| This work was funded by the National Natural Science Foundation of China?NO.31271907,31771985?.Food-derived bioactive peptides derived from food proteins by enzymatic hydrolysis have generated a lot of attentions for their potential human health-promoting functions and limited adverse effects.However,it has been found that the in vitro bioactivities of peptides do not always correlate with in vivo activity due to their low absorption and bioavailability in vivo.In fact,different from endogenous bioactive peptides,many food-derived bioactive peptides,for example,angiotensin I-converting enzyme?ACE?-inhibitory peptides,have to overcome two important physiological barriers,extensive enzymatic degradation in the gastrointestinal tract and poor permeability through the intestinal epithelium,and be absorbed in an intact form into the circulation to exert their bioactive functions in vivo after oral administration.This implies the importance of resistance to various peptidases,including brush border membrane peptidases,and absorption through the intestine for the bioavailability of food-derived bioactive peptides.Since up to now,the most of the identified bioactive peptides derived from food proteins are oligopeptides containing 3 to 10 amino acid residues.Unfortunately,the transport mechanism through the intestinal epithelium of oligopeptides is still obscure.In this study,the hydrolysis and transepithelial transport of egg white-derived peptides were determined using Caco-2 cell monolayers and everted rat sacs.The effects of FITC modification and amino acid replacement at N-or C-terminus on the transport of oligopeptides in Caco-2 cell monolayers were investigated.In addition,the relationships between the molecular properties of hydrophobicity?log D?and molecular weight?MW?of oligopeptides and transport across Caco-2 cell monolayers were analyzed to discuss their transport mechanisms.The main research work and results were summarized as follows:?1?The hydrolysis and transepithelial transport of egg white hydrolysates after simulated gastrointestinal digestion were investigated using Caco-2 cell monolayers and everted rat sacs.The peptide sequences were identified with Q Exactive mass spectroscopy.Results showed that the degradation of peptides in everted rat sacs was higher than that in Caco-2 cell monolayers.There were 56 and 28 egg white-derived peptides with MW less than 1000 Da could transport through Caco-2 cell monolayers and everted rat sacs in intact forms,respectively,suggesting that the absorption of egg white-derived peptides in Caco-2 cell monolayers was significantly higher than that in everted rat sacs.In addition,the peptides of LGAKDSTRT,VNDLQGKTS,DGSRQPVDN and GKKDPVLKD could be absorbed in intact forms in not only Caco-2 cell monolayers but also everted rat sacs.?2?The hydrolysis and transepithelial transport of egg white-derived ACE-inhibitory peptide TNGIIR in simulated gastrointestinal digestion and Caco-2cell monolayers were determined.Results showed that the degradations of TNGIIR by simulated gastrointestinal enzymes and brush border membrane peptidases were5.87%±1.92%and 17.17%±0.64%,respectively.The apparent permeability coefficients(Papp)of TNGIIR from the apical to basolateral side and from basolateral to apical side in Caco-2 cell monolayers were?4.92±0.40?×10-6 cm/s and?5.99±0.27?×10-6 cm/s,respectively.It suggested that TNGIIR can transport across Caco-2 cell monolayers in intact form,but its efflux was higher than absoption.In addition,only cytochalasin D,a disruptor of tight junctions?TJs?,changed TNGIIR transport permeability significantly,suggesting that the main transport route for TNGIIR across Caco-2 cell monolayers might be TJs-mediated paracellular pathway.?3?The effects of FITC modification at N-or C-terminus on the transport of RVPSL,RVPSP and VPP in Caco-2 cell monolayers were determined.Results showed that the Papp values were?0.88±0.11?×10-6 cm/s and?1.43±0.15?×10-6 cm/s for FITC-RVPSL and RVPSLK-FITC,?0.66±0.10?×10-6 cm/s and?2.37±0.01?×10-6 cm/s for FITC-RVPSP and RVPSPK-FITC,?4.68±0.08?×10-6 cm/s and?3.25±0.08?×10-6cm/s for FITC-VPP and VPPK-FITC,respectively.It suggested that the FITC modification at C-terminus of RVPSL and RVPSP had higher transport permeability but lower degradation in Caco-2 cell monolayers than that of N-terminus.However,a contrary result was observed for VPP.It was found that wortmannin significantly decreased the transport of FITC-RVPSL,RVPSLK-FITC,FITC-RVPSP and RVPSPK-FITC,but not of FIT-VPP or VPPK-FITC.In addition,cytochalasin D significantly increased the transport of FITC-RVPSL,FITC-RVPSP,FITC-VPP and VPPK-FITC,but not of RVPSLK-FITC or RVPSPK-FITC.All those indicated that both of paracellular pathway and transcytosis were involved in the transepithelial transport of FITC-RVPSL and FITC-RVPSP,while only transcytosis was involved in the transepithelial transport of RVPSLK-FITC and RVPSPK-FITC.The main transport route for FITC-VPP and VPPK-FITC might be paracellular pathway.Moreover,no significant relationship between the secondary structure and transport of oligopeptides across Caco-2 cell monolayers was observed.?4?The effects of terminal amino acids on the transport of oligopeptides across the Caco-2 cell monolayer were determined.Ala-based tetra-and pentapeptides were designed,and the N-or C-terminal amino acid residues were replaced by different amino acids.Results showed that the oligopeptides had a wide range of transport permeability across the Caco-2 cell monolayer and could be divided into four categories:non-/poor permeability,low permeability,intermediate permeability,and good permeability.Tetrapeptides with N-terminal Leu,Pro,Ile,Cys,Met,and Val or C-terminal Val showed the highest permeability,with Papp values over 10×10-6 cm/s.Pentapeptides with N-or C-terminal Tyr also showed high permeability levels,with Papp values about 10×10-6 cm/s.The amino acid Glu,Asn,and Thr at N-terminus or Lys,Asp,and Arg at C-terminus were also beneficial for the transport of tetra-and pentapeptides,with Papp values ranging from 1×10-6 to 10×10-6 cm/s.In addition,peptides with different N-terminal amino acids generally showed higher permeability than those with amino acids replaced at C-terminus,suggesting that N-terminal amino acids were more important for the transport of oligopeptides across Caco-2 cell monolayer.?5?The relationships between molecular properties of log D and MW of oligopeptides with different N-terminal amino acids and their transport permeability across Caco-2 cell monolayers were analyzed.Results showed that,according to the correlations of Papp values with log D or MW,the oligopeptides could be divided into three categories:good permeability,intermediate permeability,and low permeability with Papp values ranging from 5×10-6 cm/s to 40×10-6 cm/s,0.25×10-6 cm/s to 5×10-6cm/s,and 0 to 0.25×10-6 cm/s,respectively.At a good level of permeability,the permeability was positively correlated with log D,but negatively correlated with MW;at an intermediate level of permeability,the permeability was negatively correlated with log D and MW;and at a low level of permeability,the permeability was positively correlated with log D and MW.The above results may indicate that there are two or three routes for the transepithelial transport of oligopeptides across Caco-2cell monolayers.One of them is TJs-mediated paracellular pathway.Another is oligopeptide transporter-mediated pathway or transcytosis,or both of oligopeptide transporter and transcytosis were involved at the same time. |
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