Mechanism Study Of Active Compounds From Traditional Chinese Medicine Using Novel Target Identification Technologies Combined With Omic Techniques

Traditional Chinese Medicine（TCM）is a big source of new drugs,since it has a diversity of natural active compounds.However,natural active compounds usually target multiple sites through a number of physically or functionally linked molecules or pathway networks rather than an isolated protein to exert their effects,making them unsuitable for the mechanism study mode of single target drugs.Therefore,pharmacodynamic mechanism study of natural active compounds has become a major"bottleneck"on the road to modernization of TCM.In this study,a strategy based on novel target identification technologies combined with omic techniques was developed to study the mechanism of oroxylin A and salvianic acid A sodium,two active compounds from TCM.Firstly,identify the target of oroxylin A,an in vivo anti-hepatoma component screened from rat serum after oral administration of Radix scutellariae,and investigate its anti-tumor mechanism.Secondly,identify the binding proteins of salvianic acid A sodium,a new drug involved in phase I clinical trial in China for treatment of coronary heart disease and stable angina pectoris,and elucidate its cardioprotective mechanism.1.On-linecomprehensivetwo-dimensionalHepG2cellmembrane chromatographic analysis system for charactering anti-hepatoma components from rat serum after oral administration of Radix scutellariaeCell membrane chromatography（CMC）is a bioaffinity chromatography technique for characterizing interactions between drugs and membrane receptors and has been widely used to screen active components from complex samples such as herbal medicines（HMs）.In this study,a novel on-line comprehensive two-dimensional HepG2/CMC/enrich columns/high performance liquid chromatography/time-of-flight mass spectrometry system was developed to rapidly screen potential anti-hepatoma components from drug-containing serum of rats after oral administration of Radix scutellariae.A matrix interference deduction method with a home-written program in MATLAB was developed,which could successfully eliminate the interference of endogenous substances in serum.Baicalein,wogonin,chrysin,oroxylin A,neobaicalein and rivularin from Radix scutellariae extraction were significantly retained in the HepG2/CMC column.Three potential active components,wogonin,oroxylin A and neobaicalein were firstly screened from the drug-containing serum as well.The cell counting kit-8 assay demonstrated that wogonin and oroxylin A showed high inhibitory activities in a dose-dependent manner on HepG2 cells at the concentration of 12.5–200μM（p<0.05）and the IC₅₀ values were 69.83 and 16.66μM,respectively.Wogonin and oroxylin A,which were screened both from Radix scutellariae extraction and the drug-containing serum,could be selected as lead compounds to obtain good anti-hepatoma effects.The proposed comprehensive 2D CMC system and matrix interference elimination strategy have significant advantages for in vivo screening of active components from complex biological samples and could be applied to other biochromatography models.2.Oroxylin A suppresses hepatocellular carcinoma by targeting transketolase to inhibit the nonoxidative pentose phosphate pathway and activate p53 signalingIn the present study,we aimed to confirm the anti-tumor effect of oroxylin A in vitro and in vivo,an active compound screened from Radix scutellariae,and investigate its potential target protein（s）and molecular mechanisms.Oroxylin A was found to be with robust bioactivity both in HepG2 cells and mouse xenograft models.By using drug affinity responsive target stability and surface plasmon resonance（SPR）assays,transketolase（TKT）,a major enzyme of the nonoxidative pentose phosphate pathway（PPP）,which produces more than 80%of ribose for nucleoside synthesis in rapidly dividing cancer cells and is of utmost importance for cell immortalization,was found to specifically bind oroxylin A with a K_D value of 11.2μM.The interaction of oroxylin A-TKT was further confirmed by molecular docking,enzyme activity assay,protein expression and effect changes after TKT silencing.Additionally,targeted metabolites quantitative analysis showed that TKT catalytic activity was inhibited by oroxylin A along with the decrease of ribose 5-phosphate（R5P）,a critical compound for de novo ribonucleotide synthesis in tumor cells,leading to tumor cell apoptosis resulted from inadequate RNA and DNA synthesis.Furthermore,transcriptome profiling of oroxylin A-treated HepG2 cells revealed that several pathways including inducible factor-1（HIF-1）signaling and glycosphingolipid biosynthesis closely correlated with the PPP were significantly activated with p53 signaling was most frequently affected by oroxylin A,and the results were in high correlation with RT-PCR array analysis of TKT silencing-HepG2 cells.Taken together,our results indicated oroxylin A,a potent TKT inhibitor,could induce hepatoma carcinoma cell apoptosis by directly targeting TKT to inhibit the nonoxidative PPP and activate p53 signaling.This compound would be a promising lead candidate that deserves further development for TKT targeted cancer therapy.3.Salvianic acid A sodium protects HUVEC cells against tert-butyl hydroperoxide induced oxidative injury via mitochondria-dependent pathwaySalvianic acid A（Danshensu）is a major water-soluble component extracted from Salvia miltiorrhiza（Danshen）,which has been widely used in clinic in China for treatment of cardiovascular diseases（CVDs）.This study aimed to investigate the protective effects of salvianic acid A sodium（SAAS）against tert-butyl hydroperoxide（t-BHP）induced human umbilical vein endothelial cell（HUVEC）oxidative injury and the underlying molecular mechanisms.In the antioxidant activity-assessing models,SAAS pretreatment significantly ameliorated the cell growth inhibition and apoptosis induced by t-BHP.An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry（UPLC-QTOF-MS）based-metabolic profiling was developed to investigate the metabolic changes of HUVEC cells in response to t-BHP and SAAS.The results revealed that t-BHP injury upregulated 13 metabolites mainly involved in tryptophan metabolism and phenylalanine metabolism which were highly correlated with mitochondrial function and oxidative stress,and 50μM SAAS pretreatment effectively reversed these metabolic changes.Further biomedical research indicated that SAAS pretreatment reduced the t-BHP induced increase of lactate dehydrogenase（LDH）,intracellular reactive oxygen species（ROS）,malondialdehyde（MDA）and mitochondrial membrane potential（MMP）,and the decrease of key antioxidant enzymes through mitochondria antioxidative pathways via JAK2/STAT3and PI3K/Akt/GSK-3βsignalings.Taken together,our results suggested that SAAS may protect HUVEC cells against t-BHP induced oxidative injury via mitochondrial antioxidative defense system.4.Cardioprotective mechanism study of Salvianic acid A sodium based on proteome microarray approach and serum metabolomic analysis of rats after myocardial infarctionSalvianic acid A sodium（SAAS）is a new drug involved in phase I clinical trial in China for treatment of coronary heart disease and stable angina pectoris.However,its direct binding protein（s）is not understood and the broader cardioprotective mechanism still remains to be further elucidated.This study aimed to confirm the cardioprotective effects of SAAS in rats with myocardial infarction（MI）and investigate the potential molecular mechanisms.Results showed that SAAS significantly improved cardiac function and reduced the contents of lactic dehydrogenase（LDH）,creatine kinase（CK）,creatine kinase isoenzyme（CK-MB）and cardiac troponin I（cTnI）in serum of MI rats.Using a human proteome microarray,370 proteins were identified to specifically bind SAAS,which were strikingly enriched in metabolic pathways.The results were further verified by global serum metabolic profiling of MI rats treated with SAAS using an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry（UPLC-QTOF-MS）based metabolomic approach.Totally,26 potential biomarkers,including various glycerophospholipids（GPLs）and an array of fatty acids were identified from the rat serum.The results indicated increased phospholipid catabolism,sphingolipid metabolism and linoleic acid metabolism,decreased tryptophan metabolism,and impaired glycerophospholipid metabolism and primary bile acid biosynthesis in MI animals,while SAAS remarkably reversed these metabolic changes.Taken together,SAAS may protect against myocardial infarction in rats by reversing multiple metabolic changes-induced by MI injury.Our findings will shed light on the cardioprotective mechanism of SAAS and aid its clinical use.Moreover,the SAAS-binding proteins identified by the proteome microarray are expected to be a valuable resource for its greater development.5.Cardioprotective mechanisms of salvianic acid A sodium in rats with myocardial infarction based on proteome and transcriptome analysisOur previous research confirmed that salvianic acid A sodium（SAAS）,a new drug involved in phase I clinical trial for treatment of coronary heart disease and stable angina pectoris,could provide significant cardioprotection against MI injury by reversing multiple metabolic changes-induced by MI injury.This study aimed to investigate the underlying cardioprotective molecular mechanisms of SAAS based on proteome and transcriptome profiling of the myocardial tissue.Results showed that the differentially expressed genes and proteins were important transcription factors,cofactors,structural molecules and receptors,which were mainly involved in cellular process,cell and binding in the part of biological process,cellular component and molecular function,respectively.Functional enrichment analysis indicated that SAAS took active part in regulation of actin cytoskeleton,phagosome,focal adhesion,tight junction,apoptosis,MAPK signaling and Wnt signaling,at both the transcriptome and proteome levels.Taken together,SAAS may exert its cardioprotective effects by targeting multiple pathways at transcriptome,proteome,and particularly metabolome levels.This study has provided not only new insights into the pathogenesis of myocardial infarction,but also a road map to the cardioprotective molecular mechanisms of SAAS,which may provide pharmacological evidence to aid its clinical application.In summary,using novel target identification technologies such as drug affinity reaction target stability analysis and proteome microarray approach combined with omics profiling including transcriptomics,proteomics and metabonomics analysis,this study mainly investigated the target proteins of oroxylin A,a in vivo anti-hepatoma component from rat serum after oral administration of Radix scutellariae,and salvianic acid A sodium,a new drug involved in phase I clinical trial in China for treatment of coronary heart disease and stable angina pectoris,and elucidate their anti-tumor and cardioprotective mechanisms,respectively.Results indicated that oroxylin A was a potent TKT inhibitor and could induce hepatoma carcinoma cell apoptosis by directly targeting TKT to inhibit the nonoxidative PPP and activate p53 signaling,which would be a promising lead candidate for TKT targeted cancer therapy.The binding proteins of SAAS were strikingly enriched in metabolic pathways and SAAS may exert its cardioprotective effects by targeting multiple pathways at transcriptome,proteome and particularly metabolome levels.Our results could provide a road map to the cardioprotective molecular mechanisms of salvianic acid A sodium,which may shed light on its clinical application.The proposed novel target identification technologies combined with omic techniques strategy could also be used for mechanism study of other active compounds from traditional Chinese medicine.