Effects And Mechanisms Of Duck Egg White Peptides On Calcium Absorption And Bone Formation Via CaSR And Wnt/?-catenin Signaling Pathway

Calcium deficiency is a common issue in the world,and osteoporosis has become an important health concern with the increasingly serious aging problem.In our previous study,desalted duck egg white peptides?DPs?and Val-Ser-Glu-Glu?VSEE?have been proved to facilitate calcium absorption through activating TRPV6 calcium channel in vitro and in vivo.However,whether Ca SR is involved in the calcium-promoting effects of DPs,whether DPs and VSEE could directly affect bone formation metabolism,remain unclear.The present study will utilize DEAE-52 column,HPLC-ESI/MS/MS,computer molecular docking simulation,and in situ single-pass intestinal perfusion?SPIP?study to explore whether Ca SR is involved in the calcium-promoting effects of DPs,as well as to comprehensively understand the mechanism of DPs on the promotion of calcium absorption.Additionally,MC3T3-E1 cells and ovariectomized?OVX?rat models are used to evaluate the effect of DPs and VSEE on bone formation.Importantly,this study will use mimic gastrointestinal digestion in vitro,Caco-2/HT-29 cells co-culture model and pharmacokinetic study to investigate the stability,transport and absorption and related mechanisms,and pharmacokinetic parameters of VSEE,which will contribute to the application of VSEE.The main results are as follows:1.Purification and characterization of positive allosteric regulatory peptides of Ca SR from DPsDPs was preliminarily purified by DEAE-52 column.The fraction?F3?with highest calcium binding capacity was thereby chosen and analyzed by HPLC-ESI-MS/MS.Forty-nine peptides including 16 peptides enriched aromatic amino acids were identified.Then molecular docking simulation was used to predict the peptides with high binding affinities with Ca SR.FAE,FNE,INSW,FDPE,MKVY and NFE were selected for further study based on the total scores.SPIP study indicated that NFE and INSW significantly reduced the increased PTH levels by 45.80%and 48.81%,respectively?P<0.05?,and increased calcium percent absorption,calcium absorption rate constant?Ka?and calcium effective permeability?Peff??P<0.05?,as well as up-regulated m RNA levels of Ca SR?P<0.05?.Moreover,NFE and INSW could interact with the VFT domain of Ca SR via hydrogen bonds and hydrophobic interactions,which exhibited the potential activities in the regulation of Ca SR.2.Proliferation,differentiation and matrix mineralization of MC3T3-E1 cells by DPs and VSEEThe proliferation and the proportion of the cells in S phase of MC3T3-E1 cells inDPs?0.8-10mg/m L?and VSEE?0.2-2m M?with/without Ca Cl₂ groups were significantly increased for 5 and 7 days?P<0.05?.Alkaline phosphatase?ALP?activities in all treated groups except for Ca Cl₂ group were significantly elevated compared with thecontrol group for 7 or 14 days?P<0.05?,and the effects of DPs or VSEE with Ca Cl₂ were superior than DPs or VSEE alone?P<0.05?.Compared with the control group,the mineralization level in all treated groups except for Ca Cl₂ group was increased.Additionally,DPs and VSEE treatments with/without Ca Cl₂ significantly upregulated the expressions of Wnt3a,LRP5,-catenin?P<0.05?,as well as the expressions of downstream proteins including runt-related transcription factor 2?Runx2?and osteoprotegerin?OPG??P<0.05?.Moreover,DPs could partially induce the calcium influx via the activation of L-type calcium channels in the presence of 2m M extracellular calcium.VSEE is mainly through the release of endoplasmic reticulum calcium stores and activation of L-type calcium channels to induce calcium influx;While the the ability of VSEE inducing the calcium influx is better than that of DPs.The structure–activity relationship study indicated that the repeating Glu-Glu sequences exerted a critical role in the differentiation and matrix mineralization of MC3T3-E1 cells.The effect of Valine in N-terminal area of VSEE on MC3T3-E1 cells was better than Alanine in N-terminal area.Additionally,VS?P?EE could contribute a lot to the differentiation and mineralization of MC3T3-E1 cells,while the ration of calcium/phosphorus should be taken into consideration before the addition of calcium and VS?P?EE.3.Absorption and metabolism of VSEE in vitro and vivoThe results of stability test in vitro showed that the content of VSEE remained above95%after the treatment of VSEE for 1 hour at 37-100?or p H 4-10,indicating that VSEE had a high tolerance to temperature,acid and alkali.After treatment with artificial gastric juice and artificial intestinal juice for 2 hours,the content of VSEE was above80%,indicating that VSEE also had a certain tolerance to digestive enzymes.The optimal transport conditions of VSEE in Caco-2/HT-29 cells co-cultured model were observed to be 2h?transport time?and 0.05m M?concentration?,which lead to the transport rates of 16.51%.Pharmacokinetic study demonstrated that the plasma concentration–time curve of VSEE fitted two-compartment model in rats.VSEE had a low apparent volume?1.17±0.15 L/kg?,while had a long residence time?76.05 min?and half-life period?57.0±0.42 min?.These pharmacokinetic characteristics of VSEE reflected the poor lipid solubility and good stability in vivo of VSEE.4.Effect of DPs and VSEE on the osteoporosis and abnormal lipid metabolism in OVX rats and related mechanismThe serum levels of ALP and N-terminal propeptide of type I procollagen?PINP??cross-linked C-terminal telopeptide of type I collagen??-CTX?,markers for bone turnover,were significantly reduced by DPs and VSEE in OVX model?P<0.05?.DPs and VSEE could also improve the biomechanical features of femurs and tibias,and reduce the bone microstructure destruction.Simultaneously,the expressions of Wnt3a,LRP5 and-catenin in the femurs and tibias,occludin and claudin-3 in the gut were both significantly upregulated by DPs and VSEE?P<0.05?.Species composition analysis presented that the composition and proportion of intestinal microbiota were also improved by DPs and VSEE.Additionally,DPs and VSEE could significantly decrease the serum total cholesterol?TC?,triglyceride?TG?,low density lipoprotein cholesterin?LDL-C?contents?P<0.05?,and increase high density lipoprotein cholesterin?HDL-C?.Moreover,the adipocyte and lipid droplets in DPs and VSEE groups were markedly diminished compared with the model group.Overall,DPs and VSEE could improve the bone loss and abnormal lipid metabolism via the improvement of intestine barrier,gut microbiota composition and activation of Wnt/?-catenin signaling pathway.