Study On The Antihypertension Mechanisms Of Yeast-derived Peptides

With the acceleration of life pace,the incidence of cardiovascular diseases such as hypertension increased year by year,which severely affected the public health.Antihypertensive drugs were reported to exhibit some side effects.Accordingly,naturally derived antihypertensive peptides had attracted extensive attention for their mild,stable and safe features.Previously,yeast hydrolysates(YH)were produced from yeast by Bacillus subtilis derived enzymes in our laboratory.In vivo antihypertensive activity of YH was confirmed in animal model.However,the main active peptides in YH were not yet clear.In this study,the active peptides in YH were identified,and new peptides with strong ACE inhibitory activity or antioxidant activity were screened.ACE inhibition,antioxidation and endothelial function protection were proved to be involved in blood pressure-lowering mechanisms of YH,by using methods of enzyme kinetics study,cell model and high-throughput transcriptome sequencing.This study provides a theoretical basis for the development and application of yeast-derived peptides in functional foods and medicines.The specific research contents are as follows:(1)Identification and in silico screening of peptides in YH.Strong ACE inhibitory activity of YH was convinced in vitro with IC₅₀value of 26.13?g/m L,which was lower than the most protein hydrolysates derived from foods.A total of 778 peptides were identified from YH by mass spectrometry,and 91%of which were smaller than 1500 Da.Several bioinformatics methods were adopted to predict the biological activity of peptides and their ability to bind with ACE.As a result,12 peptides with potential ACE inhibitory activity were screened and synthesized by a solid-phase method.(2)ACE inhibition mechanisms of yeast-derived peptides.Among the synthesized peptides,VIPVPFF(V7),SSAPMF(S6)and FSEYPPLGRF(F10)showed the highest ACE inhibitory activity,with IC₅₀ values of 10.27?M,32.94?M and 32.07?M,respectively.Enzyme kinetics study proved that V7 inhibited ACE in a competitive manner,while S6 and F10 were non-competitive.In addition,V7 could resist hydrolysis of digestive enzymes and ACE,and was consider to be a true inhibitor of ACE.Molecular docking results predicted that these three peptides could form hydrogen bonds with ACE,which were located near the active center.Moreover,according to a factorial design experiment,the interactions between the three peptides on ACE inhibition were explored.The results showed that the combination with S6 or F10 could promote the ACE inhibitory activity of V7,which may be attributed to the different inhibitory mechanisms and different binding sites.(3)Antioxidant activity of yeast-derived peptides.Animal experiments showed that YH could significantly increase the total antioxidant capacity(TAC)of SHRs and significantly reduce the plasma malondialdehyde(MDA)after 40-day gavage at a dose of 1200 mg/kg.In vitro experiments confirmed the antioxidant activity of YH,with DPPH scavenging and superoxide anion scavenging EC₅₀values of 1.85 mg/m L and 20.27 mg/m L,respectively.YH-derived peptides ADLWPR(A6)and GFGFPR(G6)exhibited relatively high DPPH scavenging activity and superoxide anion scavenging activity.Peptide G6 could protect EA.hy926 cells against oxidative injury induced by H₂O₂.High-dose(500?g/m L)of YH and G6could significantly reduce the MDA levels in H₂O₂ oxidative damaged cell model.High-dose YH and G6 could significantly increase the superoxide dismutase(SOD)level of the H₂O₂oxidative damaged cell model.It can be concluded that the yeast-derived peptides have good antioxidant activity in vivo and in vitro,and may exert a positive effect on lowering blood pressure.(4)Effects of YH and V7 on human umbilical vein endothelial cells(HUVEC).After 48hours of incubation with low(150?g/m L)and medium(300?g/m L)doses of YH and V7,there were no significant changes in HUVEC proliferation.High dose(600?g/m L)of YH exhibited no significant effects on cell proliferation.However,V7 could significantly inhibit cell proliferation.Medium-dose and high-dose YH and V7 were found to promote the NO content in HUVEC significantly.The highest NO content increasement was found in HUVEC treated with high dose YH for 48 h.In addition,all experimental doses of YH and V7 could significantly reduce ET-1 secretion in HUVEC.Among them,the lowest content of ET-1 in HUVEC was observed after culture for 24 hours with high-dose V7,which is 8.96 ng/L.The results show that,YH and V7 could reduce blood pressure through combined mechanisms with multiple targets including regulation NO and ET-1 secretion in endothelial cells.(5)Effects of YH and V7 on the transcriptome of HUVEC.RNA-seq technology was adopted to illustrate the effects of yeast-derived peptides on genes expression and signaling pathways in HUVEC.There were 765 and 577 differentially expressed genes(DEGs)in V7and YH treated HUVEC,respectively.The most significantly up-regulated(more than 15-fold)gene in group V7 was GUCY1A1,encoding the soluble subunit?1 of guanylate cyclase 1,which could bind with NO to promote vasodilation.In YH treated group,oxidative stress related genes SOD2 and HMOX1 were found to be significantly up-regulated,which may explain the antioxidant mechanism of YH to a certain extent.Moreover,DEGs of YH and V7 treated group were mainly enriched in some hypertension-related pathways,such as“JAK-STAT signaling pathway”,“PI3K-AKT signaling pathway”and“c AMP signaling pathway”,revealing that yeast-derived peptides may reduce blood pressure by regulation hypertension-related genes and pathways.