective therapeutic drugs is important for the prevention and treatment of these diseases.Chinese medicines have multi-target and multi-pathway properties,which can intervene and inhibit viral replication and regulate inflammatory responses.New use of old drugs,i.e.,secondary development of proprietary Chinese medicines,has become one of the most rapid and efficient strategies for developing new drugs.The compound Chinese medicine Shuqing granules have been shown to have in vitro anti-influenza viral effects,suggesting that the drug has the potential to be developed as an in vivo antiviral agent.The efficacy of Chinese medicine is achieved through the overall regulation of the organism,so this thesis conducts in vivo tests based on in vitro tests,which also further corroborate the results of in vitro tests.Therefore,an in-depth investigation of whether the Chinese medicine Shuqing granules have in vivo antiviral effects and their mechanisms will provide important evidence for understanding the role of this drug in the prevention and treatment of influenza.In addition,more and more studies have shown that in viral pneumonia caused by influenza virus infection,relying solely on antiviral strategies is unable to reduce morbidity and mortality in patients due to influenza virus infection,and anti-inflammatory therapies that mitigate the cytokine storm caused by viral infection are being increasingly emphasized.With the continuous updating of databases and in-depth bioinformatics studies,many pharmacodynamic components and their targets of action in sparing pellets are yet to be explored in terms of anti-inflammatory effects and related mechanisms.The rapid development of network pharmacology and metabolomics technologies in recent years has provided powerful tools to access the molecular network mechanism of action of drugs,which are widely used in drug screening and drug pharmacodynamics research.Objective:To observe the protective effect of Shuqing granules on H1N1 influenza virus infected mice based on the network pharmacology and metabolomics techniques,and to analyze its antiviral and anti-inflammatory mechanism of action in depth.Methods:1.Pharmacological analysis of anti-influenza virus network of Shuqing granulesThe TCMSP database was used to screen the active compounds of the drug components dacryophyllum,licorice,rhizome,mulberry leaf and gypsum,and the relevant targets of influenza were searched by Mala Card,Gene Cards and other websites,and uploaded to venny 2.1.0 website to obtain the intersection targets and draw the Wayne diagram.We used STRING database to build the PPI network diagram of sparse clear particle compound targets-Influenza targets,and used Cytoscape 3.9.0software to analyze the network topologically,obtain the relevant topological parameters,and screen the mean value greater than the node connectivity(Degree),node closeness(Closeness),node intermediate degree(Betweenness)respectively to filter the key targets.The key targets were screened by Metascape,Omicshare,and the node betweenness indexes.The enrichment analysis of GO and KEGG signaling pathways was performed by Metascape,Omicshare and other databases.2.In vivo pharmacodynamic study of anti-influenza virus pneumonia with Shuqing granulesIn this thesis,the in vivo pharmacodynamics of the active ingredients of Shuqing granules and the validation of the key target genes of network pharmacology at the m RNA level were verified by constructing a mouse H1N1 infection model: the weight change and death of each group of mice were recorded by detecting Shuqing granules,and the effect of Shuqing granules on the weight change of virus-infected mice was observed;the lungs of each group of mice were dissected on the7 th day after infection,and the lung tissue sections were stained with HE to observe The histopathological changes of lung tissue were observed on day 7 after infection,and the changes of m RNA expression of relevant cytokines in mouse lung were detected by real-time fluorescence quantitative PCR.3.Metabolomic analysis and integration analysis of anti-influenza virus of Shuqing Granules By constructing a mouse H1N1 infection model,the treatment group was given different doses of Shuqing granules,blood was taken and executed on day 7,and serum samples were collected for non-targeted metabolomic analysis based on high-resolution UPLC-Q/TOF-MS.The possible metabolic pathways were also searched for.Finally,the Metabo Analyst 4.0 platform was used to integrate network pharmacology key targets with metabolomic biomarkers for preliminary prediction of the possible pathogenesis of anti-influenza-induced viral pneumonia by Shuqing granules.Results:1.There are 8 active ingredients in the sparing granules namely quercetin,kaempferol,naringenin,arachidonic acid,indocyanine,β-sitosterol,indigo and β-carotene.Topological analysis yielded 25influenza-related target genes such as TNF,IL-6,IL-1β,IL-10,CXCL10,etc.The enrichment results showed that signaling pathways such as Influenza A,TNF and NF-κB also played a dominant role in the treatment of Shuqing granules.2.The mice in the viral model group showed significant weight loss,while the high,medium and low dose groups of Shuqing granules could effectively inhibit the weight loss of infected mice at the early stage of infection.Compared with the virus model group,the expression of NF-κB,TNF-α,IL-6,IL-Iβ,PKR,CXCL10 and IL-10 m RNA was significantly reduced and PKR was significantly increased in the high,medium and low dose groups of Shuqing granules.Inflammatory exudates in the alveoli were significantly reduced in the low-dose group,and the area of alveolar infiltrates was significantly reduced compared with the viral group;alveolar lesions were still visible in the lungs of the middle-dose group,and the inflammatory lesions were reduced compared with the viral group;some infiltrates and inflammatory lesions were visible in the lungs of the high-dose group,indicating that Shuqing granules mainly reduced the inflammatory changes caused by infection through early inhibition of the expression of relevant inflammatory factors,and the inflammatory factors at the level of gene expression were consistent with the The results of the network pharmacology study were consistent with the predicted results.3.Metabolomic analysis identified a total of 147 differential metabolites in the C-M group and 48 differential metabolites in the M-H group,and enriched to the KEGG pathway identified eight significantly altered metabolic pathways: linoleic acid metabolism,arachidonic acid metabolism,retinol metabolism,linolenic acid metabolism,sphingolipid metabolism,glycerophospholipid metabolism,steroid hormone biosynthesis,and unsaturated fatty acid biosynthesis,of which linoleic acid metabolism,arachidonic acid metabolism,and retinol metabolism,underwent extremely significant changes,which were significantly associated with the development of influenza and the intervention of Shuqing granules.After integrated analysis,we found a total of 5metabolic pathways,namely arachidonic acid metabolism,retinol metabolism,α-linolenic acid metabolism,fatty acid biosynthesis,and unsaturated fatty acid biosynthesis.In addition,we found 4 related key targets namely IL-6,TNF,IL-10,CXCL10 and 2 related biomarkers arachidonic acid and α-linolenic acid,which are closely related to the process of anti-influenza virus infection by Shuqing granules.Conclusion:Shuqing granules may regulate arachidonic acid metabolism, retinol metabolism and alpha-linolenic acid metabolism by targeting inflammatory pathways(TNF signaling pathway,NF-κB pathway,etc.)through its key active ingredients(quercetin,arachidonic acid,etc.),so as to improve lung inflammation caused by influenza virus infection.