Multi-omics Joint Analysis To Explore The Mechanism Of Action Of Sanyang Combined Treatment For Influenza Virus Pneumoni

ObjectiveTo study the clinical medication rule of exogenous fever through data mining of traditional Chinese medicine prescriptions of patients with exogenous fever.To explore the effect and mechanism of Maxing ShiGan Decoction and Xiaochaihu Decoction,the representative prescriptions based on three yang combined therapy,in treating influenza viral pneumonia by using metagenome,metabolome,and transcriptome sequencing techniques.Methods1 Study on clinical medication rule of traditional Chinese medicine in treating exogenous fever based on data mining:Collect traditional Chinese medicine prescriptions from patients with community-acquired pneumonia who was consistent with clinical manifestations of exogenous fever,establish a database,analyze the frequency of medication,gender,taste,and efficacy classification,and perform association rules and cluster analysis on the high-frequency use of drugs.2 Effect of three yang combined therapy on acute lung injury in mice with influenza virus pneumonia:Six-week-old female C57BL/6J mice were divided into two large groups,each of which was divided into four groups:blank group,model group,oseltamivir group,and Sanyang Hezhi group.Except for the blank group,the other groups used nasal drops of influenza virus suspension to establish influenza viral pneumonia mouse models.The drug was administered the next day after infection,and was intragastrically administered to 7 days after infection.One large group received 900 PFU influenza virus suspension nasal drops,and recorded the survival of mice until 14 days after infection.The death protection of Sanyang Hezhi on influenza virus pneumonia mice was observed.In the other group,the model was established by nasal drip of 450 PFU virus suspension.The weight changes of the mice were recorded daily,and the mice were observed until 7 days after infection.Eyes were removed,blood was taken,and the mice were euthanized.Lung tissue was taken,lung wet weight was measured,and lung index was calculated;Take the colon and measure its length;The pathological changes of lung tissue were observed by HE staining and electron microscopic examination;The viral load of lung tissue was measured by RT-PCR method;Luminex technique was used to detect the expression of cytokines in lung tissue and serum.3 Metagenome sequencing analysis of the regulatory effect of three yang combined therapy on gut microbiota in mice:DNA was extracted from mouse faces,the library was constructed and sequenced,the original data were filtered and sequenced,the data were compared to non-redundant gene sets,the abundance information of genes in each sample was calculated,and the species diversity of each sample was analyzed.According to the abundance information of gene set,the species abundance of each group was obtained,and the composition and abundance of microflora were compared from the level of phylum,class,order,family,genus and species.KEGG enrichment of differentially expressed genes was carried out to analyze the main enrichment pathways.4 Metabonomics analysis of the effect of Sanyang Hezhi on the metabolites in the caecum contents of mice:the metabolites in the caecum contents were extracted,the original mass spectrometry data were preprocessed and qualitatively controlled,the differential metabolites were identified,and the differential metabolites were enriched by KEGG,and the pathway of significant enrichment of differential metabolites was analyzed.Select the pathway with the most significant differential metabolic enrichment,and compare the abundance of metabolites and their metabolites in the blank group,model group,and Sanyang combined treatment group based on peak area.5 Transcriptome sequencing analysis of the effect of Sanyang combined therapy on gene expression in lung tissue of mice:the total RNA of lung tissue was extracted,the library was constructed and sequenced,and the original data was filtered after sequencing and compared to the reference sequence.Calculate the amount of gene expression,screen the differentially expressed genes(DEGs)between the Sanyang combined treatment group and the model group,classify the DEGs between the two groups by KEGG classification,enrich the KEGG pathway,and analyze the enrichment pathways of DEGs and their up-and-down trends by gene enrichment analysis(GSEA).Results1 Clinical researchAmong the prescriptions of 127 patients with community-acquired pneumonia who met the clinical manifestations of exogenous fever,the most frequently used drugs were Scutellariae Radix,Armeniacae Semen Amarum,Bupleuri Radix,Pinelliae Rhizoma Preparatum Cum Alumine,Platycodonis Radix,Ephedrae Herba,Gypsum,Glycyrrhizae Radix Et Rhizoma,etc.The four properties of the drugs were mainly cold,followed by warm and peaceful;The five tastes are mainly bitter,spicy,and sweet;The meridians are mainly focused on the lung,stomach,and spleen;The main functions include relieving external symptoms,clearing heat,relieving cough,resolving phlegm,and relieving asthma.Scutellariae Radix-Bupleuri Radix,Pinelliae Rhizoma Preparatum Cum Alumine-Scutellariae Radix,Ephedrae Herba-Armeniacae Semen Amarum,Pinelliae Rhizoma Preparatum Cum Alumine-Bupleuri Radix,GypsumArmeniacae Semen Amarum are the drug pairs with higher frequency of use.According to the association rules,the highest ranking of support were Rhizoma Preparatum Cum Alumine,Bupleuri Radix and Scutellariae Radix,Gypsum,Armeniacae Semen Amarum and Ephedrae Herba,Ephedrae Herba,Gypsum and Armeniacae Semen Amarum.Cluster analysis identified five core drug formulations,two of which were based on Xiaochaihu decoction and Maxing Shigan decoction.2 Effect of three yang combined therapy on acute lung injury in mice with influenza virus pneumonia(1)Survival analysis experiment suggested that Sanyang Hezhi had a protective effect on the death of mice infected with influenza virus;(2)General state:Sanyang Hezhi could improve the mental state of mice and improve the weight loss of mice caused by influenza virus infection;(3)Lung index and colon length:Sanyang Hezhi could significantly reduce the lung index of mice and improve the shortening of colon length caused by influenza virus infection;(4)Pathology of lung tissue:the results of HE staining showed that Sanyang Hezhi could improve the pathological injury of lung tissue,reduce inflammatory cell infiltration,pulmonary interstitial edema and hyperemia;the results of electron microscope showed that Sanyang Hezhi could reduce the emptying of lamellar bodies in alveolar type Ⅱ epithelial cells caused by influenza virus infection;(5)viral load in lung tissue:Sanyang Hezhi could significantly reduce the viral load in lung tissue;(6)Cytokines in lung tissue:influenza virus infection could increase the levels of IL-6,TNF-α,IFN-γ,CCL2,CCL3,CCL5,CXCL10,ICAM1 and GMCSF in lung tissue,and Sanyang Hezhi significantly decreased the levels of IL-6,IL-10,CCL2 and CXCL10;(7)Cytokines in serum:the serum levels of IL-6,TNF-α,IFN-γ,CCL2,CCL5,CXCL10 and ICAM1 increased after influenza virus infection,and Sanyang Hezhi could significantly reduce the levels of IL-6,IL-1β,TNF-α,CCL5 and CXCL10.3 Regulatory effect of three yang combined therapy on gut microbiota in mice(1)Species diversity:the results of α-diversity showed that the species composition and abundance of intestinal microorganisms in mice decreased after influenza virus infection,and Sanyang Hezhi could improve the species composition and abundance;the results of β-diversity showed that the species composition was similar in each group,but there were differences among different groups;(2)Species composition and difference of flora:at the gate level,the relative abundance of Verrucomicrobia and Proteobacteria increased significantly in the model group,while the relative abundance of Bacteroides decreased in the model group.Sanyang Hezhi can restore the above three changed flora to varying degrees.In the class level,the relative abundance of Bacteroidia and Bacilli decreased in the model group,while the relative abundance of Verrucomicrobiae increased in the model group.Sanyang Hezhi can restore the above changed bacterial communities to varying degrees.At the order level,the relative abundance of Bacteroidales and Lactobacillales decreased and the relative abundance of Verrucomicrobiales increased in the model group,and the above-mentioned microflora could be restored in different degrees by the combined treatment of Sanyang.At the family level,the relative abundance of Akkermansiaceae,Tannerellaceae and Enterobacteriaceae in the model group increased,while the Prevodiaceae,Muribaculaceae and Lactobacillaceae decreased,and the combination of Sanyang could regulate the above flora;(3)Analysis of species differences among the three groups:at genus level:In the model group,the abundance of Bacteroides,Bacteroides,Lactobacillus and Prevotella decreased,while the relative abundance of Escherichia,Shigella,Parabacteroides,Erysipelatoclostridium and Butyricimonas increased.However,the abundance of the changed floras in the Sanyang combined treatment group was restored to varying degrees.At species level,the abundance of Muribaculaceae,Prevotella and Lactobacillus in the model group decreased,while that of Butyricimonas,Desulfovibrionaceae and Parabacteroides increased in the model group,and the Sanyang combination recovered the above flora in varying degrees.(4)Functional enrichment analysis of differential genes in species:the results of GO enrichment analysis showed that the differential genes in the Sanyang combination group and the model group were mainly enriched in the metabolic process in the biological process.The results of KEGG pathway enrichment analysis showed that the differential genes between Sanyang Hezhi group and model group were significantly enriched in phenylalanine,tyrosine and tryptophan biosynthesis pathway.4 Regulatory effect of three yang combined therapy on intestinal metabolites in mice(1)Multivariate statistical analysis:PC A and PLS-DA analysis showed that the repeatability of samples within the group was good,but there were significant differences in metabolic spectrum between different groups.(2)Enrichment of differential metabolites in KEGG pathway:differential metabolites between Sanyang Hezhi and model group were significantly enriched in tryptophan metabolic pathway.(3)Comparison of relative abundance of tryptophan and its metabolites:there was no significant difference in the level of tryptophan among the three groups,and there was no significant change in the contents of L-kynurenie and anthranilic acid in the model group,but the levels of kynurenie acid and indole were significantly up-regulated.Sanyang Hezhi could significantly down-regulate these two metabolites and significantly up-regulate L-kynurenine and anthranilic acid,which had no significant change in the model group.5 Study on the key targets and pathways of Sanyang combined therapy in the treatment of influenza viral pneumonia based on transcriptome sequencing.(1)Screening of differential metabolites:there were 2838 differential genes between the Sanyang Hezhi group and the model group,of which 1884 were up-regulated and 954 downregulated.(2)Heat map analysis of differential genes:compared with the model group,genes related to inflammation and tissue injury were down-regulated in Sanyang Hezhi group,such as pro-inflammatory factors IL-1β,IL-6,TNF-α,CCL3,CCL4,CCL9,CXCL3,pro-apoptosis factors Fas,Bax,Toll-like receptor related factors TLR1,TLR2,TLR6,TLR13,NOD receptor related factors NLRP3,NLRC4,acute lung injury related factors S100a8,S100a9.Some genes related to pulmonary physiological function,such as mucins Muc1,Muc5b related to airway defense and pulmonary immune homeostasis,surface active proteins Sfta2 and Sftpal related to reducing alveolar surface tension,secreted red proteins Scgblal,Scgblcl and Scgb3a2 related to alveolar injury and repair,and peroxisome proliferator-activated receptor PPARa are up-regulated.(3)Functional enrichment analysis of differential genes:KEGG functional classification and gene entry KEGG pathway enrichment analysis in functional classification:360 genes enriched in signal transduction function classification,these genes were significantly enriched in PI3K-Akt signal pathway,MAPK signal pathway,NF-κB signal pathway and TNF signal pathway.226 genes enriched in the immune system classification,these genes were significantly enriched in IL-17 signal pathway,NF-κB signal pathway,TNF signal pathway,NOD-like receptor signal pathway and Toll-like receptor signal pathway.51 genes enriched in amino acid metabolic classification,these genes were significantly enriched in tryptophan metabolism and other pathways.(4)Pathway of differential gene enrichment and its upward and downward trend:the results of GSEA analysis showed that tryptophan metabolism and PI3K-Akt signal pathway related to immunity and metabolism were significantly up-regulated in Sanyang Hezhi group.IL-17 signal pathway,NF-κB signal pathway,TNF signal pathway,Toll-like receptor signal pathway and NOD-like receptor signal pathway related to inflammation were significantly down-regulated in Sanyang Hezhi group.ConclusionsMaxing Shigan decoction and Xiaochaihu decoction are widely used in the treatment of exogenous fever,and their combination is a classic combination of three yang combined therapy.In clinical application,drugs such as clearing heat and detoxifying,resolving phlegm,relieving cough,and strengthening the body can be used in combination with the treatment of the combination of the three yang according to the patient’s clinical manifestations.Three yang combined therapy has a good protective effect on acute lung injury induced by H1N1 influenza virus,and its mechanism may be related to the regulation of intestinal flora-tryptophan metabolism,IL-17 signal pathway and TLR/NF-κB signal pathway.