| Objective: To screen and identify the active components of deer fetoprotein peptides with therapeutic immunity and anemia activity,and to study the network pharmacology and molecular docking of the main active components in deer fetoprotein,so as to predict its mechanism of action.Methods:1.Firstly,the whole component analysis of deer fetus was carried out,and MTT method was used to investigate the promoting effect of different polar parts of deer fetus on macrophage RAW264.7 and human bone marrow stromal cells HS-5,and the best active part was selected;2.Based on enzyme engineering technology and peptide bond thermal oscillation theory,the optimal extraction process of active fraction from deer fetus was selected;3.HPLC-DAD was used to establish the chemical fingerprints of different batches of deer fetus before and after processing.Combined with the immunomodulatory activity of each sample,the partial least squares(PLS)analysis was obtained by grey correlation analysis and SIMCA software,and the activity changes and spectrum effect correlation before and after processing were preliminarily screened out;4.Using ion exchange chromatography DEAE-52 and glucose gel column chromatography Sephadex G-25 to separate the active components of deer fetus,and speculate on its possible chemical structure through LC-ESI-MS/MS,evaluate its immunity and anemia activity by MTT method,and further identify the main active components in deer fetus.5.The immunomodulatory effect of active components of deer fetus and the determination of related factors were studied;6.Network pharmacology and molecular docking method was used to predict the anemia mechanism of the main active ingredients in deer fetus.Results:1.The proliferation of RAW264.7 cells and HS-5 cells was promoted by different polar parts of deer fetus in a dose-dependent manner,and the activity of water layer part was better than that of model group,which indicated that the main active components of deer fetus were concentrated in water layer.The results showed that the water layer part of deer fetus was less than 1KDa by membrane separation method The activity of peptide was the best;2.Taking the degree of hydrolysis as the index,the single factor experiment and response surface methodology were used to optimize the enzymatic hydrolysis process of deer fetus.The optimum conditions were determined as follows: the temperature of enzymatic hydrolysis was 45 ℃,the amount of enzyme was 3.0%,the time of enzymatic hydrolysis was 6h,and the p H was 8.0.At the same time,compared with the activity of natural < 1KDa component,the activity of natural < 1KDa component was better than that of enzymatic hydrolysis.3.Based on the thermal oscillation theory of peptide bond,the correlation between the activity and composition of deer fetus before and after processing was studied.The peaks of VIP > 1 and grey correlation coefficient > 0.8 were 7.069,7.772 and 9.471,which were positively correlated with immunomodulatory activity in deer fetus.4.DEAE-52 and Sephadex G-25 were used to separate deer fetus.The chromatographic fingerprints of deer fetus oligopeptides were established by high performance liquid chromatography(HPLC).Based on LC-MS analysis,the amino acid sequences of nine oligopeptides were determined.5.TTD database was used to predict anemia related targets,and Swiss target prediction was used to screen deer fetoprotein targets.A total of 54 common potential targets were obtained.18 biological processes were identified by cluego plug-in analysis,and 44 pathways were identified by KEGG analysis.The interaction network analysis showed that there were a lot of interactions among the targets,and seven important targets were selected.The score of ASP Tyr met,Gln Tyr and NOS2 was higher than that of natural ligands.Conclusion: In this study,the active fraction of deer fetus with immune and blood enriching effects was selected as < 1KDa component,and nine kinds of deer fetus oligopeptides were isolated and identified.Through the study of RAW264.7 cells on immune regulation mechanism,based on network pharmacology and molecular docking technology to predict the treatment of anemia in deer fetus.To provide a scientific theoretical basis for deer fetus in the treatment of anemia and immunity. |
