Study On Hepato-and Nephro-toxicity Induced By Radix Euphorbiae Pekinensis Based On Variation Of Endogenous Substances In Vivo And Cell Models In Vitro

The study was aimed at evaluating hepato-and nephro-toxicity induced by Euphorbiae pekinensis Radix based on the knowledge of pharmaceutical analysis,serum pharmacology,cytotoxicology combined with molecular biology.Small molecule metabolites changes induced by Euphorbiae pekinensis Radix toxicity in liver and urine were targeted analyzed to provide the basis for clinical early detection.Metabonomics and serum pharmacology based methods were applied to investigate related signaling pathways of hepatic and renal toxicity induced by Euphorbiae pekinensis Radix.The study screened toxicity parts of Euphorbiae pekinensis Radix according to the theories of serum pharmacochemistry and cell affinity.In addition,interaction between target cell and toxic ingredient of Euphorbiae pekinensis Radix(euphorbol and Euphorbia kansui sterol)were investigated to provide a basis for clinical rational drug use and intensive toxicity study in the future.1.Target analysis of endogenous substance variation in rats serum and urine.Rats were oral administrated with 95%ethanol extract of Euphorbiae pekinensis Radix at a dosage of 9g/kg.Inflammatory cytokines,reactive oxygen species levels and hepatic and renal function were selected to evaluate effect of Euphorbiae pekinensis Radix on hepatic and renal.A rapid and simple UFLC-MS/MS method for simultaneous determination of 10 related specific small molecule metabolites including hippuric acid(HIP),phenylaceturic acid(PA),cholic acid(CA),taurine(TA),pyroglutamic acid(5-OP),indoleacetic acid(IA),3-indoxyl sulfate(3-IS),guanidinosuccinic acid(GSA),and glycocyamine(GAA)in rat serum and urine.Bendroflumethiazide was selected as internal standard and analytes separation was performed on a CAPCELL CORE PC column with a mobile phase of acetonitrile-water(containing 5mM ammonium acetate).Protein-precipitation with methanol was used for sample pretreatment.The method show good linearity(r>0.99),selectivity,recovery,matrix effect,precision,accuracy and stability results meet requirements of methodology validation.In this study,quantitative determination and dynamic surveillance were performed to investigate variation trends of related small molecule metabolites induced after administration of Euphorbiae pekinensis Radix within 15 days.The results show that the selected metabolites levels appeared varying degrees of changes,which indicated that Euphorbiae pekinensis Radix induced hepatic and renal toxicity may be related with gut microbiota disorder,oxidative damage and function disorders.Compared with traditional clinical liver function test,serum concentrations of 5-OP,CA,3-IS and GSA show significant increase since 4th day,which earlier than ALT,AST,SCr and BUN(show significant increase since the tenth day).The results indicated that 5-OP and CA as indexes of liver function test,3-IS and GSA as indexes of renal function test could predict hepatic and renal damage more earlier which provide basis for hepato nephric toxicity discovery at early stage in clinical.2.Metabolomics study of Euphorbiae pekinensis Radix based on HL-7702 and 393T cellsHL-7702 and 293T cell model were established by rat serum after oral administration of Euphorbiae pekinensis Radix according to serum pharmacology and cytotoxicology theroies.The cell models were applied to evaluate hepato nephric toxicity and investigate possible mechanism of Euphorbiae pekinensis Radix.Observation of cell morphology change,calculation of the influence of drugs on cell proliferation,determination of oxidative damage in cell,and determination of variations of inflammatory cytokines and function structure related indexes were performed in the experiment.At the same time,an UPLC-MS/MS based metabolism analysis method was established to screen markers by comparing small molecule metabolites in cell lysis buffer variation before and after Euphorbiae pekinensis Radix administration.The results indicated that cell morphology changed significantly after 24h incubation of serum containing drug.Oxidative damage indexes including MDA,CAT,GSH and SOD levels changed.ATPase activity decreased and inflammatory cytokines(NO and NOS levels)increased.According to the score plot and loading plot of PCA from metabolism analysis of cell lysis buffer,13 biomarkers were identified in HL-7702 cell including lysophosphatidylcholine,phosphatidylcholine,sphingol,dihydroceramides,L-palmitoyl carnitine,Eicosapentaenoyl ethanolamide,5-pyroglutamic acid,phenylalanine,tryptophan,threonine,citrulline,which indicated that Euphorbiae pekinensis Radix induced liver injury may be related with sheath ester metabolism,phospholipid metabolism,amino acid metabolism,fatty acid metabolism and oxidative damage pathways.At the same time,10 biomarkers were identified from 293T cells including lysophosphatidylcholine,phosphatidylcholine,ceramide,sphingol,adenosine,glycine,homocysteine,cysteine,threonine,and threonic acid,which indicated that Euphorbiae pekinensis Radix induced renal injury may be related with sheath ester metabolism,phospholipid metabolism,amino acid metabolism,and oxidative damage pathways.3.Identification of potential toxic substances based on cell modelAn ultra high performance liquid chromatography-Q Exactive Orbitrp mass spectrometer(Q Exactive Orbitrp UPLC-MS/MS)was established to identify multi-components in HL-7702 and 293T cells after incubated with serum containing drug.Ten ingredients were identified from HL-7702 cell including quercetin,kaempferol,quercetin-3-O-glucuronide,kaempferol-3-beta-O-glucuronic acid glycosides,Corilagin,Euphol,Euphorbia kansui sterol,cycloartenol,Pekinenal,euphorpekone A.Nine ingredients were identified from 293T cell including ellagic acid,3,3-di-o-methylellagic acid,3,3-di-o-methylellagic acid-O-β-D-glucopyranoside,corilagin,geraniin,(—)-(1S)-15-hydroxy-18-carboxycembrene,Euphol,and Euphorbia kansui sterol.Then we evaluated toxicity of each ingredient including cellular proliferation inhibition rate,oxidative damage and cell function related indexes,respectively.Here 5 cell toxicity ingredients in HL-7702 were confirmed including Euphol,Euphorbia kansui sterol,cycloartenol,and Pekinenal.Three cell toxicity ingredients in 293T cell were confirmed including(—)-(1S)-15-hydroxy-18-carboxycembrene,Euphol,and Euphorbia kansui sterol.The study provide foundation for the research of material basis of Euphorbiae pekinensis Radix toxicity and intensive toxicity study in the future.4.Study of drug-organ interaction based on HL-7702 and 293T cellsA UFLC-MS/MS method was established for quantitative determination of Euphol and Euphorbia kansui sterol.Positive ion detection mode was used and oleanolic acid was selected as internal standard.Protein-precipitation with methanol was used for intracellular fluid sample pretreatment.Acetonitrile and 0.1%formic acid in water as gradient elution mobile phase.The analytes show good linearity,besides,selectivity,recovery,matrix effect,precision,accuracy and stability results meet requirements of biological sample analysis.At the same time,a UFLC-MS/MS method was established for simultaneous determination of tryptophan,pyroglutamic acid in HL-7702 and homocysteine,cysteine in 293T cell lysis buffer.Positive ion detection mode was used and tinidazole was selected as internal standard.Protein-precipitation with methanol was used for intracellular fluid sample pretreatment.The separation was performed on a CAPCELL CORE PC column with acetonitrile and 5 mM AcNH4 in water using gradient elution.The analytes show good linearity,and the method validation meet requirements of biological sample analysis.In our research,Euphol and Euphorbia kansui sterol as well as corresponding endogenous components were evaluated after Euphorbiae pekinensis Radix incubation for 0.5,1,2,3,6,9,12,24 h.The results indicated that Euphol and Euphorbia kansui sterol accumulated with the extended incubation time.At the same time,L-pyroglutamic acid in HL-7702 increased rapidly since 1h,tryptophan show rising trend since 3h.In 293T cell,cysteine decreased rapidly since 2h while homocysteine increased since 4h.Biomarkers in cells show metabolism disorder with the accumulation of Euphol and Euphorbia kansui sterol,which verified that Euphol and Euphorbia kansui sterol might be the toxic material base of Euphorbiae pekinensis Radix.