Effects And Mechanisms Of Duck Egg White Peptides On The Improvement Of Calcium Bioavailability Via TRPV 6 And Structure-activity Relationship

Administration of safe and efficient calcium supplements is a crucial method to reverse the national calcium deficient status.The usually abandoned salted duck egg white is economical source of peptides.The desalted duck egg white peptides(DPs)with high bioactivity of promoting calcium uptake has been found in our previous studies.However,the exact mechanism is not clear.The present study will utilize Caco-2 cell model,everted gut sac model and animal models to explore the interactions between DPs and TRPV6 calcium channel via molecular biology methods,molecular docking technology,spectroscopy,etc.Additionally,several pivotal peptides from DPs will be identified by HPLC-ESI/MS/MS and synthesized.The structure-activity relationship will be studied in terms of the role of repeating amino acids,such as Glu-Glu and Ser-Ser.In a word,the aim of this study is to clarify the effects and mechanisms of DPs on the promotion of calcium bioavailability in vitro and in vivo.The main results are as follows:1 Effects and mechanisms of DPs on the promotion of calcium uptake in vitroThe calcium transport in Caco-2 monolayer model and everted gut sac model increased significantly(P<0.05)after the treatments of 4mg/mL and 8mg/mL DPs.There is no significant difference between 4mg/mL and 8mg/mL groups,which indicates that the influence of DPs on the transport process was saturable.The optimal Ca2+/DPs ratio for the enhancement of Ca2+uptake was observed to be 4 mM Ca2+/4 mg/mL DPs in Caco-2 population model,indicating the importance of a suitable DPs/Ca2+ ratio for promoting calcium absorption.In the presence of oxalic acid(OA),phosphoric acid(PA)and Zn2+,the calcium transport decreased about 39.07%,47.54%and 9.56%,respectively,suggesting that in the Caco-2 cell monolayers,these dietary factors decrease the calcium bioavailability,and DPs could inhabit this negative effect.The promoting effect of calcium uptake by DPs could be inhibited by 2-aminoethoxydiphenyl borate(2-APB)which is a TRPV6 calcium channel inhibitor.The expressions of TRPV6,CalbindinD9k,and PMCAlb increased significantly(P<0.05)by 0.4mg/mL DPs treatment.The corresponding mechanism is the interaction between DPs and TRPV6,and the expressions of TRPV6,CalbindinD9k,and PMCA1b are regulated by DPs to improve calcium uptake.2 Structure characterization of DPs and stability of DPs/Ca complexThe carboxyl oxygen is the interaction site between calcium and DPs from the infrared spectroscopy.Structural folding between DPs and calcium ions,the aggregation of amino acids or oligopeptides during the chelation process were observed via the fluorescence spectra and size distribution.The most abundant amino acids are Glu+Gln and Asp+Asn,accounting for 12.14%and 7.14%,respectively.The contents of Ser,Leu,Phe,Thr and Met are each also>5%.The amino acid sequences of the seven peaks were identified as Val-Ser-Glu-Glu(VSEE),Leu-Tyr-Ala-Glu-Glu(LYAEE),Ile-Asn-Glu(INE),Leu-Ser-Tyr-Leu(LSYL),Leu-Asn-Ser-Trp(LNSW),Val-His-Gln(VHQ),and Ile-Leu-Lys-Asn(ILKN).Three amino acid sequences are synthesized and evaluated in Caco-2 monolayer models.The calcium binding capacity and the effects on the promotion of calcium uptake is ordered as follows:VHS(p)S(p)>VSEE>VHSS.VSEE functions in the promotion of calcium uptake via the interaction of TRPV6 calcium channel.The study of stability of DPs/Ca complex illustrates that DPs/Ca complex is sable for temperature(80℃)and pH.After the treatments with pepsin and trypsin,the calcium content in DPs/Ca complex is still over 50%.3 Effects of DPs on the promotion of calcium bioavailability in phytic acid-induced calcium deficiency mice model and calcium deficient diets rat model.DPs promote calcium uptake by counteracting the adverse effects of phytic acid.The accumulation calcium rate increased after the supplement with DPs.The proliferation and differentiation of intestinal enterocytes is affected by DPs and the cecal wall weights increased.The duodenum gene expression of calbindinD9k is regulated by DPs.DPs could promote calcium uptake in rats treated with calcium deficient diet or normal diet.The serum and bone parameters improved after the treatment with DPs.The TRPV6 calcium channel is regulated by DPs and VSEE in model group.The function of the promoting calcium uptake by VSEE is played by the increase of calbincinD9k expression.Additionally,the protein expression of calbincinD9k and PMCA1b increased by DPs in the normal calcium diets group.4 Effects of DPs on the promotion of calcium bioavailability in retinoic acid-induced bone loss rat model and intra-amniotic administration model.The intestinal calcium absorption is enhanced by DPs and the serum,bone parameters are improved to the normal levels significantly(P<0.05)compared to the model group in retinoic acid-induce bone loss model.Meanwhile,the osteoblast activity increased while the osteoclast activity decreased after the treatment with DPs.Intra-amniotic administration of DPs and VSEE could improve the intestinal calcium uptake and increase the bone formation activity.The beneficial bacteria increased by chickpea prebiotics,lentil prebiotics and VSEE+Ca.There are no effects observed in DPs and VSEE groups.The supplement of CaCO3 alone could destroy the intestinal environment and increase the pathogenic bacteria.Combined supplement with Ca2-lentil,Ca2++DPs、Ca2++VSEE could stabilize the bacterial population.Additionally,the intestinal viilus surface area and diameters of goblet cells increased after the treatments with DPs or VSEE,which was also observed in prebiotic groups.5 Structure-activity relationships of DPsIn order to explore the structure-activity relationships of DPs,27 peptides are synthesized based on the VSEE、VHSS 和 VHS(p)S(p).In terms of VSEE,the first acidic amino acid in C-terminal area is essential for calcium uptake.The repeating acidic amino acids like Glu-Glu are very important in the promotion of calcium uptake.Ser located in the middle sequence should be phosphorylated.In terms of VHSS,the repeating Ser-Ser sequence contributes a lot to the calcium absorption while Ser locates in the C-terminal area.In addition,the phosphorylated Ser could enhance the effects.The 3D structure of TRPV6 is modeled via the homologous modeling.Two possible active sites of TRPV6 are provided in the promotion of calcium uptake by DPs.Site 1 was in the loop section between the fifth transmembrane and sixth transmembrane domain.Site 2 was in the cavity between the first,second transmembrane and the third,forth transmembrane domain.The molecular docking results indicate that three ligands(VSEE,VHSS and VHS(p)S(p))combined with TRPV6 through hydrogen bond and van der waals forces.The combining capacity was ordered as follows:VHS(p)S(p)>VSEE>VHSS.Apart from the binding sites with TRPV6,the exposure of VSEE,VHSS and VHS(p)S(p)could provide other binding sites with calcium.The peptides might play the role as ’bridge’ between TRPV6 and calcium and promote calcium uptake.