Hepatoprotective Effect Of 6,7-dimethylesculetin Originated From Serum Constituents Of Yinchenhao Tang Protects Against Primary Hepatocytes Injury Based On Cell-metabolomics

AIM:To explore the therapeutic effect of 6,7-dimethylesculetin originated from serum constituents of Yinchenhao Tang(YCHT)against primary hepatocytes injury induced by alcohol based on cell-metabolomics approach and 6,7-dimethylesculetin affecting the metabolic pathways of the cultured primary mouse hepatocytes and related action mechanisms as well as therapeutic targets for alcoholic liver disease(ALD).Aiming to reveal the pathological mechanisms of ALD and key targets for therapeutic effect of 6,7-dimethylesculetin,finally providing new targets for ALD.METHODS:The primary hepatocytes injury alcohol-induced model was established by two-step perfusion method;cell proliferation was determined by MTT assay;cell morphology was evaluated by fluorescence microscopy,apoptosis and cell cycle was determined by flow cytometry.Based on the theory and methods of cell metabolomics,the metabolites differences of primary hepatocytes(intracellular,metabolic fingerprint)and medium(extracellular,metabolic footprint)was explored using ultra performance liquid tandem quadrupole time of flight high-resolution mass spectrometry technology combined with multivariate pattern recognition analysis(PCA,PLS,OPLS)performed by Markerlynx and EZinfo 2.0 software.The potential biomarkers were screened and selected by variable importance projection(VIP)of OPLS,and the structural identification of potential biomarkers was carried out according to mass spectrometry fragmentation and database.The potential biomarkers would be entered KEGG database to building metabolic pathways.The metabolic pathways of efficacy of 6,7-dimethylesculetin against primary hepatocytes injury were focused and found by the improved 'MetPA enriched pathways'method.Furthermore,the changes of key regulating enzymes related with the important metabolic pathways were identified by quantitative ELISA and PCR methods.RESULT:1.Establishment and evaluation of primary hepatocytes injury induced-alcoholThe high activity and continued stability of primary cultured mouse hepatocytes were obtained by the classic two-step perfusion method.The survival rate of primary hepatocytes was significantly lower after alcohol treatment,and the effect was shown dose dependent.When the alcohol treatment,the most primary hepatocytes turn out apoptosis,has significant morphological changes,such as cells smaller,surface shrinkage,cell nuclei condensation with apoptotic body formation,accompanied by necrosis.Flow cytometry results proved that alcohol damage hepatocytes mainly in the early stages of apoptosis,appeared Sub-GO/Gl peak,produce DNA strand breaks in apoptotic debris.2.Metabolic characteristics of alcohol-induced hepatocytes based on metabolomicsFirstly,alcohol-induced primary hepatocytes samples were analyzed by UPLC-SYNAPT-G2-Si-HDMS.The metabolic profiling showed the better clusters within the group and showing distinction between the control and model groups.19 specific metabolic markers were identified in alcohol-induced hepatocytes injury model.Compared with the normal group,indole,4-hydroxybenzaldehyde,L-leucine,2-phenylacetamide,phenylpyruvic acid,quinaldic acid,5-hydroxyindoleacetaldehyde,L-tyrosine,indoleacrylic acid,s-(2-methylbutanoyl)-dihydrolipoamide,lysoPC(14:1(9Z)),lactaldehyde,L-lactic acid,succinic acid,dihydrothymine,6-hydroxyhexanoic acid,N-succinyl-L,L-2,6-diaminopimelate,eicosadienoic acid showed up-regulation trend,and 1-phenylethylamine showed downward trend in model group.These biomarkers mainly involved in phenylalanine,tyrosine and tryptophan biosynthesis,valine,leucine and isoleucine biosynthesis,phenylalanine metabolism,tyrosine metabolism,pathways.3.Effect of 6,7-dimethylesculetin against primary hepatocytes injury using flow cytometry and metabolic fingerprinting methodAfter 6,7-dimethylesculetin treatment,G1 peak increased compared with the model group,diploid sub-peak decreased,indicating that 6,7-dimethylesculetin had inhibition effects on apoptosis of primary hepatocytes ethanol-induced and prevent DNA strand breaks.6,7-dimethylesculetin against primary hepatocytes injury samples at 2h,4h,8h,12h,24h were analyzed by unsupervised multidimensional analysis,PCA score plot showed the dynamic changes of overall metabolic profiling gradually deviated from the of the disease state.The center distance analysis of multivariate data analysis of overall metabolic profiles found after 6,7-dimethylesculetin treatment,the metabolite profiling of alcohol-induced primary hepatocytes changed significantly,the abnormal metabolic profiling have significant trend adjustment to the normal state,indicating that 6,7-dimethylesculetin has obvious pharmacodynamic effect on ALD.6,7-dimethylesculetin has callback effect on 19 potential biomarkers from metabolic fingerprints as an evaluation of the efficacy.Establishment the corresponding PCA model based on the potential biomarkers from metabolic fingerprints,the samples among three groups in Component 1 dimensions can significantly distinguish,the center of the treatment group is located between the control and model groups,indicating that 6,7-dimethylesculetin has therapeutic effect on ALD.Using the improved 'MetPA enriched pathways' method,it found 6,7-dimethylesculetin produced anti-ALD effects related pathways including phenylalanine,tyrosine and tryptophan biosynthesis,valine,leucine and isoleucine biosynthesis,phenylalanine metabolism,tyrosine metabolism pathways.4.Effect of 6,7-dimethylesculetin against alcohol-induced primary hepatocytes using metabolic footprinting methodCellular metabolites footprinting analysis could provide complementary information for cell metabolism fingerprinting,reflecting the metabolic state of hepatocytes.During the experiment after alcohol treatment,2h,4h,8h,12h,24h of the cell culture medium were collected,PCA analysis found that with time-course of 6,7-dimethylesculetin treatment,metabolic profile have significantly different.Compared with the previous administration Oh,2h firstly began to show significant changes in metabolic profiling,24h has significant difference with the previous treatment,also with 2h after 6,7-dimethylesculetin treatment,showing dynamic changes significantly away from hepatocytes damage state at Oh.12 differences metabolites from metabolism footprint analysis for alcohol-induced primary hepatocytes were screened and identified UPLC-G2-Si-HDMS/MS technique.Compared with the normal group,arginyl-asparagine,palmitic amide,octadecanamide,uccinic acid showed up-regulation trend;enol-phenylpyruvate,2-O-p-coumaroylhydroxycitric acid,eicosadienoic acid,oleamide,4-hydroxyphenylpyruvic acid,L-phenylalanine,L-glutamine,L-isoleucine showed downward trend in model group.These biomarkers mainly involved in ubiquinone and other terpenoid-quinone biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,valine,leucine and isoleucine biosynthesis,alanine,aspartate and glutamate metabolism.Unsupervised PCA and center distance analysis of multivariate data analysis found that 6,7-dimethylesculetin can regulate the metabolic disturbances to the normal state.6,7-dimethylesculetin has callback effect on 10 metabolites from metabolic footprint as an evaluation of the efficacy.Compared with the model group,the levels of arginyl-asparagine,palmitic amide,octadecanamide downregulated;and enol-phenylpyruvate,eicosadienoic acid,oleamide,4-hydroxyphenylpyruvic acid,L-phenylalanine,L-glutamine,L-isoleucine upregulated by 6,7-dimethylesculetin,metabolite disorders can be improved.Pathway analysis revealed that the efficacy of 6,7-dimethylesculetin with target metabolic pathways inculding phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,ubiquinone and other terpenoid-quinone biosynthesis,valine,leucine and isoleucine biosynthesis,alanine,aspartate and glutamate metabolism.5.Integrated analysis of metabolic fingerprint and metabolic footprint dataDue to the cells and extracellular medium have exchanged metabolites,the integrated analysis of intracellular metabolic fingerprint and extracellular metabolic footprint data,the cell metabolomics analysis is more complete.The intracellular metabolic fingerprint(19 biomarkers)and metabolic footprint(12 difference metabolites)input MetPA platform used for building the integrated metabolic pathways of alcohol-induced primary hepatocytes,found that mainly related with phenylalanine,tyrosine and tryptophan biosynthesis,ubiquinone and other terpenoid-quinone biosynthesis,valine,leucine and isoleucine biosynthesis,phenylalanine metabolism,tyrosine metabolism,alanine,aspartate and glutamate metabolism,etc.Using the improved 'MetPA enriched pathways' method,it found 6,7-dimethylesculetin produced anti-ALD effects related pathways including phenylalanine,tyrosine and tryptophan biosynthesis,valine,leucine and isoleucine biosynthesis,phenylalanine metabolism pathways.Furthermore,combined with significant trends of metabolites,building the regulatory network diagram of alcohol-induced primary hepatocytes,eventually focused phenylalanine metabolism that found to be the most important.Pearson correlation analysis between the intracellular and extracellular metabolites was used to explore the inherent association between these important metabolites,and it found L-tyrosine and 2-phenylacetamide and extracellular metabolites were strongly positively correlated(r>0.80).6,7-dimethylesculetin mainly regulated the metabolizing enzyme activity of phenylalanine metabolism to prevent the hepatocytes damage,determined by quantitative ELISA method,suggesting that phenylalanine hydroxylase,tyrosine aminotransferase,L-amino-acid oxidase may be used as a therapeutic target of intervention on ALD,blocking or modifying abnormal phenylalanine metabolism pathway has potential application for the treatment of liver damage caused by alcohol.CONCLUSION:This study had established the UPLC-G2-Si-HDMS/MS cell-metabolomics technology for research mechanism of alcohol-induced primary hepatocytes,and found that 6,7-dimethylesculetin regulate metabolism disorders to the normal state outcomes using unsupervised PCA and the center distance of multivariate data analysis.19 biomarkers from metabolic fingerprint and 12 difference metabolites from metabolic footprint were identified;6,7-dimethylesculetin can play a therapeutic role by reversing these metabolites;these findings help to reveal the complex pathogenesis of liver damage caused by alcohol.Using the improved 'MetPA enriched pathways'method combined with significant trends of metabolites,therapeutic effect of 6,7-dimethylesculetin related pathways focusing phenylalanine metabolism that found to be the most importantly,namely 6,7-dimethylesculetin treatment on liver damage caused by alcohol has closely related with the abnormal phenylalanine metabolism,mainly preventing hepatocytes injury by blocking the activity of the phenylalanine hydroxylase,tyrosine aminotransferase,L-amino-acid oxidase enzyme activity of phenylalanine metabolism.In this study,6,7-dimethylesculetin has significant research,development and application prospects in ALD related diseases,phenylalanine metabolism pathway may be a new target for the drug screening and treatment of ALD.