Professor Wang Shouchuan’s Experience In Treating Pediatric Viral Pneumonia Based On Theories Of ’Heat Depression With Phlegm And Stasis’ And Research On The Metabolic And Immune Mechanism Of Qingfei Oral Liquid

Objective:To summarize Professor Wang Shouchuan’s experience in treating pediatric viral pneumonia based on theory of ’heat depression with phlegm and stasis’.To investigate the clinical efficacy and safety of ’relieving lung stagnation,resolving phlegm,removing toxin’ approach in the treatment of pediatric viral pneumonia.To explore the active components and potential targets of Qingfei oral liquid(QF)in treating RSV pneumonia.To reveal the metabolic and immune mechanism of QF in treating RSV pneumonia.Methods:Systematic evaluation and data mining research:the clinical RCT study on treating pediatric viral pneumonia with ’relieving lung stagnation,resolving phlegm,removing toxin’ method was searched from Chinese and Foreign Periodical Databases.Quality of the included literature was evaluated by Risk-of-bias tool.Meta-analysis on the included data was conducted with Review Manager 5.3 statistical software.Frequency statistics and association rule analysis was conducted with Microsoft Excel and IBM SPSS statistics software,respectively.Hierarchical cluster analysis was carried out using origin Pro software.Network pharmacology and molecular docking research:TCMSP and BATMAN-TCM databases were used for searching the effective components of QF.GeneCards and OMIM databases were used for obtaining the targets related to RSV pneumonia.’Drug-ComponentDisease-Target’ and ’Protein-Protein Interaction’ networks of core targets were established with STRING and Cytoscape software.Additionally,GO and KEGG pathway analysis was also performed.Clinical trial study:According to the inclusion and exclusion criteria,plasma samples were collected from both healthy and RSV pneumonia children with phlegm heat syndrome.Lipidomic profiles were detected using ultimate 3000 ultra-high performance liquid chromatography combined with mass spectrometry(UPLC-MS/MS).Contents of fatty acids were quantified through standard comparison.Animal experimental study:Female BALB/c mice aged 6～8 weeks were divided into normal,model,low-dose QF,medium-dose QF,high-dose QF,agonist and agonist/therapy groups,respectively.The infection model was established by RSV nasal drip,and the Akt agonist model with SC-79 intraperitoneal injection.After 4-day continuous QF administration,mice were sacrificed under anesthesia.Metabolic profiles of plasma and lung tissues were also detected with UPLC-MS/MS instrument,and the contents of fatty acids in lung tissue samples were quantified through standard comparison.Besides,HE staining for paraffin section was used to observe the pathological manifestations of lung tissues.Transcriptional levels of inflammatory factors(IL-1β,IL-6,TNF-α,IFN-γ,IL-10,Arg-1,iNOS)and fatty acid metabolic enzymes(FASN,ACLY,Acaca,CPT1A,PPARα,Acox1)in lung tissues of mice were detected with realtime quantitative PCR assay.Expression of phosphorylated Akt protein,ATP citrate lyase(ACLY)and peroxisome proliferator activated receptor α(PPARα)in lung tissues of mice were detected with Western Blot assay.The number and proportion of M1 and M2 macrophages were detected with flow cytometry assay.Expression of iNOS(secreted especially by M1 macrophage)and Arg-1(secreted especially by M2 macrophage)proteins in lung tissues of mice were both detected with immunohistochemical study.Results:(1)Large samples from clinical RCT studies showed the ’relieving lung stagnation,resolving phlegm and removing toxin’ method could effectively shorten the time of fever,cough,wheezing and lung rale in children with viral pneumonia,and the total clinical effective rate was better than that of the western medicine group.Drug safety was also good.(2)Most medical herbs used in the method of ’relieving lung stagnation,resolving phlegm and removing toxin’ were pungent,cold in property,and belonged to Lung and Heart Sutra.’Maxingshigan Decoction’ was the basic prescription for treating pediatric viral pneumonia with’relieving lung stagnation,resolving phlegm and removing toxin’ method,and QF was one of the clustering prescriptions.(3)The study of network pharmacology and molecular docking technology showed that active components such as quercetin,luteolin,kaempferol and naringin in QF could dock well with Akt.PI3k/Akt signal pathway was first enriched in KEGG analysis with most significant difference.(4)The expression of FA 12:0,FA 13:0,FA 14:0,FA 14:1,FA 15:0,FA 15:1,FA 19:1,FA 20:2,FA 20:4,FA 21:1,FA 22:0,FA 22:1,FA 22:2,FA 24:1,FA 24:2,FA 27:0 was all upregulated in the plasma of children with RSV pneumonia(FDR<0.05,FC>1.2).Besides,the expression of FA 14:1,FA 16:3,FA 17:0,FA 20:4,FA 20:5;O,FA 21:5;O,FA 22:4;O,FA 22:5,FA 22:5;O,FA 22:6;O was also higher expressed in the plasma of RSV-infected mice(FDR<0.05,FC>1.2).Therefore,expression trend of myristic acid(FA 14:1)and arachidonic acid(FA 20:4)were consistent in plasma sample of both RSV-infected children and mice.(5)Contents of FA 14:0,FA 16:0,FA 16:1,FA 17:0,FA 17:1,FA 20:1,FA 20:2,FA 22:1,FA 22:2,FA 24:1 in lung tissues of RSV-infected mice were up-regulated in the model group using quantitative detection,and the medium-dose QF could significantly reduce their contents in the model group.(6)As a key molecule regulating fatty acid metabolism,the expression of phosphorylated Akt protein was increased in the model group while decreased in the therapy group.Its downstream protein ACLY was also increased in the model group while decreased in the therapy group.On the contrary,PPARα protein was decreased in the model group and increased in the therapy group.Therefore,it was suggested that QF inhibited the fatty acid synthesis(FAS)and stimulated fatty acid oxidation(FAO)processes through Akt signaling pathway.(7)Flow cytometry showed the increased number of pro-inflammatory macrophages(M1)while the decreased number of anti-inflammatory macrophages(M2)in lung tissues of RSVinfected mice.The ratio of M1/M2 macrophage was also decreased after QF intervention.Therefore,the fatty acid metabolic remodeling effect of QF further induced M1 to M2 macrophage polarization.(8)When Akt protein was activated,the regulatory effect of QF on fatty acid metabolic enzymes was weakened,accompanied by a decrease in the polarization of M1 to M2 macrophage.Besides,pulmonary inflammation was not significantly alleviated.This shows that QF stimulated fatty acid-dependent M1/M2 macrophage polarization via the Akt signaling pathway,so as to alleviate the pulmonary inflammation.Conclusions:(1)’Relieving lung stagnation,resolving phlegm and removing toxin’ is an effective and safe method in treating pediatric viral pneumonia with phlegm heat syndrome,which can effectively shorten the time of fever,cough,wheezing and lung rale in children.Qingfei oral liquid is one of the core prescriptions for this therapy.(2)Effective components of QF including quercetin,luteolin,kaempferol and naringin can directly target on Akt target,and which may play a key role for QF therapy.(3)Lipid metabolic disorder,dominated by fatty acid synthesis,was proved by lipidomic analysis based on samples from children with RSV pneumonia(phlegm heat syndrome)as well as RSV-infected mice.(4)QF can inhibit FAS and promote FAO by acting on Akt signaling pathway,thus reducing the accumulation of fatty acids.(5)The remodeling of fatty acids metabolism triggered by QF via Akt signaling pathway will further make the M1 to M2 macrophage polarization,thus alleviating the pulmonary inflammatory response caused by RSV infection.