IIdentification Of Metabolites In Mare's Milk Mechanism Of Action On Ulcerative Colitis

objective: In this study,active specific metabolites of mare's milk were mined by metabonomics.And the computer virtual screening was used to explore the working mechanism of specific metabolites treat to ulcerative colitis.Methods: 1.Based on extensive target metabolic technology screening mare s specific metabolites to camel's milk,goats' milk,cow's milk,sheep's milk group as the control group,with ultra high performance liquid chromatography(HPLC)and four bar tandem mass spectrometry(UPLC/MS/MS)method and principal component analysis(PCA)and partial least squares(PLS)-DA multivariate statistical method to find mare's milk with five differences between the milk metabolic marker,determine the mare's milk specific metabolites,and use the KEGG database on the enrichment of annotation and analysis;2.Explore the mechanism of active metabolites in horse milk based on computer virtual screening method.Firstly,Pubmed or CNKI databases were used to determine the specific metabolites of active horse milk.Secondly,the two-dimensional or three-dimensional structure of active metabolites was used in Pubchem database to establish a small molecular ligand library,and the reverse target was found in the Pharma DB module of Dicovery studio(DS)software,and the key target genes were identified by superposition with ulcerative colitis related genes found in CTD database.Then,after the corresponding protein coding was determined by Unipot,the protein crystal structure was searched on the PDB database,and the macromolecular receptor library was established after the cleaning and optimization procedures on DS.Finally,DS software CDOCKER module was applied to perform docking between small molecules and proteins,and the high-score combination was screened out,so as to analyze the mechanism of action of mare's milk in the treatment of ulcerative colitis.Results:1.A total of 563 metabolites were detected in five animal milk by screening specific metabolites uplc-ms /MS based on wide target metabolome technology,and 33 potential differential metabolites were identified by PCA,opls-da and other statistical methods,which were different from camel's milk,cow's milk,goat's milk and sheep's milk.That is,mare's milk specific metabolites.KEGG analysis showed that the metabolic pathways of these metabolites were mainly enriched to 5 metabolic pathways,including linoleic acid metabolism,biosynthetic of unsaturated fatty acids,glycine,serine and threonine metabolism,vancomycin resistance,and trace-linolenic acid metabolism.2.Based on the computer virtual screening method to explore the mechanism of active metabolites in mare's milk,21 metabolites were reported to be active by literature retrieval of 33 metabolites by Pubmed or CNKI.500-600 targets were found by reverse target search on DS.The CTD database was searched for 60 target genes associated with ulcerative colitis.The two results were combined to find two target genes,namely matrix metallopeptidase 9(MMP9)and JAK2 kinase.Through molecular docking,11 components were found to bind to MMP9 and 13 to JAK2.Of L-threonine,east belladonna lactone,L-serine,2-amino adipic acid,thymine,hypoxanthine,lactic acid,4-hydroxy 6-methyl-2-pyrone,imidazole and 1-methyl acetate,uric acid,3.such as11 ingredients amino acid and two proteins can be combined,and that these compounds may be synergies to MMP9 and JAK2 play a role.The small molecule ligand 2-aminoadipate binds the receptor proteins MMP9 and JAK2 with the lowest energy and the tightest binding,which is much lower than other ligands.There are 16 amino acid sites involved in the interaction between the small molecule ligand 2-aminoadipate and the receptor protein MMP9.Among them,the small molecule ligand has a van der Waals force effect with TYR160,and has a charge attraction with ARG158,ASP138,and ARG134.ILE125,TYR128,TRP124,GLN169,ASN127 form hydrogen bonding energy,and GLN126,PHE170,GLY171,GLU174,HIS175,VAL172 form hydrogen bonding energy.And there are 13 amino acid sites involved in the interaction between the small molecule ligand 2-aminoadipate and the receptor protein JAK2.Among them,the ligand small molecule has van der Waals force effect with ASP949,charge attraction with HIS950,and ARG1113,SER1115,VAL1110 and ASN1111 have hydrogen bonding effect,forming carbon-hydrogen bonding energy with GLY1093,ASP1118,PRO1095,PRO1114,MET1130,ALA1034,GLN1112.It suggesting that the MMP9 and JAK2 are the target proteins of 2-aminoadipate.The same result was found for the other 9 metabolites.Conclusion: 1.This study screened with wide target metabolomic technology 33 mare's specific metabolites,and the main enrichment to the metabolism of linoleic acid,unsaturated fatty acidbiosynthesis,glycine,serine and threonine metabolism,vancomycin resistance,alpha linolenic acid metabolism five metabolic pathways,such as indirect evidence mare s antioxidant and anti-inflammatory effects.2.In this study,11 active metabolites synergistically acting on MMP9 and JAK2 proteins were identified by computer virtual screening,suggesting that mare's milk mainly plays its role in the treatment of ulcerative colitis by regulating MMP9-and JAK2-related signaling pathways.That is,because mare's milk is cold,it conforms to the treatment principle of Mongolian medicine for diseases such as hotness Dachangbaoru.Indirectly also explained that the mare's milk can treat the Dachangbaoru such as disease.This study provides a scientific basis for proving the nourishing and strengthening effects of mare's milk from the perspective of metabolites,and provides theoretical support for the follow-up research and development of mare's milk and the dietary therapy of Mongolian medicine.