| Purpose:To explore the mechanism of "lung-intestinal co-treatment" of influenza virus and streptococcus pneumoniae co-infected pneumonia with xuanbai Chengqi Decoction.Ⅳ/Spn induced mouse pneumonia model was used to explore the relationship between the pathogenesis of pneumonia in coinfected mice and the theory of "lung and intestine damage by poison" in Traditional Chinese medicine.Tag(TMT)using tandem mass spectrometry(ms)technology to the lung tissue of mice differences in protein bioinformatics analysis,combined the targeted metabolomic screen potential target protein biomarkers and key,cross validation Xuan Bai Cheng gas soup regulation related pathways were pneumonia,and use parallel reaction monitoring(PRM)targeted candidate protein expression in intestinal tissue,Elucidate the mechanism of Xuanbai Chengqi Decoction to open lung and regulate intestine;Based on the screening of multiple omics techniques,the characteristic indicators of the theory of "poison damage lung and intestine" in the incidence of Ⅳ/SPn co-infection pneumonia and the treatment process of TRADITIONAL Chinese medicine were explored to provide experimental evidence support for clinical treatment.At the same time,the alcohol extraction process of xuanbai Chengqi decoction was optimized.Material and method:1.64 BALB/C mice aged 4-6 weeks were randomly divided into normal group and infected group,including 32 normal group and 32 infected group.Mice in the infected group were inoculated with 50μ L influenza A virus(Ⅳ)and 50μ L Streptococcus pneumoniae(Spn)rat lung adapted strains in nasal cavity to induce co-infection model.Mice in the normal group were given 0.9% normal saline nasal drops(100ul/ mouse)for 1 nasal day.8 mice in each group were killed at 6h,24 h,48h and 72 h after infection.In lung tissue,in combination with general status in mice,behavior,pathological lung tissue,lung tissue morphology,lung index in mice,intestinal trilobites factor(TFF3)observe all aspects of the difference between normal group and model group mice,by detecting the lung tissue of mice in different time points of viral load and bacteria load,evaluate model from multiple angles.2.Eighty clean GRADE BALB/C mice aged from 2.4 to 6 weeks were randomly divided into4 groups according to body weight by random number table: blank control group(group ⅰ),model group(group ⅱ),Xuanbai Chengqi decoction supplemented group(group ⅲ)and oseltamivir + cephalosporin group(group ⅳ),with 20 mice in each group.In addition to the normal group,the other 4 groups were modeled for co-infection,which were given intragastric administration in accordance with the corresponding groups at 6h after modeling.Samples were taken on the third day of administration and pharmacodynamics were evaluated according to the indexes in model evaluation.3.Lung tissue samples of blank control group,model group and Xuanbai Chengqi Decoction supplemented group were tested by tandem mass tag(TMT)to screen for differential proteins;And l C-MSMS untargeted metabolomics detection to screen differential metabolites.R-4.0.1 and Cytoscape3.8.2 were used to analyze the differential metabolites and differential proteins.PRM parallel response monitoring(PRM)was used to detect and validate the key biomarkers involved in regulation in mouse intestinal tissue samples.4.orthogonal experiment design method,ethanol concentration,extraction time,extraction times to examine factors,to Xuan Bai Cheng tonga nine extract rate of the taste,and application of HPLC analysis of 9 laetrile signature in the active component of the group,the content of baicalin,emodin OD value of evaluation index based on weighting comprehensive rating,The analysis results were used as process evaluation index to screen the best alcohol-extraction method in this experiment.Results:1.Study and identification of Ⅳ/SPn co-infected mouse pneumonia model1.1 Comparison results of lung index in each group:Compared with the blank control group,the lung index of the model group showed a trend of daily increase.After 48 h of infection,there was a significant difference compared with the blank control group at the same time point(p < 0.05),and reached the peak at 72 h.Compared with the previous time point,there was a significant difference within the model group at 48 h from the previous time point(p < 0.05).1.2 Comparison results of intestinal trefoil factor TFF3 in each group of mice:Compared with the blank control group,the TFF3 index increased 6h in model group,and there was a significant difference(p< 0.05).The infection decreased at 24 h and was lower than that of the normal group at the same time point,with significant difference(p < 0.05);the infection decreased continuously at 24--48 h,with significant difference(p< 0.05),and decreased slightly at 48--72 h,but with no significant difference(p>0.05).1.3 Pulmonary pathological changes after co-infection:The pathological changes of lung were observed under light microscope.After modeling,the normal group had intact bronchi,respiratory bronchi,alveolar tube,alveoli,alveolar sac and bronchiole in lung tissue at any time point,and no lesions in the whole lung field.In model group,with the advance of time,normal tissue structures such as alveoli,alveolar sac,alveolar tube and bronchiole disappeared,connective tissue grew,large lung interstitium hyperemia,edema and inflammatory cell infiltration dominated by lymphocytes and monocytes,alveolar wall widened significantly,and macrophages and red blood cells exudated in the cavity.With the prolonged infection time,the infiltration of inflammatory cells and red blood cells in the alveolar cavity increased,the alveolar cavity gradually narrowed,and the alveolar wall thickened.Bai Cheng compared with model group,western medicine group and combination group after treatment,the alveolar lumen cellulose out less,groups of alveolar consolidation gradually reduce,alveolar interval gradually clear,alveolar gas filling,pressure loss,pulmonary interstitial only a handful of congestion,and a small number of inflammatory cells infiltration,part of the alveolar damage,just saw a blue inflammatory cell aggregation,reduced compared with model group.1.4 Pathological changes of intestinal tissues after co-infection:The simple columnar epithelial cells of colon tissue of mice in blank control group were closely arranged,and the shape of intestinal gland was intact.The simple columnar epithelial cells were rich in goblet cells.The mucosa,submucosa and muscularis were clearly visible with distinct texture.Compared with blank control group,simple columnar epithelial cells of colon tissue in model group were loosely arranged and the morphology of colon gland was atrophic.The muscularis and mucous muscularis became thinner,the monolayer columnar epithelium and the goblet cells of the intestinal gland decreased,and the microstructures were disarranged or disappeared over time.Inflammatory cell infiltration,ulcer,vasculitis and other injuries appeared.Compared with model group,xuanbai Chengqi decoction treatment group reduced the infiltration range of inflammatory cells in the lesion site of intestinal mucosa,and reduced vasculitis and microstructure destruction.In the combination group,a large number of inflammatory cell infiltration was limited to the point membrane and the sublayer of the point membrane,and the destruction of the epithelium and glands was reduced.In the western medicine group,the colonic lesions were not improved,and the main symptoms were chronic ulcerative inflammation,with a large number of inflammatory cells infiltrating the mucosa and submucosa,ulcer formation and vasculitis.1.5 Determination results of bacterial and viral load in lung tissues at different time points:No pathogens were detected in lung tissues of mice in blank control group at each time point.Iv-rna amplified expression was found in the lung tissues of mice in the model group from 6h after infection,and the expression increased with the increase of viral load,peaked at 48 h,and decreased slightly at 72 h.Meanwhile,bacterial load increased with the progress of time from24 h,suggesting that the amount of viral and bacterial infection was proportional to the damage of lung tissue..。2.Metabonomics and proteomics combined analysis and verification of xuanbai Chengqi Decoction for the intervention of co-infected mouse pneumonia model2.1.Compared with the model group,there were 473 significant metabolites in the blank group,among which 12 were up-regulated and 18 were down-regulated in the top30.After KEGG enrichment,93 metabolic pathways were obtained.259 proteins were significantly differentially expressed between the model and the normal group,including 140 up-regulated proteins and 110 down-regulated proteins.GO enrichment analysis showed that the differentially expressed proteins were mainly distributed in the cytoplasm,cell membrane and extracellular region of co-infection pneumonia.Mainly involved in various metabolic processes;The main functions of differentially expressed proteins are binding activity and enzyme activity.After KEGG enrichment,53 protein pathways were obtained.After joint analysis,a total of 19 KEGG pathways were discovered.Among them,the biological processes that have a common relationship with the two levels of protein-metabolism are metabolism,pathway signal transduction,cell transport and catabolism,and are also closely related to the endocrine system.There were significant differences(p <0.05)in amino sugar and nucleotide sugar metabolism,alanine,aspartate and glutamate metabolism,beta alanine metabolism,oxidative phosphorylation,neuroactive ligand-receptor interaction,butyric acid metabolism,taste conduction,arginine and proline metabolism,respectively.The protein-metabolism-pathway network map was established.2.2 compared with the model group,367 metabolites with significant difference were found in the TCM group,16 of the top30 metabolites with significant difference were up-regulated and14 down-regulated.120 metabolic pathways were obtained after KEGG enrichment.In the process of intervening in the model of co-infection pneumonia,the 13 significantly different proteins in the Chinese medicine group and the model group were up-regulated by 5 and down-regulated by 8.The total number of differentially expressed proteins was 13,including5 up-regulated proteins and 8 down-regulated proteins.GO enrichment analysis showed that the main molecular functions of differentially expressed proteins of Xuanbai Chengqi Decoction in co-infected pneumonia were enzyme catalysis,transformation and transfer.Eleven protein pathways were obtained after KEGG enrichment.After joint analysis,a total of 10 KEGG pathways were discovered.Among them,the biological processes that have a common relationship with the two levels of protein-metabolism are metabolism,genetic information transcription,pathway signal transduction,cell transport and catabolism,and are also closely related to the biological systems of endocrine,digestion and excretion.Among them,fatty acid degradation,butyric acid metabolism,beta alanine metabolism,glutathione metabolism,lysine degradation,tryptophan metabolism,galactose metabolism and propionic acid metabolism were significant(p <0.05).The protein-metabolism-pathway network map was established.2.3 In the analysis of the direct effect of intervention regulation in the Chinese medicine group,it was found that 19 pathways were up-regulated in the protein level co-infection pneumonia,and 11 of them showed a trend of adjustment after xuanbai Chengqi Decoction intervention.There were 94 pathways that produced regulatory changes in the metabolic level xuanbai Chengqi Decoction supplementation in the mouse model of co-infected pneumonia.Combined with the differences between the pathogenic and therapeutic groups,the target pathways and key influencing factors were obtained.The results showed that the GO annotation was basically consistent with the KEGG annotation.The target pathways are fatty acid degradation,valine,leucine and isoleucine degradation,lysine degradation,tryptophan metabolism,β-alanine metabolism,propionic acid metabolism,butyric acid metabolism,PPAR signaling pathway,interaction in vesicle transport,peroxisome,rig-I like receptor signaling pathway.The key influencing factors are Propachlor ESA,Diferuloyl Putrescine and Bavachinin AEhhadh,Vamp4,Gosr1,Ehhadh,Dhx58,SUOM,Acly,Gart,Malate,3-hydroxybutanoatepyruvate,Maleate,L-2-aminoadipate,Succinate,Glu Tarate,Pipecolate,Glutamate,Anserine,Uracil,Quinolinate,Pantothenate,Histidine,2-hydroxybutanoic Acid,Hexadecanoate,Coenzyme A,Palmitoylcarnitine,Tryptamine,Indole-3-acetaldehyd,3-hydroxyanthranilate,2.4 Cytoscape software was used to map to protein-metabolization-pathway network to analyze these potential biomarkers,and acly,Gart,Ehhadh and Dhx58 were selected as the key lung-intestinal homology proteins of Xuanbai Chengqi Decoction for the intervention of co-infection pneumonia for PRM verification in mouse lung-intestinal tissues.Results The differences of candidate peptides of each protein were all around 2,and the P value was less than 0.05,indicating that the differences were significant.3.Orthogonal design study on extraction method of Youxuanbai Chengqi Decoction with gourmet alcohol3.1 Orthogonal design Preparation of Xuanbai Chengqi Decoction with gourmet alcohol extractEthanol concentration,extraction time and extraction times were taken as factors to evaluate the paste yield.The paste yield was: No.1 sample,12.25%;No.2 sample,20.81%;No.3 sample,15.99%;No.4 sample,7.00%;No.5 sample,16.96%;No.6,10.34%;No.7 sample,11.71%;No.8,11.85%;Sample No.9,13.43%.3.2 High performance liquid chromatography for the determination of signature components into the formula to calculate the scoreOD value of comprehensive score =30%×(emodin content/emodin content Max)+30%×(amygdalin content/amygdalin content Max)+30%×(baicalin content/baicalin Max)+10%×(dry cream rate/dry cream rate Max).After calculation,the comprehensive score is: no.163.508 points;No.2 66.980;No.3 77.350;No.4 58.173;No.5,80.284;No.6,62.489;No.7,68.998;No.8 73.549;No.9 60.954;Conclusion:1.By observing the lung index of mice in general state under light microscope,lung and intestinal tissue pathology and pathogen load in lung and intestinal tissues were detected to verify that intranasal inoculation of influenza A virus and Streptococcus pneumoniae under ether anesthesia and 48 h after infection could successfully establish the virus-damaged pneumonium-enteropneumonia model of BALB/C mice coinfected with Ⅳ/Spn meeting the requirements of this experiment2.Through the comparative analysis to KEGG enrichment of differentially expressed proteins involved in model group and Chinese medicine group and metabolic target path,show Xuan Bai Cheng gas mixed infection in mice poison damage lung soup intervention mainly through the mechanism of intestinal butyric acid metabolism Lysine degradation beta alanine metabolism Propionic acid metabolism of fatty acid degradation of article 5 of the regulation of metabolic pathways to realize select SUOM Acly Gart Ehhadh Dhx58,Bavachinin A and other potential biomarkers strongly correlated with the lung-intestinal homology intervention of Xuanbai Chengqi Decoction in the treatment of virus-damaged lung-intestinal pneumonia3.The visual analysis and variance analysis results of amygdalin,baicalin and emodin,the iconic active ingredient in xuanbai Chengqi Decoction,showed that the sequence that had the greatest influence on THE OD value was extraction times,extraction time and ethanol concentration.Therefore,the optimal extraction scheme in this design was No.5(A2B2C3)65% ethanol reflux for three times in 120min... |
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