The Mineralization Promotion And Related Mechanism Of Peptides Derived From Eggshell Membrane And Phosvitin

With the increasing degree of ageing society,the incidence of osteoporosis is increasing every year and the disease has become one of the chronic diseases affecting the quality of life of the elderly population.Therefore,it is important to develop on the food-derived active ingredients that are safe and can prevent osteoporosis.Phosvitin Phosphopeptides（PPPs）are hydrolysates of Phosvitin,they have high antioxidant and antibacterial properties as well as metal chelating activity,due to the presence of many phosphorylated serine clusters in their primary structure.Eggshell Membrane Peptides（EMPs）are hydrolysates obtained by enzymatic digestion of eggshell membranes and are rich in collagenous substances.EMPs have been found to have antibacterial and antioxidant activities.Therefore,the functional activity of PPPs and EMPs might have a modulating effect on alleviating the imbalance of bone metabolism.Therefore,in this study,EMPs and PPPs were selected as research objects to analyze the roles of PPPs and EMPs on promoting calcium deposition and improving bone microstructure in vivo.Further studies on the promotion of osteoblast differentiation and mineralization by PPPs and EMPs and their mechanisms of action based on the in vitro MC3T3-E1 osteoblast model were carried out.In addition to low calcium diet as a risk factor,oxidative stress is also one of the important factors causing the development of osteoporosis.Therefore,based on the oxidative stress MC3T3-E1 osteoblast model,the role of PPPs and EMPs in inhibiting oxidative stress-mediated osteoporosis and their mechanisms were investigated.Details of the study are as follows:（1）The role of PPPs and EMPs in improving the absorption and bioavailability of eggshell calcium（ESCa）was investigated based on a low-calcium C57BL/6 mice model.The results revealed that there was no significant difference between the apparent calcium absorption rate and bone calcium content of mice in the ESCa group（36.13%,79.86 mg/g）and the Ca CO₃ group（34.34%,73.29 mg/g）,while the intake of ESCa+PPPs and ESCa+PPPs+EMPs was able to significantly increase the apparent calcium absorption rate（52.78%,55.01%）and bone calcium content（85.81 mg/g,88.32 mg/g）.Compared with the ESCa group,the intake of ESCa+PPPs and ESCa+PPPs+EMPs significantly increased femoral bone mineral density from 0.13 g/cm³ to 0.14g/cm³,0.15g/cm³,respectively.The three-dimensional reconstructed images of mice femur showed that the ESCa+PPPs+EMPs group had a more complete,compact and thicker trabecular structure than the low calcium model group,calcium carbonate and ESCa group.These results suggested that both PPPs and EMPs could promote the absorption of calcium ions from ESCa,improve the destruction of bone microstructure caused by calcium deficiency and promote calcium ions deposition.（2）The MC3T3-E1 osteoblast model was constructed to analyze the role of PPPs and EMPs in promoting osteoblast osteogenic function and their mechanisms of action.We found that PPPs and EMPs might significantly increase cell proliferative activity.Compared to the control group,the cell activity increased by 1.92 times and 1.86 times after 72 h at 0.05 mg/m L PPPs and EMPs.Then,PPPs,EMPs increased the enzymatic activity of ALP in a concentration-dependent manner.In addition,PPPs and EMPs at 0.05mg/m L could promote the formation of mineralized nodules in osteoblasts.However,the two peptides did not show significant differential activity or significant synergistic effects.Finally,Real-time PCR analysis revealed that compared with control group,PPPs could upregulate the expression of Wnt3a,Trp5 andβ-catenin by 2.39 folds,2.38 folds and 3.66folds,respectively.Therefore,PPPs were able to activate the Wnt classic pathway and thus improve osteoblastic mineralization activity,however,the influence of EMPs and PPPs+EMPs on this pathway was not statistically significant.（3）In addition to calcium intake factors,oxidative stress is also a risk factor for inducing imbalance in bone remodelling and causing bone loss.Therefore,in this chapter,the antioxidant activity of PPPs and EMPs were evaluated,and the oxidative-stress osteoblast model was constructed using H₂O₂.Firstly,the metal ions-removing PPPs（PPPs-）was preparated after treatment with Na₂EDTA buffer solution.Then,the antioxidant activity of PPPs and PPPs^-was measured by total antioxidant capacity and radical scavenging capacity of ABTs,and the results showed that PPPs^-had higher antioxidant activity than PPPs,the total antioxidant activity of 10 mg/m L PPPs^-（193.95μg/m L）was higher than PPPs（163.18μg/m L）.The in vitro antioxidant activity of PPPs^-,EMPs and PPPs^-+EMPs were determined.It was found that EMPs had higher antioxidant activity at the same concentration（P<0.05）,and the two peptides did not show synergistic effects.Subsequently,H₂O₂ concentrations were selected for the induction of oxidative stress in MC3T3-E1 osteoblasts.The CCK-8 assay test,cell morphology and intracellular ROS content were measured to determine the concentration of H₂O₂ at 400μmol/L.It was found that after 24 h of 400μmol/L H₂O₂ treatment,the cell viability decreased from 100%to59%,and a slight change in cell morphology compared to the normal group,with a small number of cells starting to undergo disintegration and apoptosis.In addition,the determination of ROS content also revealed that 400μmol/L H₂O₂ treatment significantly increased the fluorescence intensity of the cells（P<0.05）.Therefore,this concentration of H₂O₂ was able to induce the formation of oxidatively stressed osteoblasts and could be used for subsequent studies.（4）Based on the oxidative stress MC3T3-E1 osteoblast model,the effects of PPPs^-and EMPs on inhibiting oxidative stress-induced osteoporosis and their mechanisms were studied.It was found that PPPs^-and EMPs could effectively interfere with cell activity reduction and apoptosis caused by H₂O₂ in a concentration-dependent manner.After 72 h of treatment with 0.05 mg/m L of PPPs^-and EMPs,the activity of cells increased from 56%（H₂O₂ group）to 95.05%and 102.61%.DCFH-DA fluorescence assay showed that PPPs^-and EMPs could significantly reduce ROS levels in cells.The ALP activity assay found that H₂O₂ reduced the differentiation ability of osteoblasts,decreasing ALP activity from 3.39Kim’unit/100 m L（Control group）in to 2.36 Kim’unit/100 m L（H₂O₂ group）,but PPPs^-and EMPs were able to significantly increase ALP activity.Alizarin red staining tests revealed that H₂O₂ inhibited the mineralizing activity of osteoblasts,while PPPs^-and EMPs were able to alleviate this inhibition and increase the number and area of mineralized nodules after 21 d of action.Real-time PCR assays showed that H₂O₂ significantly down-regulated the Wnt pathway,but up-regulated the Fox O signaling pathway.In contrast,0.05 mg/m L of PPPs^-,EMPs significantly down-regulated the expression of Fox O signaling pathway-related factors but promoted levels of the Wnt/β-catenin-related factors OPG and Runx-2,indicating that PPPs^-,EMPs alleviated the decrease in osteoblast activity and function mediated by oxidative stress through the Fox O/Wnt-related pathway.Furthermore,by analyzing the activity of PPPs^-,EMPs and PPPs^-+EMPs at the same concentration,it was found that the difference in activity or synergistic effect of the two peptides was not statistically significant.