Study On Anti-RSV Of Flavonoid Components In Qingfei Oral Liquid Based On Type ? Interferon Signaling Pathway

Objective:The material library of flavonoids in Qingfei oral liquid was established.Based on the method of network pharmacology,the main active components and potential targets of anti-RSV of flavonoid components were studied.To study the preventive and therapeutic effect of flavonoids on mouse model of RSV pneumonia.Molecular biological techniques combined with target metabolomics were used to focus on the type ? interferon signal pathway to explore the possible mechanism of flavonoid components against RSV.Methods:The purification process and qualitative and quantitative detection methods of flavonoid components:the mass concentration of solution,the mass ratio of resin,pH,eluent volume,alcohol elution volume fraction and elution volume flow rate were used as the parameters.Taking the content of lavonoids as the main evaluation index,the purification process of polyamide resin was optimized by orthogonal test.The MS/MS data of samples were collected by electrospray ion source(ESI),positive and negative ion switching scanning mode,and the flavonoid components in Qingfei oral liquid were qualitatively analyzed by MS-FINDER platform combined with manual check method.26 flavonoids were quantitatively analyzed by UPLC-MS/MS,with CSH-C18 column and ESI,anion mode SRM.Network pharmacological analysis of flavonoid components:the composition targets of 76 flavonoids identified were predicted by Swiss Target Prediction and STITCH data platform.The RSV-related targets were obtained based on the Phenolyzer platform,and the drug-component-target-disease interaction network was constructed by STRING and Cytoscape software.GO analysis and KEGG pathway enrichment were studied at the same time.In vivo experiment:The virulence of RSV was determined by plaque test,and the mouse model of lung tissue infection was induced by RSV nasal drip.Twelve hours after the establishment of the model,the low and high dose groups of flavonoids were given intragastric administration,and ribavirin was used as the positive control,and flavonoids were given prophylactic 5 days before nasal drip modeling.The pathology of lung tissue was analyzed on the 4th and 6th day after RSV infection,and the relative expression of Caspase-3 was analyzed by IHC method to evaluate the protective effect of flavonoids on lung tissue.The levels of IL-1?,TNF-?,TGF-?1 and IL-10 were detected by ELISA and qPCR.The ratio of CD4+,CD8+T lymphocytes and macrophages was analyzed by flow cytometry to evaluate the effects of flavonoids on inflammatory response and inflammatory cell infiltration in RSV infected mice.The nucleic acid transcription level of F,G,NS1 and the expression of F protein in RSV were detected by qPCR and Westernblot,respectively.At the same time,the effect of flavonoids on RSV replication was verified by IHC for F protein and RSV.The levels of serum IFN-?,IFN-?and IFN-? mRNA were detected by ELISA and qPCR,respectively.MDA5,RIG-1,TBK1,IRF3,JAK1,TYK2,MX1,TRIM5,ISG15,IP-10 mRNA were detected by qPCR,TBK1,IRF3,p-TBK1,OAS1 levels were detected by Western Blot,MX1 and OAS1 protein levels were detected semi-quantitatively by IHC.The levels of glucose metabolism and TCA related metabolites in serum and lung tissue of RSV infected mice were detected by target metabolomics technique to explore the effect of flavonoids on energy metabolites of RSV infected mice and the relationship between flavonoids and innate immunityResults:(1)The best purification technology of flavonoids involved 24 mg/mL crude drug solution,mass ratio of the polyamide resin to the medicine of 25:3,pH 4.0,5 BV water for removing the impurities,6 BV 80%ethanol for eluting flavonoids and 3.0 mL/min elution flow rate.After purification,the flavonoid content increased from 18.5%to 68.9%,and the transfer rate was 78.7%.(2)A total of 440 flavonoids were preliminary identified,including 266 negative ion patterns and 234 positive ion patterns.There were 60 kinds of flavonoids with good response in both modes.It is known that there are 146 kinds of flavonoids in Qingfei oral liquid,and 78 kinds of flavonoids had been detected.26 flavonoids such as catechin and luteolin could be detected quantitatively at the same time,and there was a good linear relationship between their mass concentration and peak area(R square>0.9978).(3)A total of 368 targets of 76 flavonoids were mined and 87 common targets with RSV infection were obtained.Quercetin,luteolin,apigenin,kaempferol and luteolin were the main active components of flavonoids.The results of target function enrichment showed that the anti-RSV mechanism of Qingfei flavonoids was mainly related to inflammatory pathway,immune regulation,energy metabolism,anti-viral infection and so on(4)The results of lung pathology showed that there was severe inflammation in the lungs of mice on the 4th and 6th day of infection.There were consolidation lesions of different sizes,significantly infiltrated inflammatory cells and obvious vascular and perialveolar edema.The inflammation score of lung tissue in high dose flavonoid group and pretreatment group was lower than that in model group.After RSV infection,the expression of Caspase-3 in lung tissue was up-regulated,and flavonoid group could partially inhibit the level of Caspase-3.(5)The results of ELISA,qPCR and flow cytometry showed that high dose and pretreatment of flavonoids could effectively reduce the levels of IL-1?,TNF-? and TGF-?1 inflammatory factors,and up-regulate the anti-inflammatory factor IL-10.Flavonoids had a tendency to reduce the proportion of infiltration of CD4+,CD8+T lymphocytes and macrophages after treatment or pretreatment.(6)RSV specific nucleic acid could be detected in lung tissue,but not in normal group.There was strong RSV replication in the model group,but the IFN-? and IFN-? mRNAs in the lung tissue were not significantly up-regulated.After pretreatment or treatment,flavonoids could inhibit the levels of F protein and mRNA,inhibit the transcription of G and NS1 nucleic acid,and effectively up-regulate the levels of IFN-?,IFN-? protein in serum and lung tissue,and the level of IFN-? mRNA.(7)Compared with the normal group,the level of TBK1 and IRF3 mRNA in the model group was up-regulated similar to that of RIG-1 mRNA.At the same time,IRF3 and p-TBK1 proteins were also significantly up-regulated,and TBK1 tended to be up-regulated.RSV had no effect on the transcription of JAK1 and TYK2,but could increase the transcription level of MX1,TRIM5,ISG15,IP-10 and the content of OAS1 protein.Medium dose and pretreatment of flavonoids could promote the phosphorylation of TBK1 and increase the levels of JAK1,IP-10 and ISG15 mRNA,but had no effect on TYK2 mRNA,and OAS1 tended to be upregulated.(8)Compared with the normal group,the serum pyruvate,lactic acid and cis-aconitic acid in the model group were up-regulated,succinic acid and 3-phosphoglyceric acid were up-regulated,and a-ketoglutaric acid was down-regulated.The high dose of flavonoids could reverse the upregulation of pyruvate,lactic acid,cis-aconitine and 3-phosphoglyceric acid induced by RSV infection,down-regulate the level of serum malic acid and decrease succinic acid.The intervention of ribavirin resulted in further up-regulation of serum lactic acid,succinic acid,fumaric acid,malic acid,a-ketoglutaric acid,phosphoenolpyruvate and citric acid.Compared with the normal group,11 target metabolites such as pyruvate and lactic acid in the model group were significantly up-regulated.The high dose of flavonoids could reverse the upregulation of lactic acid,3-phosphoglyceric acid,fumaric acid,malic acid and glucose in lung tissue induced by RSV infection,further increase the level of pyruvate in lung tissue,and decrease phosphoenolpyruvate and succinic acid.Compared with the model group,the intervention of ribavirin could down-regulate the levels of glucose and phosphoenolpyruvate,but caused a higher level of lactic acid accumulation in lung tissue.Conclusion:(1)The purification of flavonoids from Qingfei oral liquid by polyamide resin is stable,reliable and effective.The identification of flavonoids by MS-DAIL combined with MS-FINDER analysis is simple and efficient,and can be used as the first choice for the identification of flavonoids in TCM.(2)UPLC-QE-Orbitrap-MS combined with MS-FINDER can quickly and qualitatively analyze flavonoids in Qingfei oral liquid.Using UPLC-MS/MS technology and CSH-C18 column,26 flavonoids in Qingfei oral liquid can be determined with high efficiency and repeatability.(3)Through LC-MS and network pharmacology,it was found that the anti-RSV mechanism of flavonoids in Qingfei oral liquid is mainly related to inflammatory pathway,immune regulation,energy metabolism,anti-viral infection and so on,which confirmed the therapeutic characteristics of compound multi-target and multi-pathway.(4)Nasal drip of 5�10^5 PFU RSV per BALB/c mouse may cause significant inflammatory reaction and pathological injury of lung tissue.Flavonoid may effectively inhibit inflammatory reaction,improve pathological injury of lung tissue,and reduce inflammatory cell infiltration and apoptosis.At the same time,RSV nasal drip model may be detected RSV specific nucleic acid and protein in lung tissue,while flavonoid treatment may effectively inhibit RSV replication.(5)RSV infection may inhibit the transcription of type ? IFN in host cells,interfere with the normal antiviral immune response,and cause the disorder of energy metabolism in host serum and lung tissue and the accumulation of lactic acid.Flavonoids may promote the phosphorylation of TBK1,up-regulate the level of antiviral protein,activate type ? IFN signal pathway and improve the metabolic disorder induced by RSV.At the same time,flavonoids may effectively prevent RSV infection,activate host type ? interferon signal pathway,and prevent the imbalance of host energy metabolism.(6)The activation of type ? interferon signaling pathway by flavonoids may be achieved indirectly by antagonizing TGF-?1 activity,binding to viral non-structural proteins,improving host energy metabolism and down-regulating lactic acid levels.(7)Ribavirin may effectively inhibit the replication of RSV,but it is not as good as flavonoids in improving lung pathological injury and inflammation.Ribavirin caused a stronger disorder of energy metabolism in the host,induced the increase of lactic acid levels in serum and lung tissue,and suppressed the normal antiviral immune function of host cells,which may be the internal reasons for the lack of clinical benefit.