Active Mechanism On The Combined Constituents In Vivo Of Yinchenhao Tang Protect Against Alcoholic Liver Injury

AIM:To reveal the metabolic profile and metabolic biomarker compounds of urine, blood, liver tissue and primary hepatocytes on alcoholic liver injuried model mouse and cells.Forthermore, to illustrate therapeutic effect of principle components from serum constituents of Yinchenhao Tang (YCHT) against liver injury induced by alcohol.The study provided the technique support and experiment basis for traditional Chinese medicine (TCM) innovation.METHODS:Mouse were treated with 60% ethanol to duplicated alcoholic liver injured model.Firstly,physiological and biochemical indexes of histopathological were also monitored. We have analyzed the urine samples,serum samples and liver tissue samplies of model rats and cell samples using UPLC-HDMS, and an integrative pattern recognition approach of principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) was also applied for markers discovery. The metabolic pathways of efficacy of alcoholic liver injury were focused and found by the improved ’MetPA enriched pathways’method. In this study, the model mouse and cells were treated with ingredients combination of YCHT, and the effects of the combination was also evaluated.RESULT:1. Establishment and evaluation of alcoholic liver injured modelICR mouse were treated with 60% ethanol to duplicated ralcoholic live injured model. The levels of AST, ALT,MDA,TG,y-GT were increased significantly, and the level of GSH was decreased significantly indicating that the dysfunction of liver was changed. All the biochemical indexes of histopathological were changed significantly at 7th day. For HE, massoning analysis, we found that central vein and hepatic sinus significant expansion, congestion, swelling of the liver cells, crowded,and a certain degree of inflammatory cell infiltration and focal necrosis of liver cells and intracellular vacuoles, liver injured occurmd at 7th day.In TUNEL detection, compared with the blank group, the apoptotic cells in the model group were densely stained and stained deeply, ia significant difference (P<0.01). CASPASE-3 activity detection showed that the model group had strong CASPASE-3 activity, the distribution range was wide, the surface density was high, and the activity expression surface density was significantly different from the blank group (P<0.01). This experiment successfully duplicated the alcoholic liver damage model of mice, which lay the foundation for the following experiment.2. Identification and analysis of the metabolic markers of alcoholic liver injury(1) Analysis of metabolic markers and metabolic markers in urine of mice with alc oholic liver injuryA metabolomic approach based on UPLC-HDMS and integrative pattern recogniti on approach was developed for investigating the urine samples of alcoholic liver inju red model mouse. The urine metabolic profiling was obviously disturbed at 0th day, and the long time has passed, the far the urine samples of model mouse from origin al condition, and the samples of 7th day were the furthest.59 biomarkers connected with injury were confirmed and identified combined with the database for the cause of metabolic changes in the path of potential biomarkers characterized.Compared with normalgroup,the level of Methylglyoxal,Glycine,Glycolic acid,Betaine aldehyde,1-Pyrrol ine-2-carboxylic acid,L-Proline increased, and the level of L-Valine,Pyroglutamic acid, Pipecolic acid,Ureidopropionic acid,Phenylacetamide,Gentisic acid,L-Phenylalanine,Uric acid,L-Tyrosine,3-Methoxy-4-hydroxyphenylglycolaldehyde,N-(o)-Hydroxyarginine,Pheny lacetylglycine,5-Hydroxykynurenine,D-Glucosamine 6-phosphate,Saccharopine,Pantetheine, Deoxyuridylic acid,5-Thymidylic acid,Deoxyadenosine monophosphate,S-Hydroxymethyl-glutathione,Maltose,Adenosine monophosphate,S-Adenosylmethioninamine,Deoxycytidine diphosphate,Guanosine diphosphate, Se-Adenosylselenomethionine,Oxidized glutathione decreased. Significantly.These markers mainly are related with Phenylalanine, tyrosine and tryptophan biosynthesis,Phenylalanine metabolism,Pyrimidine metabolism,Purine me tabolism,Valine, leucine and isoleucine biosynthesis,Tytosine metabolism, Glycine, seri ne and threonine metabolism,Alanine metabolism,Histidine metabolism,Pentose and glu curonate interconversions,Alanine, aspartate and glutamate metabolism,Selenoamino aci d metabolism,Starch and sucrose metabolism,Arginine and proline metabolism,Pantothe nate and CoA biosynthesis.(2) Analysis of metabolic markers and metabolic markers in serum of mice with al coholic liver injuryA metabolomic approach based on UPLC-HDMS and integrative pattern recogniti on approach was developed for investigating the serum samples of alcoholic liver inj ured model mouse. The results showed that the blank group and model group mice s erum metabolic profile clearly separate PCA analysis showed that sample.29 biomark ers connected with injury were confirmed and identified combined with the database for the cause of metabolic changes in the path of potential biomarkerscharacterized.C ompared with normalgroup,the level of D-Maltose,Thromboxane B2,Sulfolithocholylgly cine,LysoPC(22:5(4Z,7Z,10Z,13Z,16Z)),LysoPC(22:5(7Z,1 OZ,13Z,16Z,19Z)),L-Urobilin,C oproporphyrin I,Trihexosylceramide(d18:1/22:0),Phenylacetaldehyde,Pipecolic acid,L-Leu cine,3-Hydroxyanthranilic acid,Norepinephrine sulfate increased, and the level of Crea tine,5,10-Methylene-THF,Glycocholic Acid,LysoPC(20:5(5Z,8Z,11Z,14Z,17Z)),Trimethyla mine N-oxide,1-Phenylethylamine,4-Guanidinobutanoic acid,Phosphorylcholine,Citrulline, 3-Methoxytyrosine,l-Methylguanosine,Taurocholic acid,LysoPC(20:3(5Z,8Z,11Z)),PC(18:0 /16:0),PI(18:0/20:4(8Z,11Z,14Z,17Z)),3a,7a-Dihydroxy-5b-cholest-24-enoyl-CoA decrease d. Significantly,These markers mainly are related with Methane metabolism,Valine,leuc ine and isoleucine biosynthesis,Glycerophospholipid metabolism,Phenylalanine metaboli sm,Primary bile acid biosynthesis,Starch and sucrose metabolism,Tryptophan metabolis m,Glycine, serine and threonine metabolism,Arginine and proline metabolism. (3) Analysis of metabolic markers and metabolic markers in liver tissue of mice wi th alcoholic liver injuryA metabolomic approach based on UPLC-HDMS and integrative pattern recogniti on approach was developed for investigating the liver tissue samples of alcoholic live r injured model mouse. The results showed that the blank group and model group m ice liver tissue metabolic profile clearly separate PCA analysis showed that sample.3 0 biomarkers connected with injury were confirmed and identified combined with the database for the cause of metabolic changes in the path of potential biomarkers chara cterized.Compared with normalgroup,the level of Pelargonidin,15-Keto-prostaglandin E2, 11-Hydroxyprogesterone 11-glucuronide,LysoPC(O-18:0),Taurocholic acid,3-Butylidene-1 (3H)-isobenzofuranone,dCMP,AICAR,7-Methylguanosine 5’-phosphate,Gamma-Tocotrien ol increased, and the level of Adenine,Guanine,Cysteinylglycine,Butyrylcarnitine,S-Ac etyldihydrolipoamide,Gamma-Glutamyl Glutamine,3’-AMP,Cervonoyl ethanolamide,LysoP C(22:2(13Z,16Z)),Uridine diphosphate-N-acetylgalactosamine,PE(15:0/14:1 (9Z)),Dipalmit oylphosphatidylserin),3-O-Sulfogalactosylceramide(dl8:1/14:0),PE(22:4(7Z,10Z,13Z,16Z) /22:6(4Z,7Z,10Z,13Z,16Z,19Z)),N-Succinyl-2-amino-6-ketopimelate,(S)-Succinyldihydrolip oamide,Androsterone glucuronide, (2-(S-Glutathionyl)acetyl glutathione,PC(18:4(6Z,9Z,1 2Z,15Z)/P-16:0),PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/P-18:1 (11Z)) decreased. Significantly, These markers mainly are related with Glycerophospholipid metabolism,Ether lipid me tabolism,Pentose and glucuronate interconversions,Purine metabolism,Glycosylphosphati dylinositol(GPI)-anchor biosynthesis,Starch and sucrose metabolism,Primary bile acid b iosynthesis,Pyrimidine metabolism,Glutathione metabolism.(4) Analysis of metabolic markers and metabolic markers in cell of mice with alcoh olic liver injuryA metabolomic approach based on UPLC-HDMS and integrative pattern recogniti on approach was developed for investigating the cell samples of alcoholic liver injure d model mouse. The results showed that the blank group and model group mice cell metabolic profile clearly separate PCA analysis showed that sample.30 biomarkers co nnected with injury were confirmed and identified combined with the database for th e cause of metabolic changes in the path of potential biomarkers characterized.Compa red with normalgroup,the level of Indole,4-Hydroxybenzaldehyde,L-Leucin,2-Phenylacet amide,Phenylpyruvic acid,Quinaldic acid,5-Hydroxyindoleacetaldehyde,L-Tyrosine,Indole acrylic acid,S-(2-Methylbutanoyl)-dihydrolipoamide,LysoPC(14:1(9Z)),Lactaldehyde,L-L actic acid,Succinic acid,Dihydrothymine,6-Hydroxyhexanoic acid,N-Succinyl-L,L-2,6-dia minopimelate,Eicosadienoic acid increased, and the level of 1-Phenylethylamine decre ased. Significantly,These markers mainly are related with Phenylalanine,tyrosine and tr yptophan biosynthesis,Valine, leucine and isoleucine biosynthesis,Phenylalanine metabol ism,Tyrosine metabolism,Glycerophospholipid metabolism,Citrate cycle (TCA cycle),Py rimidine metabolism,Tryptophan metabolism.3. Effects of YCHT effective constituents compatibility on liver injuredThe alcoholic liver injured model mouse were treat with oral administration of in vivo ingredients combination of YCHT. The results showed that the level of AST, ALT, MDA、 TG、γ-GT were obviously decreased, while GSH were increased in the treatment groups. HE and Masson staining analysis demonstrated that the treatment group liver cells arranged regularly, the structure of hepatic lobule close to normal, the central vein and hepatic sinus were slightly dilated, periportal vascular hyperplasia and other symptoms disappeared gradually. TUNEL detection showed that the end of the treatment group, reversal effect of a certain degree of liver cell apoptosis alcoholic liver injury, the surface density of apoptotic cells reaching the blank group, compared with the model group with significant difference (P<0.01). CASPASE-3 activity detection showed that the activity of CASPASE-3 in the treatment group had a certain degree of inhibition, and the expression of the activity of the expression surface density was significantly different from the model group. Unsupervised PCA and center distance analysis of multivariate data analysis in urine found that in vivo ingredients combination of YCHT can regulate the metabolic disturbances to the normal state. in vivo ingredients combination of YCHT has callback effect on metabolic footprint as an evaluation of the efficacy.Abnormal metabolic profile clearly to the normal outcome, significantly improve the alcohol induced liver cell damage. In metabolic markers level,59 biomarkers of the alcoholic liver damage potential biomarkers are found in urine samples in ingredient formula treatment.there are 49 showed callback function.19 biomarkers callback of 29 alcoholic liver damage potential biomarkers were found in serum samples 18 callback of 30 alcoholic liver injury potential biomarkers in liver tisse samples.19 alcoholic liver injury potential in biomarkers Samples of primary hepatocytes, there are 17 biomarkers callback function. 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 in vivo ingredients combination of YCHT has therapeutic effect on the primary hepatocytes injury caused by ethanol.CONCLUSION:The study duplicated alcoholic liver injury model and developed for investigating the metabolic biomarker of alcoholic liver injury model asXXX and their interrelated metabolic pathway was aslo founded asXXX. The results demonstrated that in vivo ingredients combination of YCHT could reduce certain biochemical factors of liver injury, protect and restore damaged hepatocytes, pulled back the trend of urine metabolic profile, affect the expression level of the metabolic biomarker of alcoholic liver injury. All statistical results proved in vivo ingredients combination of YCHT which composed by 6,7-dimethoxy coumarin, Geniposide, Rhein was very efficacy for treating alcoholic liver injury. It provided experimental and theoretical basis for the further investigation of the mechanism of treating alcoholic live rdesease and drug discovery.