ACE-renin Inhibitory Mechanism And Absorption Pathway Of Egg White Derived Peptides

This work was funded by the National Key R&D Program of China(2018YFD0400301).Hypertension is the main factor which leads to cardiovascular diseases and threatens human health.Moreover,the pathogenesis of hypertension is complex.It is an urgent problem to seek safe and effective methods to treat hypertension.Although commercially available antihypertensive drugs can effectively reduce blood pressure,a series of side effects will threaten human health.Food derived bioactive peptides have the characteristics of mild action and non-toxic side effects.It is an effective strategy to use food derived bioactive peptides to inhibit angiotensin converting enzyme(ACE)in the prevention and treatment of hypertension.In fact,in renin-angiotensin system,the key system of human blood pressure regulation,renin is the source protease that controls the reaction rate,and the development of its food derived inhibitory peptide is often ignored.If we can develop bioactive peptides with both ACE and renin inhibitory activities,it will provide experimental basis for the development of bioactive peptides.Using egg white protein as raw material,the dual bioactive peptides with ACE and renin inhibition was successfully identified by liquid chromatography tandem mass spectrometry(LC-MS/MS).The bioactivities of the peptides were improved by sequence modification,and the preference of terminal amino acids was confirmed.The mechanism of food derived bioactive peptides was explored by molecular docking,molecular dynamics simulation and isothermal titration calorimetry.Network pharmacology was used to explore the potential targets of bioactive peptides,and the activity and potential targets of bioactive peptides were verified by human umbilical vein endothelial cell model.Finally,Caco-2 monolayer model was used to explore the absorption pathway of the bioactive peptides,and BSA nano carrier was used to successfully improved the absorption efficiency of the bioactive peptides.The main research contents and results are as follows:(1)Using egg white protein as raw material and degree of hydrolysis as index,the enzymatic hydrolysis process was optimized by single factor and response surface methodology.The optimal enzymatic hydrolysis process was as follows:neutral protease was selected for enzymatic hydrolysis,substrate concentration was 3 g/100m L,p H was 6.5,enzyme substrate ratio was 5.32%,enzymatic temperature was 47?,enzymatic time was 4 h.The enzymatic hydrolysates were purified by ultrafiltration and Sephadex gel chromatography,and the fraction with the molecular weight<1k Da was selected.Peptide sequences were identified using LC-MS/MS.The peptide sequences LAPYK and ADWAK were obtained.Among them,LAPYK had both ACE and renin inhibitory activities,and IC₅₀ for ACE was 12.65±1.34?M.At the concentration of 2 m M,the inhibitory activity of renin was(40.01±1.56)%.The interaction mechanism of LAPYK with ACE and renin was analyzed using molecular docking.It was found that LAPYK could form electrostatic interaction with Glu411,the active center of ACE,and thus exert ACE inhibitory activity.The inhibitory activity of renin is mediated by hydrophobic interaction with Pro111 in the S3 active pocket of renin.(2)Four modified peptides were obtained by replacing the C-terminal amino acid of LAPYK with phenylalanine(F),tryptophan(W),proline(P)and glutamic acid(E).Furthermore,the N/C-terminal amino acids of these peptides were exchanged.Finally,ten modified peptides were obtained.The ACE inhibitory activity of the ten modified peptides was determined,the IC₅₀ of LAPYW was 5.42±0.12?M for ACE.The IC₅₀of LAPYE for ACE was more than 1000?M.The ACE inhibitory activity of peptides with different structures was determined.It was confirmed that hydrophobic amino acids and amino acids with cyclic structure were beneficial to the ACE inhibitory activity of peptides.Then,three peptides,LAPYK,LAPYW with the highest activity and LAPYE with the lowest activity were selected to explore their mechanism of inhibition using molecular dynamics simulation and isothermal titration calorimetry.The results showed that for the charged peptides LAPYK and LAPYE,the free energy of polar solvent greatly limited their ACE inhibitory activity.The thermodynamic parameters showed that the reactions between LAPYW,LAPYK and ACE were driven by enthalpy change,while that of LAPYE was driven by entropy change.(3)Using the same substitution strategy,ten modified peptides were obtained by replacing the N-terminal and C-terminal amino acids of LAPYK,and their renin inhibitory activity was determined to explore the terminal amino acid preference of renin inhibitory peptides.The results showed that LAPYW had the highest activity,and the inhibitory activity for renin reached(57.62±2.10)%at the concentration of 2m M,and the activity of PAPYK was the lowest.At the concentration of 2 m M,the inhibitory activity to renin was only(15.42±0.92)%.The interaction between these three peptides and renin was dynamically observed by molecular dynamics simulation.It was found that LAPYW could gradually approach to the active center,Asp32 and Asp215 of renin,thus exerting its inhibitory activity.However,the peptide PAPYK with poor renin inhibitory activity was far away from the active center of renin.(4)Using Pharmmapper database to predict the possible targets of LAPYK and LAPYW,and comparing with the hypertension related gene library in Gene Cards,we found that LAPYW had higher correlation with hypertension and higher matching with hypertension related targets.Through STRING database and Cytoscape software analysis of protein network interaction,it was found that FN1,TP53,REN,MMP9and ACE were the main frontal targets in the interaction network,indicating that the bioactive peptide may play a role in tumor inhibition and blood pressure regulation in vivo.Through GO and KEGG pathway analysis,the potential targets of these two peptides were REN,ACE,MME,ANP,EDN1 and AGT.The results showed that both LAPYK and LAPYW had ACE and renin inhibitory activities,and the activities of LAPYW was higher than that of LAPYK.It was found that these two peptides could regulate the expression of endothelin(EDN1)and atrial natriuretic peptide(ANP).(5)The absorption efficiency and mechanism of LAPYW was measured by Caco-2 cell monolayer model.The results showed that LAPYW could be completely absorbed through endocytosis pathway,and its P_app value was(3.25 1±0.91)×10^-7cm/s.BSA,liposome and?-Cyclodextrin were selected as nano carrier to entrap the bioactive peptide LAPYW.The formation of nanoparticles was confirmed by the change of particle size and transmission electron microscope.The intrinsic fluorescence of LAPYW was measured by fluorescence spectrometer.The entrapment efficiency of different nano carriers was evaluated.The results showed that the entrapment efficiency of BSA and liposome was good,and the entrapment efficiency was(34.54±1.07)%and(35.99±0.43)%,respectively.Finally,Caco-2 cell model was used to evaluate the absorption promotion effect of different nano carriers.The results showed that BSA nano carrier could successfully improve the absorption efficiency of LAPYW by about 1.8 times.