Investigation Of The Mechanism Of Hepatocyte Injury Caused By Oleanolic Acid Based On The Cell Metabolomic

Objective:To explore the mechanism of oleanolic acid-induced liver cell damage based on the cell metabolomics.Methods:The primary hepatocytes were cultured to the 8th hour and the 3rd day then treatment with oleanolic acid（0,20,40,60,80,100μmol/L）to determine cell viability.The primary hepatocytes of wild-type mice and FXR knockout mice were cultured to 8 h and to the 3rd day,and were given OA control group（iso-volume solvent）,low-dose group（20μmol/L）,medium-dose group（40μmol/L）and the high-dose group（60μmol/L）,samples were collected after administrated 48 hours;Using UPLC-MS/MS detected cell metabolites and observing the different metabolites of wild-type and FXR knockout primary mice hepatocytes after oleanolic acid administration,and further conduct pathway analysis.Results:（1）MTT and LDH together suggested that dose-dependent decrease in cell viability,the cell viability of primary hepatocytes was only 53%when the OA concentration was 60μmol/L.（2）The metabolomics study found that OA was given to WT and FXR^-/-PMHs at the 8th hour,the identified differential metabolites involved pyrimidine metabolism,purine metabolism,nicotinate and nicotinamide metabolism,glutathione metabolism and other pathways.The disordered metabolic pathways between WT PMHs and FXR^-/-PMHs are mainly pyrimidine metabolism,purine metabolism,riboflavin metabolism and citric acid cycle.It shows that oleanolic acid may damage hepatocytes through oxidative stress and energy metabolism.This oxidative stress damage has nothing to do with the regulation of FXR,and there may be through another way;（3）The metabolomics study found that OA was given to WT and FXR^-/-PMHs on the 3rd day,the identified differential metabolites involved glutathione metabolism,purine metabolism,nicotinate and nicotinamide metabolism,amino acid metabolism and other metabolic pathways.The disordered metabolic pathways between WT PMHs and FXR^-/-PMHs are mainly glutathione metabolism,purine metabolism,nicotinate and nicotinamide metabolism,and amino acid metabolism.It further shows that oleanolic acid may damage hepatocytes through oxidative stress and energy metabolism.This oxidative stress damage has nothing to do with the regulation of FXR,and there may be through another way;and this damage is caused by OA directly injury on hepatocytes rather than OA induced cholestasis and then damage to hepatocytes.Conclusion:OA-induced hepatocyte injury is caused by OA directly injury on hepatocytes rather than OA induced cholestasis and then damage to hepatocytes,and the mechanism may be closely related to oxidative stress,energy metabolism,and cell apoptosis.And this hepatocytes damage caused by oxidative stress and energy metabolism is not regulated by FXR,and there may be through another pathway.This research provides a basis for future research on related mechanisms of oleanolic acid.