Network Pharmacology-based Approach For Screening TCM Against Coinfection Of MG And E.coli And Preparation Technology Of Granules

Respiratory disease of poultry is one of the most complicated diseases in poultry breeding industry,which is notorious for its high infection rate,high mortality rate and complicated etiology.It is an important factor that affects and restricts the healthy growth of poultry.The pathogen s that cause poultry respiratory disease including several etiologies such as bacteria,mycoplasma,virus and immune suppression of the pathogen.Among them,Mycoplasma gallisepticum(MG)and Escherichia coli(E.coli)infection rates were generally high.Coinfection is more common than single pathogen infection.So,it is often misdiagnosed and mistreated in clinic,which also increases the risk of overuse of antibiotics.Moreover,with the reduction and limitation of antibiotics in the breeding industry,traditional Chinese medicine(TCM)have shown great potential for developmen t due to their integrity,multi-component and multi-target characteristics.The network analysis of TCM and complex diseases through systems biology and polypharmacology can better understand the behavior of cells and organs at the molecular level,which a ccelerate the confirmation of active ingredients in TCM and discover new biomarkers.Therefore,the multi-gene-multi-target-complex disease"model can more conform to the trend of new drug research and development.(1)Transcriptomics and metabolomics sequencing analysis of MG and E.coli infection modelIn this study,the coinfection model of MG and E.coli was established based on previous laboratory studies.The key targets and differential metabolites of coinfection were screened by combined transcriptomics and metabolomics analysis.Then,the whole data of TCMSP was collected to construct a TCM network.Transcriptomics results showed that a total of 3115differentially expressed genes were found between the coinfection group and the control group,among which 1456 genes were up-regulated and 1659 genes were down-regulated.These DEGs were widely distributed in 44 subclasses of 6 categories in KEGG database,and 57 key target genes were screened by further protein interaction analysis,with a total of 54 ge nes after removing duplicates,which were used for TCM screening.The serum samples of coinfection model were collected for non-targeted metabolomics study under the positive and negative ion mode of UPLC-QTOF/MS.A total of 928 differentially expressed metabolites were screened by univariate analysis combined with fold change and q-value for omics combined analysis.A total of 53 potential biomarkers were identified,including leukotriene C4,leukotriene D4,linoleic acid and a large number of energy-metabolism-related products.(2)Construction of target network based on network pharmacology and reverse screening of TCMThe key targets were substituted into the network for reverse screening to obtain the total network of"target-ingredient-TCM".The 54 target genes were substituted into the DRUGBANK database for information conversion,and 25 effective drug targets were obtained.The drug targets were substituted into the TCMSP network for reverse screening,and the top 20 Chinese medicines with node correlation were obtained.Combined with the compatibility law of Chinese medicine,six Chinese medicines were obtained such are:Isatdis Radix and Forsythia Fructus;Minister drug:Ginkgo Folium and Mori Cortex;Supplementary drug:Licorice and Radix Salviae.After screening out the six ingredients from the TCM total network,the network of"target-component-TCM"of the compound was constructed.(3)Study on optimization of proportion of NCMC by uniform designThe uniform design was used to study the proportion of six herbs in NCMC,and the proportion was determined by the method of pharmacodynamics evaluation.In this study,Isatdis Radix(X ₁),Forsythiae Fructus(X₂),Ginkgo Folium(X₃),Mori Cortex(X₄),Licorice(X₅)and Radix Salviae(X₆)were chosen to be the principal constituents by UD.The U×₁₀(10⁶)UD table was used to design an experiment of 10 combinations.The composite score as a dependent variable,represented by Y.The multivariate quadratic equation was established by DPS:Y=-223.2+57.8×X₂+0.5×X₄+46.4×X₅+0.1×X₁²-2.9×X₂²-2.0×X₅²+0.002×X₂×X₃-4.1×X₂×X₅,R²=0.999998,F value=62499.9063,Regression significance test(P=0.0031).The results showed that the ratio of Isatdis Radix:Forsythiae Fructus:Ginkgo Folium:Mori Cortex:Licoric e:Radix Salviae were 14:7:11:12:5:3,which has the best treatment effect.(4)Analysis of targeted therapeutic effect of NCMC aqueous extract on coinfection of MG and E.coliFurthermore,according to the targets in the complex network of coinfection obtained by the group,multi-dimensional targeting verification was carried out using a variety of molecular biology and polypharmacology methods.The pharmacodynamic results of the treatment trial:Under the light microscope,ciliary shedding and goblet cell proliferation were observed in the upper mucosa of the coinfection group.After NCMC treatment with aqueous extract,the tracheal cilia were well arranged,but there was still a small amount of inflammatory cells infiltration.Scanning electron microscopy(SEM)showed that the tracheal cilia were more abundant and intact after treatment.By transmission electron microscopy(TEM),the cilia ruptured,inverted,swelling of cytoplasm and umbrella-like shapes were observed in the coinfection group.After tr eatment,symptoms improved significantly in the treatment group compared with the coinfection group,although some cilia were shed.The key targets and metabolites of coinfection were obtained by multi-omics joint analysis network,and the targeted verification experients of NCMC aqueous extract were carried out.RT-PCR results showed that,except for CHRM4,MMP2 and VEGFA,the chemokines and mucins secreted by the coinfection group all had outbreak.After treatment,the target genes were significantly down-regulated(P<0.05).At the protein level,the expression of these five target proteins(BDKRB1,EDN1,MMP2,TLR4 and VEGFA)were significantly down-regulated by treatment compared with the coinfection group.At the metabolite level,compared with the co ntrol group,the expressions of three key metabolites were significantly up-regulated between the coinfection groups(P<0.01).Compared with coinfection group,the expressions of dopamine and?-aminobutyric acid after treatment were significantly decreased(P<0.05).In vitro molecular docking simulation,the docking results reflect the characteristics of multiple targets and multiple pathways of aqueous extract.The compounds with the lowest scores were salvianolic acid A(-10.1kcal/mol),licorice(-9.3 kcal/mol)and isoglycyrrhiza(-8.7 kcal/mol),and we speculated that they had the highest binding potential with the active site of target protein.(5)Establishment of material basis and fingerprint of NCMC aqueous extractIn order to clarify the composition of the compound,we carried out non-targeted metabolomics study on the NCMC,and constructed the fingerprint of the aqueous extract of the compounds.The results showed that 260 compounds(including positive and negative modes)were detected by UPLC-QTOF-MS/MS at the receiving end.There were 85 flavonoids,64 organic acids,17 phenyl-propyl compounds,etc.In order to identify the main components in the compound,we screened 21effective substances with high content from the total ion flow diagram u nder positive and negative ion mode.The similarity calculation was carried out by“The Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition”.The results showed that the similarity of each batch of NCMC aqueous extract was between 0.9 and 1.0,which indicating that the preparation process was stable,and the quality could be better evaluated by fingerprint similarity.(6)Optimization of extraction technology of NCMCThe extraction and preparation process of the NCMC were established respectively by using orthogonal test and response surface analysis.The Design-expert software was used to optimize and predict the extraction parameters,the best extraction temperature was 89.075?,the ratio of liquid to solid was 20.264:1,and the extraction time was 3.208 h,the optimal predi cted OD value was0.753.In consideration of the convenience of clinical operation,and through single factor investigation,response surface analysis,and extraction times,the optimal e xtraction process was finally determined as follows:extraction temperature:89?,liquid-solid ratio:20:1,extraction time:3.2 h,extraction cycles:2.(7)Results of preparation technology of granulesBased on the results of orthogonal experiment,it is found that the ratio of main and auxiliary has a significant effect on the formability of granules.In this study,the optimal prescription conditions were as follows:the ratio of dry paste powder and exclusives was 1:1.5(exclusives were prepared by 1:1 mixing sucrose and starch),the concentration of PVP was 4%,and the concentration of ethanol was 80%.In conclusion,transcriptomics and metabonomics were used to mine the target network of coinfection,and the mapping between key targets and TCM networ k were used to conduct reverse screening of TCM,and the ratio was optimized by uniform design.Based on the joint analysis network,we carried out targeted verification from multiple dimensions,and preliminarily revealed the multi-target-multi-pathway targeting effect of NCMC.Meanwhile,in order to clarify the substance basis of Chinese medicinal compounds,this study carried out non-targeted metabolomics analysis and fingerprint construction.Furthermore,the extraction process and granules preparation process were optimized and verified,which laid a foundation for the development of drugs for the clinical treatment of respiratory diseases in veterinary medicine.