Protective Effects Of Aqueous Extract From Gei Herba And Gallic Acid On AML12 Hepatocyte Injury Induced By Cyclophosphamide

Objective:According to metabolomics and other methods,the protective effect and mechanism of aqueous extract from Gei Herba and gallic acid against AML12 hepatocyte injury induced by cyclophosphamide(CP)were studied.Methods:A high performance liquid chromatographic method was established to simultaneously determine the content of gallic acid and ellagic acid in Gei Herba and then the methodology was verified.Firstly,the injury model of AML12 cells induced by CP was established and the effect of aqueous extract from Gei Herba and gallic acid on cell survival rate were investigated.Secondly,the comprehensive protective effect of aqueous extract from Gei Herba and gallic acid on AML12 cell injury model was studied including cell morphology observation,hepatic enzymes,anti-apoptosis and anti-oxidation activities.Thirdly,the expression of oxidative and apoptosis-related genes by moducation of aqueous extract from Gei Herba and gallic acid were studied by RT-qPCR and Western blot(WB).Finnally,a metabolomics method for the analysis of hepatocyte disruption was established by reversed phase high performance liquid chromatography-tandem mass spectrometry(RPLC-MS/MS).Metabolic profile analysis was performed on different groups.Pattern analysis was used to identify differential metabolites and their associated metabolic pathways were analyzed to preliminarily explain its mechanism.Results:A method for simultaneously determining the content of gallic acid and ellagic acid in aqueous extract from Gei Herba was established,which showed good linearity,good precision,good repeatability,high accuracy and good stability within 24 h.The average content of gallic acid and ellagic acid measured in 6 batches of medicinal herbs was 0.033% and 0.192%,respectively.The AML12 cell survival rate of CP model group significantly decreased and thecontent of ALT,AST,LDH significantly increased(P<0.05);cell apoptosis significantly increased(P<0.05);SOD and MDA significantly increased(P<0.05);the content of GSH significantly decreased(P<0.05)when compared with the normal control group.Compared with the CP model group,administration of aqueous extract from Gei Herba and gallic acid,he survival rate of AML12 cells was increased,the content of ALT,AST,and LDH in the cell supernatant were decreased and the apoptosis of AML12 cells was inhibited(P<0.05).The levels of SOD and GSH in the cell disrupting fluid were increased(P<0.05),and the level of MDA was decreased(P<0.05).In the metabolomics analysis,six metabolites such as Niflumic acid,Palmitoylethanolamide,and 4-oxo-Retinoic acid were found in this study by regulation of aqueous extract from Gei Herba,which mainly may involve into amino acid metabolism and arachidonic acid metabolism.Gallic acid mainly regulates 17 metabolites such as L-Threo-3-Phenylserine,L-Leucine and Met His Lysm,which mainly related to amino acid metabolism,phospholipid metabolism,sphingolipid metabolism and so on in the analysis of metabonomics.Determination of apoptosis and oxidative stress gene by RT-qPCR method: the mRNA expression levels of Bad,Bax,caspase3,caspase9 and P53 in CP model group were significantly increased(P<0.05)and the mRNA level of Bcl-2,Keap1,Nqo1 were decreased(P<0.05).Compared with the CP model group,the expression of Bad,Bax,caspase3,caspase9 and P53 mRNA were down-regulated(P<0.05)and Bcl-2,HO-1,Keap1,Nqo1,Nrf-2 mRNA were significantly up-regulated(P<0.05).At the same time,Compared with the normal control group,the protein level of Bax in CP model group was up-regulated(P<0.05)and the protein level of Bcl-2 was significantly decreased(P<0.05).Compared with the CP model group,aqueous extract from Gei Herba and gallic acid could promote Bax protein expression and inhibit Bcl-2 protein expression and there were significant differences(P<0.05).Conclusion:Aqueous extract from Gei Herba and gallic acid could reduce the damage of AML12 cells cause by CP and the mechanism may related to improving cell function,reducing cell apoptosis,resisting oxidative stress and regulating cell metabolic network.