| At this stage,chemotherapy is still the critical part of treatment of malignant tumor.However,the serious side effects of chemotherapeutics in most cases limited their usage in clinical.Mitomycin C(MMC)is a kind of broad spectrum chemotherapeutic whose usage was limited by the serious toxic effects.Sijunzi decoction(SJZD)is a chemotherapy assistant traditional Chinese medicine,and many litteratures showed that SJZD used combination with MMC could produce effect-enhancing and toxicity-reducing effects to the latter.The research related to SJZD was mainly involved in chemical classification characterization in vitro at present,the specific chemical consitituents in vitro and metabolites in vivo were not conducted in-depth study.As for the research related to the combination usage of SJZD and MMC was mainly involved in the confirming of effect-enhancing and toxicity-reducing effect,do not touch the mechanism.For this instance,this research studied on the chemical consitituents of SJZD in vitro and metabolites of SJZD in SJZD single dose group and SJZD combination with MMC group in vivo by using the UHPLC-FT-ICR-MS technology.And in addition,the MMC-induced toxicities and the toxicity-reducing effect of SJZD were systematically researched.Based on 1H-NMR and LC-MS/MS technology,the metabonomics analysis platform was established to illustrate the whole metabolism characteristics and abnormal metabolic pathways in MMC treatment rats.The changes of endogenous metabolites was also monitored before and after the treatment of SJZD to find the deeper relationship between these factors and correlation.This paper illustrated the treatment mechanism for toxicity-reducing effect of SJZD,and laid the foundation for the further research of the combination usage of SJZD and MMC and supply the scientific basis for the clinical treatment.1.The characterization of chemical consitituents of SJZD in vitro.SJZD was produced by traditional water decoction method and made into powder by lyophilization.An Universil XB C8(2.1×150 mm,1.8 μm)column was used and the mobile phase was consisted of acetonitrile and 0.2%formic acid.The UHPLC-FT-ICR-MS technology was used and the detection modes were set both positive and negative.119 compounds were characterized in total and 9 of them were accurate comfirmed by standard matierials.The key constituents among them included triterpenesaponins(in Radix Ginseng and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle),triterpene carboxylic acids(in Poria),sesquiterpenes(in Rhizoma Atractylodis Macrocephalae),flavonoids(in Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle and Rhizoma Atractylodis Macrocephalae)as well as coumarins(in Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle)in Sijunzi decoction.2.The study on metabolites in rats treatment with SJZD used before and after combination with MMC.The UHPLC-FT-ICR-MS technology was used in the characterization of metabolites.SJZD was delivered at a dosage of 10 g/kg by i.g.and MMC was delivered at a dosage of 0.25mg/kg by i.p.The plasma,bile,urine and feces samples were collected in 24 after treatment.An Universil XB C8(2.1×150 mm,1.8 μm)column was used to separate the metabolites and the mobile phase was consisted of acetonitrile and 0.2%formic acid.ESI ion source and positive ion mode were selected.As a result,a total of 38 prototypes and 66 metabolites were characterized in plama,bile,urine and feces samples.25 phase I metabolites and 41 phase II metabolites were observed.The result showed that after using combination with MMC,the number of prototypes detected was declined and the number of metabolites in urine detected was declined markedly.3.The study on the toxicities of MMC and the toxicity-reducing effect of SJZD.The research were done on the SD rats,and the determination of body weight change and mortality,histopathological observations,sternum micronucleus assay,hematological analysis,serum biochemical indices(ALT,AST,BUN,CR)and relative viscera weight were used to systematically evaluate the toxicities induced by MMC.The results showed that MMC induced immunotoxicity,hepatotoxicity,mutagenicity(genotoxicity)and nephrotoxicity,and SJZD demonstrated attenuation effects on all the toxicities above.Among the toxicities detected,immunotoxicity was the most significant one and SJZD demonstrated markedly attenuation effect on it.4.The study on the preventive effect of Sijunzi decoction on mitomycin C induced immunotoxicity in rats by untargeted metabolomic analysisNMR and MS-based metabolomics approaches were combined for monitoring MMCinduced immunotoxicity and the protective effect of SJZD.An OPLS-DA approach was used to screen potential biomarkers of immunotoxicity and the MetaboAnalyst and KEGG PATHWAY Database were used to investigate the metabolic pathways.As a result,8 biomarkers in plasma samples,19 in urine samples and 10 in spleen samples were identified as being primarily involved in amino acid metabolism,carbohydrate metabolism and lipid metabolism.5.The study on the preventive effect of Sijunzi decoction on mitomycin C induced immunotoxicity in rats by targeted metabolomic analysisA targeted metabolomic analysis was used to quantification with the metabolite types identified by the untargeted research.34 metabolites were targeted quantified on MRM detection with the methods validation.An Universil C18(2.1×150 mm,1.8 μm)column and acetonitrile and 0.1%formic acid moble phase were used to detect the amino acids.An Universil C8(2.1×150 mm,1.8 μm)column was used to detect other metabolites.Acetonitrile and 0.1%formic acid moble phase was used in positive ion mode while acetonitrile and 0.05%ammonia in negative ion mode.OPLS-DA analysis was performed to select the critical biomarkers.ELISA method was used to quantify the three critical enzymes in rat plasma.As a result,aspartate,isoleucine,succinate,asparagine and pyroglutamate were detected as critical biomarkers.Alanine,aspartate and glutamate metabolism were inferred as critical metabolic pathway and ASNS was the critical enzyme by which MMC induce immunotoxicity. |
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