| Semen Strychni is the seeds of Strychnos nux-vomica L.It is a famous Traditional Chinese Medicine in of Materia Medica,which is recorded in Chinese Pharmacopoeia.Recent studies have reported that Semen Strychni have anti-tumor,analgesic and anti-inammatory angiogenesis effects.However,a high dose of Semen Strychni would result in severe kidney damage,which strictly limits its clinical use.In order to explore the nephrotoxicity of Semen Strychni and the intervention effect of Radix Glycyrrhizae,a comprehensive biomarker investigation was conducted in this study.Firstly,the potentical nephrotoxicity biomarkers in rat serum and urine was evaluated by the metabolic target analysis;secondly,the potential nephrotoxic substances were identified based on the developed cell model;thirdly,the metabolites in cell lysate were analyzed by the metabolomics study to reveal the related metabolism pathways;finally,the endogenous and exogenous biomarkers were simultaneously determined to discuss the relationship between nephrotoxicity and the changes of biomarkers.This investigation would help in rational drug use and further toxicology study.1.Metabolic target analysis of potential biomarkers in rat serum and urineRats were orally administrated of Semen Strychni at the dosage of 0.2 g/kg/d for 10 d,then oral administrated of Radix Glycyrrhizae at the dosage of 5 g/kg/d for 42 d.The physiological status and tissue pathological section were used to assess the animal models of different experiment groups.An LC-MS method was developed to simultaneous determination of 12 potential biomarkers,including citric acid(CA),guanidinosuccinic acid(GSA),taurine(Tau),guanidinoacetic acid(GAA),uric acid(UA),creatinine(Cr),hippuric acid(HIP),xanthurenic acid(XA),kynurenic acid(KA),3-indoxyl sulfate(3-IS),indole-3-acetic acid(IAA),and phenaceturic acid(PA).Sulbactam was used as the internal standard,and a methanol-5 mM ammonium acetate mobile phase system was use for the gradient separation.A Phenomenex Luna C18(250 mm～4.6 mm,5 μm)column was used in this study for the separation of the analytes.Samples were prepared by using protein precipitation with acetonitrile before analysis.All the analytes showed good linearity,selectivity,precision,accuracy,absolute recovery,matrix effect and stability.This method was applied to continuously detect the potential biomarkers in serum and urine samples for 52 d.Results showed that the significant changes of some biomarkers(3-IS,GSA and GAA in serum;3-IS,IAA,GSA and GAA in urine)were found in Semen Strychni group.The metabolic target analysis results indicated that several amino acid metabolism pathways(especially tryptophan metabolism pathway and nitrogen metabolism pathway)might be disturbed by Semen Strychni nephrotoxicity.In addition,the levels of the potential biomarkers were recovered to normal after Radix Glycyrrhizae treatment,indicating that Radix Glycyrrhizae might have intervention effect for Semen Strychni nephrotoxicity.Results also showed that 3-IS,IAA(changed from 3rd day)and GSA,GAA(changed from 6th day)were more sensitive than the traditional clinical indexes(serum creatinine and uric acid,changed from 10th day).These biomarkers would provide basis for nephrotoxicity discovery at early stage in clinical.2.Identification of the nephrotoxic substances in HEK293T cellsCells were incubated with medium containing Radix Glycyrrhizae for 12 h,then exposed to the replaced medium containing Semen Strychni for another 12 h.Cell morphology,cell viability and cell injury indexes were used to assessed the cell models of different experiment groups.An ultra high performance liquid chromatography-Q Exactive Orbitrp mass spectrometer(Q Exactive Orbitrp UPLC-MS/MS)method was developed to identify the nephrotoxic substances in HEK293T cell lysate.Acetonitrile-0.1%formic acid was used as the mobile phase sysem for the gradient elution.The chromatography separation was conducted in a CAPCELL CORE C18(100 mm × 2.1 mm,2.7 μm)column.A total of 7 nephrotoxic substances were identified in cell lysate after treating with Semen Strychni,including 19-N-methyl-strychnine,strychnine,strychnine-N-oxide,brucine,hydroxystrychnine and α(β)-colubrine.Strychnine and brucine showed relative high contents in cell lysate.Thus,cell morphology,cell viability and oxidative stress indexes were used to verify the nephrotoxicity of strychnine and brucine.Results showed that Strychnine and Brucine were of significant nephrotoxicity,which could be selected as the representative exogenous biomarkers.This study revealed that alkaloids compounds were the material basis of Semen Strychni nephrotoxicity and provided the foundation for the further study.3.Metabolomics study of Semen Strychni nephrotoxicity based on cell modelMetabolomics strategy was used in this study to investigate the change of endogenous metabolites and the disturbance of metabolism pathway.A UPLC-MS/MS method was developed with acetonitrile-0.1%formic acid as the mobile phase system.An ACQUITY UPLC HSS T3(100 mm × 2.1 mm,1.8μm)column was applied for the gradient separation.The object of this study was to investigate the changes of endogenous metabolites in cell lysate,then screen the potential endogenous biomarkers and the related metabolism pathways.The results of Principal Component Analysis(PCA)and Partial Least Squares-Discriiminate Analysis(PLS-DA)showed that the metabolome of Semen Strychni group was significantly changed than that of blank group.And the metabolome of Radix Glycyrrhizae group showed the recovered tendency than that of Semen Strychni group.A total of 10 potential endogenous biomarkers were screened,including spermine,adenosine,tryptophan,5-oxoproline,phenylacetylglycine,creatinine,tyrosine,oxoglutaric acid,taurine and uric acid.Metabolism pathway analysis revealed that 7 metabolism pathways were related to Semen Strychni nephrotoxicity,including taurine and hypotaurine metabolism(1),nitrogen metabolism(2),phenylalamine,tyrosine and typtophan metabolism(3),glutathione metabolism(4),arginine and proline metabolism(5),purine metabolism(6)and TCA cycle(7).Considering the results of metabolic target analysis in rats,three representative amino acids(tryptophan,phenylalamine and tyrosine)were chosen as the endogenous biomarkers for the further study.4.Quantitative analysis of endogenous and exogenous biomarkers in cell lysateBased on the developed cell models,the exogenous alkaloids(strychnine and brucine)and endogenous amino acids(tryptophan,phenylalamine and tyrosine)were simultaneous determined.AHILIC-ESI-MS/MS method was developed with acetonitrile-0.1%formic acid as the mobile phase system.A CAPCELL CORE PC(150 mm × 2.1 mm,2.7 μm)column was applied for the gradient separation.Samples were prepared by using protein precipitation with methanol before analysis.All the analytes showed good linearity,selectivity,precision,accuracy,absolute recovery,matrix effect and stability.Cell lysate samples of different time points(0,3,6,9,12 h)were determined for revealing the changes of endogenous and exogenous biomarkers.In Semen Strychni group,the levels of strychnine and brucine were continuously increasing,while tryptophan,phenylalamine and tyrosine were continuously decreasing during the experiment.Compared with the biomarker levels in Semen Strychni group,the levels of endogenous and exogenous biomarkers in Radix Glycyrrhizae group showed the recovered tendency.Results indicated that alkaloids might accumulate in cell and amino acids might drop after Semen Strychni treatment,and Radix Glycyrrhizae showed the intervention effect for the nephrotoxicity.In correlation analysis,endogenous and exogenous biomarkers showed a significant negative correlation,indicating that there was a close connection between the accumulation of exogenous alkaloids and the changing of endogenous amino acids. |
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