| Objective: To establish the cell and serum metabolomics methods to explore the intervention effects of curcuminon triptolide-induced liver damage,providing a basis for illumination of mechanism of curcumin effects.Methods:The concentration of triptolide was investigated to establish L02 hepatocyte injury model.Three concentrations of curcumin and 9 natural components were used to intervene thehepatocyte injury induced by triptolide,respectively.The cell survival rate was determined by CCK-8 method to compare the effect of curcumin and 9 natural componentson hepatocyte injury.L02 cells were used for cell metabolomics study and they were divided into five groups includingcontrol group,triptolide group,low-concentration curcumin group,medium-concentration curcumin group and high-concentration curcumin group.The cell samples in each group werequenched with 80% cold methanol,sonicated,centrifuged,and then the supernatant was evaporated to dryness,and reconstituted in 50% acetonitrile for determination.Serum metabolomics experiments were performed with C57BL/6N mice and five mice groups were used including control group,triptolide group,low-concentration,medium-concentration curcumin and high-concentration curcumin groups.The effect of curcumin on liver injury was evaluated by AST,ALT levels and liver staining results.The blood of the rats was collected by plucking the eyeballs,then the serum samples were obtained.The acetonitrile was added into the serum sample to get the mixture.The cell and serum samples were analyzed by metabolomics study based on UHPLC-Q/TOF-MS.The Luna Omega 1.6? Polar C18(100*2.1mm)column was used to detect the samples in positive and negative ion modes,and the mobile phase was 0.1% formic acid aqueous solution(v/v)–acetonitrile in gradient elution.Statistical methods such as PCA,OPLS-DA,venn ananlysis,k-means cluster analysis and Pearson correlation analysiswere used to screen potential biomarkers.The potential biomarkers were identified through some online databases such as Human Metabolome Database(HMDB)and METLIN.The pathway enrichment analysis was processed by an online software Metabo Analyst 4.0 and the online database KEGG.Results:Curcumin was found to alleviate cytotoxicity more than other components.A total of 43 metabolites were discovered as potential biomarkers in cell metabolomics study and 19 potential biomarkers were identified.Pathway analysis revealed that curcumin mainly regulated glutathione metabolism,TCA cycle,purine metabolism and glycerophospholipid metabolism to reduce the metabolic disorders caused by triptolide.A total of 52 metabolites were discovered as potential biomarkers in serummetabolomics study,among them,21 potential biomarkers were identified.Pathway analysis revealed that curcumin mainly regulated D-glutamine and D-glutamic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,glycerophospholipid metabolism,arginine biosynthesis,?-alanine metabolism,arachidonic acid metabolism to reduce the metabolic disorders caused by triptolide.The comparison of the two results revealed 7 common potential biomarkers and 2common important pathways.Conclusion: In the presentstudy,non-targeted cell and serum metabolomicsmethods were established based on UHPLC-Q/TOF-MS.It was found that curcumin could improve triptolide-induced liver damage by changing the levels of L-glutamine,xanthine,guanine,2-oxyglutarate,docosahexaenoic acid,glutathione and citric acid and regulating the TCA cycle and glutathione metabolism.This study provided new theoretical basis for the molecular mechanism research of curcuminand improved drug discover for preventingtriptolide-induced toxicity. |
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