Regulation Mechanisms Of Phosvitin Peptides On Osteoblast And Osteoclast Differentiation

Normal metabolism of bone tissue is essential for maintaining normal bone mass,which is mediated by the balance between osteoblast bone formation and osteoclast bone resorption.Phosvitin and its hydrolysate have been proved to have the potential benefit in bone therapy by chelating metal ions and promoting mineralization,but the regulatory mechanism remains unclear.In this paper,cellular experiments were carried out to investigate the regulation mechanism using the nonapeptide DEEENDQVK(named DK),which was selected from phosvitin and had a high calcium binding capacity,and the pentapeptide EDD-pSer-pSer(named ES),which was selected according to the protein sequence and phosphorylated.The research contents and experimental results are as follows:(1)The two peptides(DK and ES)were applied to MC3T3-E1 cells,respectively.MTT assay,ALP activity analysis,and alizarine red S staining were used to detect the effects of peptides on the proliferation,differentiation and mineralization of osteoblasts,and transcriptomic analysis was applied to investigate the mechanism of these two peptides regulated osteoblast differentiation.The results showed that the two peptides had no significant effect on the proliferation of MC3T3-E1 cells.When the concentration of DK was higher than 100 μg/mL,DK could promote the ALP activity and mineralization of MC3T3-E1 cells in a dose-dependent manner.While ES inhibited the ALP activity and mineralization of MC3T3-E1 cells when ES was 50 μg/mL,but promoted when higher than200 μg/mL without significant difference between groups.Transcriptomic analysis revealed that DK significantly affected the genes associated with the function of promoting ossification,bone development,skeletal system development,biomineralization and regulating cell differentiation through regulation of ribosome and metabolic pathways.ES achieved this function mainly through phosphatidylinositol signaling system,inositol phosphate metabolism,adherens junction,cell cycle and Hedgehog signaling pathway.(2)The two peptides were then applied to RAW264.7 cells,respectively.MTT assay,TRAP staining,TRAP activity assays,and RT-qPCR were used to examine the effects on the proliferation,differentiation and relative mRNA expression of related factors.The results showed that both the two peptides had no significant effect on the proliferation of RAW264.7 cells.TRAP activity in RAW264.7 cells was effectively inhibited by DK at lower concentrations,with the best effect at 200-300 μg/mL.While ES showed significant inhibition at 200-500 μg/mL,with no difference in effect between groups.The relative mRNA expression of CTR,CTSK,MMP-9,TRAP and TRAF6 were significantly reduced in osteoclast cells at the corresponding concentrations.(3)A co-culture system was established,and osteoclast cells were identified.RT-qPCR was used to detect the relative mRNA expressions of the relevant factors in osteoblast and osteoclast.The results showed that these two peptides significantly upregulated the relative mRNA expression of ALP,BMP2,COL I,Runx2 and OPG in osteoblast cells,and inhibited the differentiation of osteoclast by regulating the MAPK signaling pathway in an inhibitor-like manner to suppress the expression of related genes in osteoclast.In conclusion,DK and ES can promote biomineralization and have the potential to be new functional foods such as calcium supplements,at the same time,this study provides a new direction for the study of the mechanism of other peptides and proteins with similar structures.