Study On The Antioxidative Mechanism And Screening Of Antioxidant Small Peptides From Egg White Via Keap1-Nrf2 Pathway

Antioxidant peptides as an important antioxidant,has the advantages of safety,good activity,easy to absorb,becoming a research hotspot.However,there are still some problems to be solved in the research of antioxidant peptides,such as deficiency in study on the molecular mechanism of antioxidant peptides.In order to solve these problems,egg white protein was used,a high quality of food protein,as protein source of antioxidant peptide.Then small peptides,the sum of dipeptides and tripeptides,are used as experiment research goal,because they can be fully absorbed and their activity will hardly be affected.Next,all experiments of this thesis suggest that the effect of small peptides from egg white on Keap1-Nrf2 interaction via Keap1-Nrf2 pathway that associated with cell antioxidant.What is more,they clarify the antioxidant effect of small peptides based on Keap1-Nrf2 pathway,and elucidate the antioxidant mechanism of small peptides from the molecular level.Finally,they lay the foundation for the research and application of antioxidant peptides.The main conclusions obtained were as follows:Based on molecular docking technology,small peptides from egg white which can directly inhibit Keap1-Nrf2 interaction were screened.First,extended egg white small peptide library which consist of 8400 small peptides was established and optimized and used as ligand library.The structure of the Keap1 Kelch domain,a docking receptor,bound to the Nrf2 16-mer peptide(PDB ID: 2FLU)was obtained from the RCSB database.Three binding sites: site 1(center coordinates: x:-4,y: 6,z: 0,radius: 21 ?),site 2(center coordinates: x: 5,y: 9,z: 1,radius: 15 ?),and site 3(center coordinates: x: 7.36,y: 8.33,z: 1.77,radius: 15 ?)were selected according to the Kealp1 structure and receptor binding site.To perform molecular simulations,Discover Studio for the semi-flexible docking program CDOCKER was used.Finally,20 small peptides which had strongest abilities of directly inhibit Keap1-Nrf2 interaction were screened out,including 6 dipeptides: EK,DK,WE,DW,EY,EW and 14 tripeptides: DKE,QKE,DKD,EDW,DWE,DKK,EEW,EWE,ECD,DET,DEW,DWD,DDW,DKQ.Based on fluorescence polarization technology,20 small peptides from molecular docking which can directly inhibit Keap1-Nrf2 interaction were screened and the antioxidant mechanisms of these peptides were analysed.First,fluorescent polarization experimental method for small peptides was established through relevant literature and analysis of the experimental system.Then,these 20 peptides was tested by this fluorescence polarization experimental method,and small peptides DKK and DDW which had strongest abilities of directly inhibit Keap1-Nrf2 interaction in non cellular environment were screened out.Next,the possible molecular levels antioxidant mechanism of these two peptides was obtained is that these peptides form hydrogen bonds with the residues which were key residues in interaction of Keap1 Kelch domain and Nrf2 peptide including ETGE motif in the Keap1 Kelch domain.And a conclusion was obtained that Arg380 and Asn382 likely had the greatest influence of Keap1-Nrf2 interaction in Keap1 protein.Besides,it is meaningful to design multi sites for small peptides molecular docking experiments by analysis of molecular docking and fluorescent polarization experiments.And a possible conclusion was obtained that this method was possible to reduce the adverse effects on the test results caused by the difference in the molecular size of test ligands and ligand attached to receptor files and improve accuracy of test results.Based on cell experiments,small peptides DKK and DDW which can directly inhibit Keap1-Nrf2 interaction in cellular environment were studied and the antioxidant mechanisms of these peptides were analysed.First,the H2O2-induced Hep G2 cell oxidative damage model was established.Then,small peptides DKK and DDW were tested for cytotoxicity and the working concentration of small peptides DKK and DDW in the subsequent experiment was determined.Next,it was found that small peptides DKK and DDW could improve antioxidant capacity of H2O2-induced Hep G2 cell though the tests about effect of small peptides DKK and DDW on H2O2-induced Hep G2 cell.What's more,it was found that small peptides DKK and DDW could improve the activities of CAT and SOD of H2O2-induced Hep G2 cell though the tests about the activities of CAT and SOD s on H2O2-induced Hep G2 cell treated with small peptides DKK and DDW respectively.Based on the above results,it was clear that the small peptides DKK and DDW had the abilities to inhibit Keap1-Nrf2 interaction in the cellular environment.Finally,the mechanism of the small peptide DKK and DDW to improve the activities of CAT and SOD in H2O2-induced Hep G2 cell was obtained through the analysis.The small peptides DKK and DDW partially occupy the Keap1 binding site for Nrf2 through hydrogen bonding with the key residues,thus preventing Nrf2 proteasomal degradation.The accumulated Nrf2 then translocates to the nucleus,where it forms heterodimers with the Maf protein and binds to Antioxidant Response Element(ARE),activating gene expression of phase II detoxification enzymes,including CAT and SOD.Finally,the activities of CAT and SOD were improved.In summary,small peptides DKK and DDW were screened out through the above experiments from egg white peptides,which could directly inhibit Keap1-Nrf2 interaction and played antioxidant roles in the cell.Besides,cell and molecular levels antioxidant mechanisms of small peptides were obtained,to lay the foundation for the research and application of antioxidant peptides.