| Objective:To predict the target and main components of Qian-Jin-Wen-Wu Decoction(QWD)in the treatment of diabetic nephropathy(DN)by using network pharmacology analysis,and to reveal the mechanism of QWD intervention in DN.The HPLC-ESI/MS method for the chemical composition of QWD in vitro and in vivo was established to identify and analyze the chemical constituents of QWD in vitro test solution and rat plasma after administration.Methods:1.Obtain all the chemical components of QWD through TCMID database and literatures;use BATMAN-TCM,STITCH database and Swiss TargetPrediction Webserver to predict the potential target according to the similarity of 3D chemical spatial structure.2.Known DN-related targets were obtained from 9 existing resources such as DrugBank,the Online Mendelian Inheritance in Man(OMIM)Database and TCMGeneDIT database.Mapping of disease targets and drug targets,the potential targets of QWD intervention in DN were obtained.3.Use Cytoscape software to retrospect the ingredient and herb of the targeted pharmacodynamic target.4.Cytoscape was used to perform functional annotation and enrichment analysis of all pharmacodynamic targets of QWD in the treatment of DN.The mechanism of QWD intervention in DN was preliminarily revealed.5.10 batches of QWD samples were prepared by the same preparation process and uniform quality standards as standard samples.The test solution is obtained by ultrasonic extraction of standard sample solution with methanol.6.Based on network pharmacology analysis,43 standards were selected as qualitative alignment compounds for QWD chemical composition..Use the LC-MS technology to identify the components of QWD test solution.A reversed-phase Cis column was selected as a fixed phase,and 0.1%formic acid acetonitrile solution-0.1%formic acid aqueous solution was used as mobile phase,and the components were separated by gradient elution;SCAN was performed in a positive ion mode by electrospray ionization mass spectrometry(ESI-MS).Extracted-ion chromatography was used to analyze the spectra.Firstly,the reference compounds in the mixed standard solution were identified,with this as a reference,some compounds in QWD test solution were preliminary identification.7.Rats were given QWD standard samples by gavage and plasma was taken.The blank plasma and medicated plasma were separately detected with the same LC-MS instrument under the same analysis conditions,and compared with liquid mass spectrum of QWD test solution in vitro,the prototype components of QWD in rat plasma were initially identified.Results:1.A total of 533 ingredients of QWD were collected,the clustering results show that these ingredients could be roughly classified into five categories:flavonoids,phenylpropanoids,triterpenoids,steroidal saponin and organic acids.533 chemical components were targeted at 2 279 potential targets.2.64 of all predicted potential targets of QWD are involved in the treatment of DN.3.The 21 ingredients of Radix Puerariae are medicinal ingredients in the treatment of DN,and these 21 ingredients target 23 pharmacodynamic targets;57 medicinal ingredients of Scutellariae Radix target 41 pharmacodynamic targets;10 medicinal constituents of Ligustici Rhizoma Et Radix act on 11 DN-related targets;16 medicinal constituents of Rhizoma Dioscoreae act on 21 DN-related targets;82 medicinal constituents of Schisandrae Chinensis Fructus target 45 DN-related targets;29 medicinal constituents of Platycodon Grandiforus target 11 pharmacodynamic targets;12 medicinal ingredients of Cimicifugae Rhizoma act on 17 DN-related targets;48 medicinal constituents of Angelicae Dahuricae Radix act on 42 DN-related targets and 28 medicinal constituents of Ophiopogonis Radix may act on 20 DN-related protein targets.4.Functional analysis showed QWD could play a role in the treatment of DN through transporters,hormones,protein kinases,protein kinase activators,phospholipase C,cytokines,copper ions,receptors and other molecules at the molecular functional level;Cellular component analysis revealed that pharmacodynamic targets were mainly located in platelet alpha granule and caveola;The biological process revealed that QWD mainly involved in these biological processes to play pharmacological effect:improve glucose/lipid metabolism disorders,inflammatory immune response,improve blood circulation,renal morphological repair and redevelopment,regulate the salt and water balance,maintain calcium homeostasis,mediate apoptosis,reverse development of the insulin resistance and regulate reactive oxygen metabolism;The pathway enrichment analysis results indicated that QWD involved multiple complex pathways,and regulated epithelial cell,mesenchymal cell,leukocyte,cytokine,islet cell,smooth muscle cell,fibroblast,etc,from multiple aspects including synthesis,proliferation,differentiation,transport,metabolism,response,immunity,secretion,apoptosis.activation and migration.The mediated pathways mainly include AGE-RAGE signaling pathway to diabetic complications,type Ⅱ diabetes,HIF-1 signaling pathway,insulin resistance,FoxO signaling pathway,fluid shear stress and atherosclerosis.MAPK signaling pathway,etc.5.The 10 batches of QWD standard samples had the similar characteristic peaks of HPLC spectrum,indicating that their main chemical components are stable and consistent.6.13 compounds were identified in QWD test solution in vitro,respectively puerarin,ferulic acid,isoferulic acid,baicalin,platycodin D,daidzein,wogonoside,bergapten,wogonin,schisandrol A,oroxylin A,imperatorin and schisandrin B.7.9 prototypical components were identified in QWD test solution in vivo,including puerarin,isoferulic acid,baicalin,daidzein,wogonoside,wogonin,oroxylin A,imperatorin and schisandrin B.Conclusion:1.It was found that Angelicae Dahuricae Radix,Angelicae Dahuricae Radix and Scutellariae Radix had clear therapeutic targets and exert a wide range of anti-DN pharmacological effects by network prediction analysis,the three could be designated as the main drug of QWD prescription;Dioscoreae Rhizoma,Puerariae Lobatae Radix,Ophiopogonis Radix and Cimicifugae Rhizoma also covered a wide range of pharmacodynamic effects,which can be regarded as the auxiliary drugs in QWD;Platycodonis Radix and Ligustici Rhizoma Et Radix targeted the least therapeutic targets and involved the least biological processes,mainly play a synergistic role in anti-DN effect,and they can be designated as a synergistic drug.2.The study showed that network pharmacology can be used as a method to preliminarily clarify the mechanism of action of QWD in the treatment of DN.The predicted ingredients and anti-DN pharmacodynamic components in the network analysis results provide some guidance for the research of QWD material basis,64 DN-related potential targets of QWD provide theoretical and experimental basis for further mechanism research.3.A HPLC-ESI-MS analysis method of the identification of QWD in vivo and in vitro was established by the chemical substance group study.The blood migration components are the pharmacodynamic components of QWD acting directly in vivo.The results laid the theoretical and experimental basis for the study of the pharmacodynamic substance basis and action mechanism of QWD. |
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