Mechanism Of Wogonin In The Treatment Of Streptococcus Pneumoniae Infection Based On Multi-omics Analysis

Part Ⅰ Wogonin attenuates the pathogenicity of Streptococcus pneumoniae through dual target inhibition of PLY and SrtAObjective: To explore the mechanism of wogonin in the treatment of S.pneumoniae infectionMethods:1.The main active ingredients of the scutellaria baicalensis georgi including baicalin,baicalein,wogonoside,wogonin,which has the inhibitory effect on the pneumolysin and sortase A of the S.pneumoniae,is screened out through hemolysis test and SrtA peptidase activity inhibition test.2.S.pneumoniae D39 was incubated with different concentrations of wogonin,and the expression of PLY oligomer was detected by Western blot.3.To establish cell model and evaluate the inhibitory effect of wogonin on the adherence of S.pneumoniae.4.To evaluate whether wogonin can improve the pulmonary inflammatory reaction and the expression of inflammatory factors in mice infected with S.pneumoniae.Results:1.Wogonin significantly inhibited the hemolytic activity of PLY with IC50 of 52μg/ml.Wogonin also inhibited SrtA peptidase activity and biofilm formation.2.Wogonin inhibited the oligomerization of PLY,but did not affect the transcription and expression of PLY.3.Wogonin could reduce the injury of A549 cells induced by PLY and the ability of S.pneumoniae to adhere and colonize epithelial cells.4.Wogonin can improve the survival rate of mice infected with S.pneumoniae D39,reduce the expression of TNF-α,IL-6 and IL-1β,reduce the number of strains colonized in lung tissue,and reduce the pathological reaction of lung tissue in mice infected with S.pneumoniae D39.Conclusion:1.Wogonin is a dual target inhibitor of PLY and SrtA.2.Wogonin reduces pathogenicity by inhibiting the oligomerization of PLY.3.Wogonin reduced the cell injury induced by S.pneumoniae.4.Wogonin has protective effect on lung tissue of mice infected with S.pneumoniae.Part 2 Transcriptomic study of wogonin in mice infected with S.pneumoniaeObjective: To explore the mechanism of wogonin on the transcription level in mice infected with S.pneumoniaeMethods:1.The left lungs of mice in healthy group,model group and wogonin group were taken out for transcriptomic sequencing.2.To compare the difference expression between healthy group and model group,model group and wogonin group.3.Enrichment analysis was performed between healthy group and model group,model group and wogonin group.Results:1.There were 2394 differential genes between healthy group and model group,1317 up-regulated genes and 1077 down-regulated genes.There were 263 differential genes between model group and wogonin group,79 of which were up-regulated and 184 down-regulated.2.KEGG pathway enrichment analysis of model group and wogonin group,the first 20 pathways were: IL-17 signaling pathway 、 Complement and coagulation cascades 、Chemokine signaling pathway、Antigen processing and presentation、Cytokine-cytokine receptor interaction、Cell adhesion molecules(CAMs)、MAPK signaling pathway、Jak-STAT signaling pathway、NF-kappa B signaling pathway、Neuroactive ligand-receptor interaction、Legionellosis、Transcriptional misregulation in cancer、Epstein-Barr virus infection、Chronic myeloid leukemia、Chagas disease(American trypanosomiasis)、Thyroid cancer、HTLV-I infection、Glioma、p53 signaling pathway.Conclusions:1.Wogonin can affect gene expression in mice infected with S.pneumoniae at transcriptional level.2.After wogonin intervention,KEGG enrichment analysis was mainly related to tumor,cancer,inflammatory signal and other signal pathways.Part 3 Metabonomic study of wogonin on S.pneumoniae infected miceObjective: To explore the mechanism of wogonin on the metabolism level in mice infected with S.pneumoniaeMethods.1.The serum of mice in healthy group,model group and wogonin group were taken out for metabolomics sequencing.2.According to VIP>1 and P<0.05 as the screening standard,to find out the different metabolites between the healthy group and the model group,the model group and the wogonin group.3.KEGG enrichment analysis was used to compare the healthy group with the model group,the model group with the wogonin group.Results:1.In the negative ion mode,PCA,OPLS-DA and PLS-DA analysis showed that the healthy group and the model group,the model group and the wogonin group were significantly separated;In positive ion mode,PCA analysis,OPLS-DA analysis,PLS-DA analysis found that the healthy group and model group,model group and wogonin group can also be significantly separated.2.There were 128 different metabolites between the healthy group and the model group,31 of which were up-regulated and 97 down-regulated;There were 134 differential metabolites between model group and wogonin group,of which 64 were up-regulated and 70 were down-regulated.3.KEGG pathway enrichment analysis of model group and wogonin group,the top 20 enrichment pathways were Protein digestion and absorption,Central carbon metabolism in cancer,ABC transporters,Aminoacyl-t RNA biosynthesis,m TOR signaling pathway,Tyrosine metabolism,Mineral absorption,Pathways in cancer,Pyrimidine metabolism,Lysine degradation,Thermogenesis,Prostate cancer,Retinol metabolism,Intestinal immune network for Ig A production,Glucagon signaling pathway,Arginine and proline metabolism,Amoebias,Regulation of lipolysis in adipocytes、Small cell lung cancer、Rheumatoid arthritis.Conclusions:1.Compared with the model group,there were many different metabolites after wogonin treatment,among which tyrosine,lysine,arginine,proline,leucine and other amino acids were up-regulated.2.After wogonin intervention,the enrichment pathway is mainly related to protein digestion,absorption and transport,inflammatory reaction,cancer,lysine,arginine,proline and other amino acid metabolism.Part 4 Gut microbiota of wogonin in S.pneumoniae infected miceObjective: To explore the mechanism of wogonin on S.pneumoniae infection in mice by microbiomeMethods:1.Intestinal contents of mice in healthy group,model group and wogonin group were taken out for microbiome sequencing.2.Using Illumina Novaseq high-through sequencing technology,using QIIME2 software,the OTUs were finally obtained by DADA2 de-priming,mass filtration and other steps.The effects of wogonin on the species diversity,structure and function of intestinal microflora in mice infected with S.pneumoniae were investigated.Results:1.There were 5627 OTUs in the healthy group,7124 OTUs in the model group,5764 OTUs in the wogonin group,1628 OTUs in the normal group and the model group,1624 OTUs in the model group and the wogonin group,1278 OTUs in the healthy group and the wogonin group,and 943 OTUs in the three groups.2.S.pneumoniae infection and wogonin treatment did not affect the diversity and richness of mouse species,but significantly changed the structure of microbial community.3.At the phylum level,Firmicutes,Bacteroidetes and Proteobacteria were the dominant species,accounting for more than 80% of the total in all three groups.The proportion of Firmicutes(31.01%)in the model group was reduced compared to the healthy group(45.64%).The proportion of Firmicutes in the wogonin group(49.64%)increased compared to the model group(31.01%)and was similar to that of the healthy group.In contrast,the proportion of Bacteroidetes increased in the model group(58.18%)and decreased in the wogonin group(19.35%)compared to the healthy group(45.98%).The proportion of Proteobacteria in the wogonin group was 14.19% compared to 3.82% and 5.44% in the healthy and model groups respectively.The proportion of Verrucomicrobia in the wogonin group was(12.54%)compared to 0.04% in the healthy group and 0.08% in the model group.The distribution of gut microbiota in S.pneumoniae infected mice was altered by wogonin at class,order,genus,and family level.Conclusions:1.Wogonin did not affect the species homogeneity and richness in the intestine of mice infected with S.pneumoniae,but it could significantly change the structure of the intestinal microbial community.2.After wogonin treatment,the abundance of Firmicutes increased,while the abundance of Bacteroidetes decreased,the ratio of Firmicutes to Bacteroidetes(F/B)increased,and the proportion of Verrucomicrobia increased.Wogonin can also change the abundance of intestinal flora in mice infected with S.pneumoniae at the level of family and genus.Part 5 Transcriptomics and metabolomics of wogonin in S.pneumoniae infected miceObjective: To analyze the mechanism of wogonin on S.pneumoniae infected mice by combined transcriptomics and metabolomicsMethods: Same as parts Ⅱ and ⅢResults:1.In positive ion mode,we found 28 significant enrichment pathways by KEGG enrichment analysis of metabolites and transcripts between model group and wogonin group.2.In the negative ion mode,we found 23 significant enrichment pathways by KEGG enrichment analysis of differential genes and metabolites between model group and wogonin group.Conclusion:After wogonin intervention,in positive and negative ion mode,transcriptomics and metabolomics KEGG combined analysis found that the enrichment pathway was mainly related to protein digestion,absorption and transport,inflammatory response,cancer and some amino acid metabolism pathways.