Study On The Cell Metabolomics Method And Hepatocytotoxicity Of Triptolide

Objective:To establish a Quasi-target Identification cell metabolomics method based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry（UHPLC-Q/TOF-MS）technique,and to apply this method in the study of HepG2 and L02 hepatocytotoxicity induced by triptolide.Methods:HepG2 cells were induced by triptolide as an example to establish a Quasi-target Identification metabolomics method.Luna Omega 1.6Polar C₁₈（100×2.1mm）chromatographic column was used to detect the positive and negative ion patterns of ESI.The samples were first analyzed by non-target full scanning,and then the identified compounds were analyzed by target analysis.The established method was used for metabolomics analysis of80 nmoL/L triptolide induced HepG2 cells and L02 cells at different times.Differential metabolites were screened by PCA,OPLS-DA,and identified by combining with HMDB,METLIN and other online databases differential metabolites were identified.MetaboAnalyst 4.0 and KEGG were used to analyze the pathway of differential metabolites.According to K-Means clustering analysis combined with SVM classification method and Pearson correlation analysis,the biomarkers of the toxic effects of triptolide on the two kinds of cells were screened.Results:Compared with the conventional metabolomics results,for the differential metabolites identified before and after triptolide induction in HepG2 cells,the Quasi-target Identification method newly identified 7compounds,found 16 compound information,and corrected 2 compounds;For pathway studies,the new metabolic pathways of alanine,aspartic acid and glutamate were found in the Quasi-target Identification method,and the contribution rate of TCA cycle and sphingolipid metabolism pathway was increased.A total of 140 differential metabolites were identified in the metabolomics study of HepG2 cells induced by triptolide at different times,and 5 potential biomarkers were selected,mainly involving amino sugar and nucleotide metabolism,starch and sucrose metabolism,glycerophospholipid metabolism,purine metabolism,arginine and ornithine metabolism.A total of 82 different metabolites were identified in the metabolomics study of L02 cells induced by triptolide at different time,and 10 potential biomarkers were selected,mainly involving purine metabolism,glutathione metabolism,glycerophospholipid metabolism,alanine,aspartic acid and glutamate metabolism.There were significant differences in the results of the two cell metabolomics studies,including 17 identical differential metabolites and 205different differential metabolites,lipids as the significantly different components,and glutathione is a potential biomarker for co-existence.The results involved 7 different metabolic pathways and 2 same metabolic pathways.Conclusion:This study successfully established the UHPLC-Q/TOF-MS Quasi-target Identification metabolomics method,which can enrich the identification types of differential metabolites,improve the identification accuracy,and is conducive to the study of action pathway.Both HepG2 cells and L02 cells induced by triptolide are related to purine metabolism and glycerophospholipid metabolism,but there are significant differences in the metabolic pathways involved,indicating that there are differences in the mechanism of action of triptolide on HepG2 cells and L02 cells,suggesting the importance of cell selection in the study of drug hepatotoxicity.