The Experimental Study Of Liver Metabolites Spectrum Changing In Realgar-induced Mice And Interventional Effect Of Glycyrrhetinic Acid Based On LC-MS Metabonomics

Objective: In order to study the changes of small molecule metabolites spectrum in livers of mice induced by realgar and the protective effect of glycyrrhetinic acid,we established a metabolomics method based on LC-MS.The changes of antioxidants and Nrf2 pathway in mice’s livers were measured to investigate the possible toxic mechanism,while providing theoretical basis and research ideas for the oxidative damage induced by realgar,the protective effect of glycyrrhetinic acid and the detoxification mechanism of liquorice and realgar.Mehtods: Healthy ICR male mice were selected as the research objects,90 ICR mice were random divided into control group,low（0.15 g/kg）,medium（0.45 g/kg）and high（1.35 g/kg）dosage realgar group,glycyrrhetinic acid group（48 mg/kg glycyrrhetinic acid）,low and high dosage glycyrrhetinic acid group（1.35 g/kg realgar+16 mg/kg glycyrrhetinic acid,1.35 g/kg realgar+48 mg/kg glycyrrhetinic acid）,gavaged once a day for 8 consecutive weeks,after the treatment,the mice were sacrificed by ice-bath and the livers were removed from mice immediately.The samples were stored at-70 ℃ before use.The metabonomics based on LC-MS was used to detect changes of small molecule metabolites spectrum in mice’s livers after exposing to realgar and combining realgar with glycyrrhetinic acid.After the spectrum was processed by Markerlynx software,the data was subjected to PLS-DA by SIMCA-P11.5,the differential metabolites were screened according to the load map and the value of the importance of the variables.One-way ANOVA was used to analyze the differences of the selected metabolites by SPSS 17.0 software to find out statistically significant substances,then identifying differential metabolites by combining literature and online databases.ELISA was used to observe the contents of GSH,GSH-Px,SOD and CAT in the livers of mice.Western blot was used to measure the relative protein levels of Nrf2,NQO1,HO-1 and Keap1 in the livers of mice.Results: 1.LC-MS was used to determine the changes of small molecule metabolites in the livers of each realgar exposed group and glycyrrhetinic acid intervention group,the results showed that the relative intensity of the nicotinamide,tyrosine,LPC18:2and the retention time-mass-to-charge ratio of 1.09₁16.0,1.58₁19.0 were significantly increased in realgar exposed group comparing with the control group,the relative intensity of the metabolite with the retention time-mass-to-charge ratio of1.16₁33.0 was significantly decreased and choline of the high-dose realgar exposed group was significantly lower than that in the control group.Then,the relative intensity of nicotinamide,tyrosine and the metabolites with retention time-mass-to-charge ratio of 1.09₁16.0 and 1.58₁19.0 were significantly decreased in mice treated with glycyrrhetinic acid comparing with the realgar-treated group,the relative intensity of LPC18:2 and choline were significantly increased,while the relative intensity of metabolite with retention time-mass-to-charge ratio of 1.16₁33.0had no significant change.2.Realgar could significantly reduce the levels of GSH,GSH-Px and SOD in mice’s livers（P<0.05）,but had little significant influence on the level of CAT（P>0.05）,suggesting that realgar could cause oxidative damage in liver.The high dosage glycyrrhetinic acid group could obviously antagonize the decrease of GSH,GSH-Px and SOD in the liver caused by realgar（P<0.05）,suggesting that glycyrrhetinic acid had a protective effect on realgar-induced liver injury.3.The relative protein levels of Nrf2,NQO1 and HO-1 in the livers of realgar exposed groups increased significantly with the increase of realgar dosage.The relative protein levels of Nrf2,NQO1 and HO-1 in the livers of mice treated with glycyrrhetinic acid also showed an upward tendency,in which high dosage glycyrrhetinic acid group increased most.However,there were little significant differences among all these groups as to the relative expression levels of Keap1.Conclusion: Realgar can cause liver oxidative damage in mice,while glycyrrhetinic acid has a protective effect on the realgar caused liver damage.Its specific mechanism is related to the level of small molecule metabolites spectrum,the level of antioxidants and the activation of Nrf2 pathway.