Study On The Mechanism Of Rapeseed Protein-derived Peptides Mediating Glucagon Like Peptide-1 In Regulating Blood Glucose

Type?diabetes mellitus?T2DM?accounts for about 90%of the total number of diabetes patients worldwide,and is recognized as one of the world's three largest non-communicable fatal diseases.Due to the side effects,adverse reactions and drug resistance of drug therapy,its wide application has become increasingly controversial.Studies have shown that food protein and its peptidyl protein hydrolysates play an important role in the prevention and treatment of T2DM.Among them,rapeseed meal is rich in food protein resources,and its biological potency and nutritional value are comparable to soybean protein.It is worth noting that the primary structure of rapeseed protein contains peptide sequence fragments that potentially inhibit DPP-IV enzymes and regulate intestinal endocrine hormones such as GLP-1 and CCK.Reports have shown that such hormones directly controlled the intake and metabolism of blood glucose.However,the structure-activity relationship of food protein derived peptides including rapeseed in the prevention and treatment of T2DM is still unclear.In particular,there are still many doubts regarding the regulation of intestinal endocrine hormones that control human blood sugar.On the other hand,how to improve the bioavailability of rapeseed protein-derived peptides that regulate blood sugar is also the primary problem faced by whether they can be widely used.Therefore,the objective of this study is to evaluate the blood glucose regulation function of rapeseed protein-derived peptides based on the intestinal endocrine hormones;the nano-gelation was used under the covalent action of polysaccharide-type nanoparticles and ion-complementary peptides to improve its bioavailability;in vivo and in vitro experiments to study the molecular mechanism of its synergy.It is urgent that it can be used as a dietary supplement to prevent and relieve T2DM.8 kinds of peptidyl rapeseed protein hydrolysates?RPHs?were prepared by a step-by-step two-enzyme method,and two of them,RNPH-1 and RCPH-3 had the best hypoglycemic effects,which were used to mediate GLP-1 in regulating blood glucose and liver's gluconeogenesis.RNPH-1 and RCPH-3 could regulate liver gluconeogenesis?G-6-Pase and PEPCK?through the insulin metabolism pathway of PI3K-Akt,and alleviate liver fat accumulation and pathological changes in ICR mice with T2DM.Transcriptome high-throughput sequencing results of gastrointestinal tissue in SD rat showed that RNPH-1and RCPH-3 could significantly increase the expression of 16 and 20 intestinal endocrine hormone genes,including Ca SR,GLP-1,and CCK,respectively?p<0.005?.Single gavage experiment and in situ ileal administration experiment found that the RNPH-1 and RCPH-3could significantly improve the glucose tolerance,insulin level,GLP-1 secretion,CCK secretion and DPP-IV inhibition?p<0.05?,the inhibitory activity of RNPH-1 on DPP-IV enzyme in rat ileum tissue was higher than that of RCPH-3.Western-blot and immunofluorescence experiments proved that RNPH-1 and RCPH-3 can mediate GLP-1 to regulate blood glucose levels in rats.The elution fraction G2-R3,which promoted the highest GLP-1 secretion in rat ileum tissue,was separated and purified,and the amino acid residue sequence of the rapeseed protein-derived Ca SR agonist peptide was identified from this fraction.The separation,purification and structure-activity relationship of DPP-IV inhibitory peptides in peptidyl rapeseed protein hydrolysates?RPHs?were studied.The DPP-IV IC50value of RNPH-1 was the lowest measured by in vitro substrate chemistry method,which was0.68±0.09 mg/m L.After further separation and purification of RNPH-1,it was found that the eluted fraction G2-R2 had the highest inhibitory activity of DPP-IV.Ten DPP-IV inhibitory peptides were identified from this fraction,of which 4 oligopeptides?PAGPF,IPQVS,Q?-17.03?KTMPGP and ELHQEPL?had the highest DPP-IV inhibitory activity.For the DPP-IV,both of the PAGPF and ELHQEEPL showed a competitive-non-competitive mixed inhibition,Q?-17.03?KTMPGP showed a non-competitive inhibition,and IPQVS showed a competitive inhibitor.Molecular docking and synchronous fluorescence spectroscopy studies showed that the absolute value of the binding energy between ELHQEEPL and DPP-IV enzyme was the highest?-9.27 k J/mol?;after spatial structure optimization,IPQVS showed more binding sites with DPP-IV than ELHQEEPL,where Glu205,Glu206 and Tyr547 are consistent with the positive control Diprotin A.With the increasing concentration of IPQVS and ELHQEEPL,the fluorescence intensity of DPP-IV gradually decreased and the fluorescence spectrum showed a slight red shift.Based on the Caco-2 cells,the activity and the intestinal absorption mechanism of the DPP-IV inhibitory peptides IPQVS and ELHQEEPL in peptidyl rapeseed protein hydrolysates?RPHs?were studied,and the effects of intestinal absorption and degradation on the DPP-IV inhibitory activity of parent peptide were evaluated.The apparent absorption rate?Papp?values of IPQVS,ELHQEEPL and their degraded peptide fragments were measured.IPQVS is mainly transported across Caco-2 cell monolayers through endocytosis,and ELHQEEPL is mainly transported across Caco-2 cells through paracellular pathway.Both IPQVS and ELHQEEPL inhibited the DPP-IV activity in the Caco-2 cell monolayer in a dose-dependent manner,with IC₅₀values of 101.66±6.02?M,and 112.29±5.27?M,respectively.The values of them were higher than the in vitro chemical substrate method,but the expression of DPP-IV m RNA effected by different concentrations of IPQVS and ELHQEEPL were unchanged?p>0.05?.The results of molecular docking showed that the binding of DPP-IV and degradation products of IPQVS and ELHQEEPL was weaker than that of the parent peptide,and the binding sites were also significantly reduced,which greatly restricted the DPP-IV inhibitory activity of the parent peptide.Nanoparticle CS/ALG-RPHs loaded with peptidyl rapeseed protein hydrolysates?RPHs?were prepared using natural degradable polysaccharide chitosan?CS?as membrane material and sodium alginate?ALG?as wall material.Nanoparticles could effectively avoid the destruction of digestive enzymes in the gastrointestinal tract,meanwhile,achieve the purpose of sustained release of RPHs?RNPH-1 and RCPH-3?.Nano CS/ALG-RPHs particle was prepared using three-channel mixing reactor device,ion cross-linking and electrostatic combination.When the ratio of CS/ALG/RNPH-1 and CS/ALG/RCPH-3 was 1:3:1,the Zeta-potential test showed that the two nanoparticle aqueous solutions were negatively charged,which were-40.27 m V and-39.75 m V,respectively.Under these conditions,the average particle size of CS/ALG-RNPH-1 was 141.8±1.75 nm,the load was 15.38%,and the embedding rate was 90.7%;the average particle size of CS/ALG-RCPH-3 was 220.2±2.11 nm,the load was 16.63%,and the embedding rate was 91.4%.The experimental results of infrared spectroscopy and nuclear magnetic resonance spectroscopy showed that the RNPH-1 and RCPH-3 were successfully loaded into the nanocarriers where CS and ALG were associated by electrostatic interaction.Drug release kinetics experiments showed that both CS/ALG-RNPH-1?n=0.7121?and CS/ALG-RCPH-3?n=0.7685?were unconventional transportation?0.43<n<0.85?.The amount of RNPH-1 or RCPH-3 in the intestinal fluid released from the two kinds of nanoparticles was controlled within 75%.Single gavage experiment in SD rats showed that CS/ALG-RNPH1 and CS/ALG-RCPH3 nanoparticles can improve the body's glucose tolerance and GLP-1 secretion level,and the duration of action was significantly increased?p<0.05?.Based on the covalent coupling between ion-complementary polypeptide RADA16 and functional peptide region,the nano-gelation and synergistic effects of rapeseed derived DPP-IV inhibitory peptides IPQVS and ELHQEEPL were studied.RADA16-GG-IPQVS?RADA16-RAP1?and RADA16-GG-ELHQEEPL?RADA16-RAP2?could form a stable nanohydrogel in a PBS solution or DMEM solution under mild conditions.The best condition of RADA16-RAP1 and RADA16-RAP2 was weak acidity?p H=6.5?and neutral?p H=7.0?,respectively;RADA16-RAP1 and RADA16-RAP2 can self-assemble to form a stable?-sheet structure in an aqueous solution with a certain ionic strength.The mutually exclusive positive and negative charges on the inner and outer ends of RADA16 drove the?-sheet structure to form nano-packed fibers,thereby forming nano-scale hydrogels.Both RADA16-RAP1 and RADA16-RAP2 have good thixotropy,and the G'value at different frequencies is 10 times higher than G'',showing the characteristics of typical soft hydrogels.Compared with RAP1and RAP2,RADA16-RAP1 and RADA16-RAP2 had lower DPP-IV IC₅₀values and longer duration of action,respectively,in addition,RADA16-RAP2 enhanced Ca SR activation at a concentration of 250?M?p<0.05?.