Study Of Efficacy Of Flos Lonicerae Japonica-Fructus Forsythiae Herb Pair On H1N1-Induced Pneumonia By Lung-Gut Axis Metabolomics

Influenza,caused by influenza virus,is the most prominent annually recurring global respiratory infection because of its wide range of hosts and its ability to mutated and spread across species,which has not yet been efficiently controlled.In recent years,influenza virus has frequently erupted and in severe cases,such as the new type influenza virus A(H1N1)originated in North America in 2009,has caused ten thousands of deaths annually,which makes H1N1 one of the main epidemic strains spread in humans.HlN1 infection can lead to severe pneumonia,acute lung injury and respiratory failure,and even mortality.In addition,H1N1 infection can also cause intestinal inflammation."Flos Lonicerae Japonicae-Fructus Forsythiae"herb couple(Yin Qiao in Chinese,YQ)has been widely used for treatment of epidemic febrile disease due to its detoxification effect.Yinqiao powder,a traditional Chinese medicine,which is mainly consisted of YQ extract,has been clinically used for the treatment of HlN1-induced pneumonia for a long time.However,its underlying mechanism needs further study.In this study,the H1N1 induced pneumonia model was established by intranasal dropping H1N1 virus chicken embryo allantoic fluid in C57BL/6 mice.The body weight,lung index,lung histopathology and the level of Tumor necrosis factor(TNF-?)and(Interleukin)IL-6 were determinate to evaluate the efficacy of the viral pneumonia model and the pesticide effect of YQ extract.The results of pesticide effect experiment showed that the YQ extract has a good therapeutic effect on influenza virus A(H1N1)pneumonia.The second part of this study analyzed the composition and endogenous metabolites of intestinal flora.First,the short chain fatty acids(SCFAs)were analyzed by using GC-MS platform and the results showed that YQ reduced the concertation of SCFAs significantly compared with H1N1 group.It may suggest that YQ did not cure the H1M1 pneumonia by reregulating the concertation of SCFAs.Besides,the analytical results of intestinal flora sequencing showed that flora abundance and composition changed significantly.The flora in cecel contents consisted of Bacteroidetes,Firmicutes,Proteobacteria,Cyanobacteria and others at the phylum level.In detail,there is no significance between HIN 1-pneumonia group,normal group and YQ group when it comes to Bacteroidetes,Firmicutes,Proteobacteria and others.While we can easily find that the amount of Cyanobacteria reduced significantly in cecel contents when YQ extract has been applied.At the genus level,the amount of bacteria Bilophila,[Eubacterium]coprostanollgenes group,Family XIII UCG-001,Oscillibacter,Rhodospirillaceae_uncultured,Ruminiclostridium and Ruminiclostridium 9 changed significantly in H1N1 group compared to normal group.In addition,expect Family XIII UCG-001,the amount of other six bacteria turned back to normal level when YQ extract applied,which suggested that YQ extract was able to play a useful role in gut bacterial composition in order to cure H1N1-induced pneumonia.On the other hand,results of fecal metabolomics reflected that 24 metabolites in feces changed obviously of H1M1-infected mice and YQ administration help recover the metabolic levels of 10 metabolites of them.That is to say,the metabolism of inosine,xanthine,uric acid,tryptophan,uracil,ethanolamine,phosphate,glycine,oxalic acid and thymine was regulated by YQ administration,which then took effects on amino acid catabolism,tryptophan metabolism,phospholipid metabolism.Last but not least,we performed spearman correlation analysis between metabolites and microbes which we have screened primarily above according to their significances.From the results we can easily found out that glycine,stearic acid,uric acid,uracil,thymine,xanthione,ethanolamine,oxalic acid,tryptophan and phosphate strongly related to all seven bacteria.So we considered these metabolites as key metabolites.In the third part of this study,non-targeted metabolomics analysis of plasma were performed based on GC-MS platform.The levels of plasma cytokine were significantly increased after H1N1 infection and significantly turned back to normal level after administration of YQ extract.Partial Least Squares-Discriminant Analysis(PLS-DA)was used to describe the metabolic profiles of each group.We chose the potential biomarkers of H1N1-induced pneumonia based on three rules:for one rule,fold change(FC)value between H1N1 group and control group must be higher than 1.5;for the second rule,the p value between H1N1 group and control group must smaller than 0.05;last but not least,VIP value derived from PLS-DA must be higher than 1.0.The results showed that 17 plasma metabolites which can be considered as potential biomarkers significantly altered in HIN1-infected mice,namely xanthosine,xanthine,4-hydroxyproline,threitol,talose,ribose,proline,phosphate,myo-inositol,L-cysteine,lactose,isopropyl beta-D-glucoside,glyceric acid,cycloleucine,beta-hydroxybutyric acid,aminomalonic acid,5-methoxyindoleacetate.Additionally,YQ extract could regulated 14 of 17 disordered metabolites and 4 other metabolites which are caffeic acid,beta-mannosylglycerate,glycine and hydrocinnamic acid to normal level.These metabolites in plasma affected by YQ extract administration mainly involving in three metabolic pathways,namely "galactose metabolism" "glycine,serine and threonine metabolism"and "synthesis and degradation of ketone bodies" pathways,which may be related to the underlying mechanisms of YQ extract's pesticide effect on H1N1-induced pneumonia.The last part of our research focused on the changes of endogenous metabolites such as lipids and small molecular subjects in lung by performing LC-MS based lipidomics and GC-MS based metabolomics.Principal component analysis(PCA)and cluster analysis were used to analyze data.The metabolites whose FC>1.5 and P<0.05 were selected as potential biomarkers in lung of H1N1-induced pneumonia mice.When it comes to metabolomics,the concentration of 2-hydroxypyridine,3-phosphoglycerate,arabitol,asparagine,caffeic acid,L-carnitine,dehydroascorbic acid,glycine,lauric acid,L-kynurenine,myo-inositol,ornithine and sorbitol disordered observably under the influence of HIN1-induced pneumonia.It has showed that YQ extract can also correct the disordered state of metabolites,which involved in energy metabolism pathways,oxidative stress and inflammatory pathways and cell repairing pathways.While when it comes to lipidomics,several lipids showed significantly changed trends between control group and H1N1 group,such as acylcarnitine(ACar),hexose ceramide non-hydroxy fatty acid-sphingosine(HexCer-NS),lysophosphatidylcholine(LPC),phosphatidylcholine(PC),phosphatidylethanolamine(PE),phosphatidylglycerol(PG),triglyceride(TAG),free fatty acid(FA),hexose ceramide non-hydroxy fatty acid-sphingosine(HexCer)-NS)and oxidized fatty acids(OxFA).YQ extract could protect the pulmonary surfactant lipid homeostasis by regulating various lipid levels such as TAG,PC,PE,PG and unsaturated fatty acids,which could be underlying mechanisms to explain the drug effects.This proj ect proposes the following hypothesis:"There are several metabolic disorders on the lung-blood-gut axis of H1N1 infected mice,and YQ extract can be able to regulate the metabolic level of glycine,which may ameliorate the composition of the intestinal Flora of HIN1-infected mice,systemic inflammation and the function of lung.Finally,YQ extract will be able to treat HIM1-induced pneumonia.