Exploring The Mechanism Of Tanreqing Injection Against Staphylococcus Aureus Based On Network Pharmacology

Objective: The aim of this study was to explore the mechanism of Tanreqing injection against Staphylococcus aureus based on network pharmacology,Looking for the ingredients of Tanreqing Injection and entering the blood ingredients,Summarize the results of network pharmacology and design experiments to verify its antibacterial mechanism.Methods: The first chapter introduce the antibacterial mechanism of antibiotics and the antibacterial effect of Tanreqing injection.The second chapter uses high performance liquid chromatography-ion trap mass spectrometry(HPLC-MS/Trap)method to identify the ingredients of Tanreqing injection,five Chinese herbs in the prescription(Scutellaria baicalensis,bear gall,cornu caprae hircus,honeysuckle,Forsythia suspensa Vahl)and each single liquid ingredients.The third chapter uses high performance liquid chromatography-ion trap mass spectrometry(HPLC-MS/Trap)method to identify the ingredients of Tanreqing injection into rat blood.The groups were divided into experimental group(after injecting the drug 15,30,60 min)and control group(without drugs),By comparing the experimental group and the control group,in order to remove the interference of endogenous components,thereby confirming the ingredients of Tanreqing injection into rat blood.The ingredients of Tanreqing Injection was not detected after 15 minutes of administration in rats blood,believe it is rapidly metabolized by the body,it does not have pharmacological activity and is not considered to be a blood active ingredient.The fourth chapter,exploreing the antibacterial mechanism of Tanreqing injection,it can be divided into five steps:(1)Using Stitch,ScPDB,Discovery Studio and Pharm Mapper database to construct the ingredients-target database of Tanreqing injection.(2)Constructing the Staphylococcus aureus(SA,strain NCTC 8325)target database by using the DrugBank database and literature search.(3)Using the String database to perform protein-protein interactions(PPI)on(1)and(2)database targets,and establish a target interaction network.(4)Using Cytoscape software to intersect the two networks in the(3),thereby obtaining core targets.(5)Enrichment analysis of core targets by using ClueGo software,including signaling pathway(KEGG)and gene ontology(GO).The fifth chapter verifies the antibacterial activity of Tanreqing injection.include detecting the growth curve of SA(strain NCTC 8325)by turbidimetry,detecting the minimum inhibitory concentration(MIC)of Tanreqing injection in 96-well plates by micro-broth dilution method,take 1 ?L of each concentration well of 96-well plate and streak it on TSA plate.After culture,the minimum drug concentration of bacterial colony<5 is determined as the minimum bactericidal concentration(MBC).And detecting experimental group(bacteria at the MIC dose of Tanreqing injection)and the control group(non-medicated bacteria)shape by transmission electron microscopic.The sixth chapter to design experiments through peptidoglycan synthesis,fatty acid synthesis,pyruvate metabolism,and purine metabolism.The content of peptidoglycan monomer,fatty acid,pyruvate,purine and its intermediates(nucleosides,nucleotides)was detected before and after bacterial administration.Among them the bacterial peptidoglycan monomer and pyruvate by using the test kit;after extracting bacterial fatty acids,methyl esterification,gas chromatography-mass spectrometry(GC-MS)was used to calculate the fatty acids content of the cells;Extracting bacteria's purine and its intermediates,and detected by ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry(UPLC-MS/MS).Results:The total of 51 ingredients were identified in the Chinese herbal extract,43 ingredients identified in the single liquid,Tanreqing injection contains 37 ingredients: including organic acids,flavonoids,iridoids,phenylethanoid glycosides,lignans,bile acids,it contains 5 ingredients of Scutellaria baicalensis,4 ingredients of bear gall,10 ingredients of cornu caprae hircus,13 ingredients of honeysuckle and 5 ingredients of Forsythia suspensa Vahl.As a result,there were 23 ingredients of Tanreqing injection into rats blood.The network pharmacology results include 7 KEGG signaling pathways in 6 classes,involving fatty acid synthesis,peptidoglycan synthesis,pyruvate metabolism,purine metabolism,etc;24 GO-biological processes in 8 classes,involving Cell morphology adjustment,ribosome large subunit assembly,DNA synthesis,fatty acid synthesis,nucleotide metabolism,glycine metabolism,starvation response,etc;13 GO-molecular functions in 7 classes,involving rRNA binding,GTP diphosphate kinase activity,UDP-N-acetylglucosamine 2-epimerase activity,3'-5' exonuclease activity,large ribosomes subunit rRNA binding,vitamin binding,etc.The bacterical delay period is between 0-2h,the logarithmic period is between 2-12 h,the equilibrium period is after 12 h,and the decay period is not obvious.As the antibacterial potency,MIC=1/4 Tanreqing Injection,MBC=1/2 Tanreqing Injection.the morphological results showed that the control group had a smooth cell wall and cell membrane,and the whole was rounded,the cell wall of experimental group was rough,loose and not tight,the shape of the cells was irregular,it was not spherical,the cells were abnormally divided.The mechanism verification result showed that the content of peptidoglycan monomer content,fatty acid content,purine and its intermediates changed significantly after the action of Tanreqing injection,the current method found that the pyruvic acid content did not change significantly,but it has a certain trend.So,it shows that the antibacterial mechanism of Tanreqing injection may affect the synthesis of peptidoglycan,affect fatty acid metabolism and purine metabolism.However,it was found that the content of certain fatty acids,purine and its intermediates increased in the experimental group,need to be further studied.Conclusion: This study preliminarily verified the antibacterial mechanism of Tanreqing injection through multiple pathways,explaining that the results of network pharmacology predictions are reliable.The confirmed results need to be further studied.