| Osteoporosis is a congenital or secondary metabolic disorder.More than 200 million women worldwide suffer from osteoporosis,which increases the risk of fracture due to increased bone fragility.The essential cause of osteoporosis is that the rate of osteoblast bone fomation is lower than that of osteoclast bone resorption.Studies have shown that bioactive peptides can not only be used as therapeutic drugs for osteoporosis,but also as a functional food to promote bone synthesis.Oyster active peptide is an important component of marine natural products.The preparation,purification and biological activities of oyster peptide have been widely studied.What' more,the shell formation mechanism of oyster is similar to the biological signaling pathway of bone formation mechanism in mammals to some extent.However,studies on the role of functional foods related to oyster peptide in promoting bone formation are relatively lacking.Therefore,the purpose of this study was to screen out the bioactive peptide with osteogenesis from oyster protein and clarify its regulatory mechanism.In this study,the hydrolysates and peptides of Crassostrea gigas were obtained from simulating gastrointestinal digestion of Crassostrea gigas protein.The digestion of Crassostrea gigas was determined by hydrolysis degree,molecular exclusion and SDS-PAGE.Using UPLC-Q-TOF and CESI-Q-TOF to identify the peptide sequence of Crassostrea gigas,a total of 1,908 peptide sequences were identified,which increased the number of peptide sequences.In order to screen bioactive peptide,the influence on osteoblast proliferation activity of oyster hydrolysate and 3000 Da as the boundaries of peptide component in simulated intestinal digestion 1-4 h(SID 1-4 h),and its impact on osteoblast secretion of alkaline phosphatase activity were determined.Finally,the results showed that the proliferation rate of the hydrolysates in SID 3 h on osteoblasts was up to 168%.The peptides less than 3000 Da,which were derived from SID 3 h,had pro-osteogenic activity,and promoted the proliferation rate of osteoblasts up to 135%.Then,molecular docking was used to predict the four peptides from the 1,908 peptides with the strongest binding ability to the receptor associated with proliferation of osteoblasts.After chemical synthesis,it was finally confirmed that peptide P-CG-01 had the strongest effect on the proliferation of MC3T3-E1 before osteoblasts.P-CG-01 at 100 nM significantly promoted the proliferation of MC3T3-E1 cells and increased alkaline phosphatase activity.In animal experiments,ovariectomized mouse model was used to investigate P-CG-01 with 3,10,30 mg/kg·BW concentration after gavage intervention(OVX+P3,OVX+P10,OVX+P30 group).Micro-CT scan was performed on mice two months later,and it was found that BMD value of trabecular bone in OVX+P30 mice recovered to the level of Sham group.Moreover,the BV/TV ratio,trabecular separation and the trabecular number in OVX+P30 group have all recovered to the level of Sham group.The serum activity of ALP in OVX+P30 group was higher than OVX,but TRAP was lower.In addition,the ratio of RANKL/OPG content in serum was also determined.The OVX+P30 group was significantly lower than OVX.The above results indicated that peptide P-CG-01 had a proliferation effect on osteoblasts and a certain effect on promoting bone formation in ovariectomized osteoporosis mice.Transwell cell transport model and mouse absorption experiments were used to study the absorption and transport of peptide P-CG-01,which revealed that the peptide could be absorbed into the blood circulation through intestinal epithelial cells and could still be identified in the blood after 4 h of gavage.After 500 ?g/mL of peptide was added to the apical chamber(AP)covered with Caco-2 cells on the Transwell plate,culture media on the AP and basolateral chamber(BL)were taken at 15,30,60 and 120 min,respectively.With culture time to 120 min,the peptide concentration on AP decreased to 193.19±11.47 ?g/mL,while that on BL increased to 6.77±0.47 ?g/mL.Peptide was intragastric intragastric at 30 mg/kg·BW concentration in the blood absorption test of mice.The peptide was identified in serum after 10 min and peaked immediately at 2.3610.71?g/mL.The peptide could still be identified in the blood circulation of mice for 4 h,and could not be identified completely at 12 h.Fluorescent isothiocyanate(FITC)labeled peptides were added to plate for culture.The localization analysis of osteoblasts treated with peptide was conducted by laser confocal microscopy.It was proved that the peptide was abundant in the cytoplasm of osteoblasts,indicating that the peptide P-CG-01 could cross the osteoblastic cell membrane and enter into the cell.Finally,the molecular simulation algorithm CDOCKER,Gold and LibDock were optimized to predict the binding of peptide P-CG-01 with integrin alpha 5 beta 1(PDB ID:3VI4)and parathyroid hormone receptor(PTHrP),respectively.Then the expression of ERK1/2,p38,ALP,RUNX2,BMP2,OCN,OPN and other cell proliferation or differentiation related genes were quantitatively analyzed by reverse transcriptase polymerase chain reaction(RTQPCR).Compared with the control group,the expression of BMP2 increased 3.8 times.ERK2 gene was regulated by about 1.4 times,p38 expression was negatively regulated by 0.5 times,ALP and RUNX2 genes were regulated by about 2 times,BMP2 gene expression was increased by 3.5 times,OCN and OPN were positively regulated by about 2 times and 1.8 times respectively.In addition,Western blotting was used to analyze the protein levels of ERK1/2,JNK1/2,BMP2 and RUNX2.BMP2 and RUNX2 also showed a significant trend of increased expression after 100 nM peptide treatment.This indicated that peptide P-CG-01 mainly enhanced the expression of ERK and JNK in the MAPK pathway,so that cells entered into the cycle of proliferation and differentiation,and further promoted the improvement of bone morphology by increasing the expression of BMP2.Therefore,P-CG-01 can promote the osteogenic activity of osteoblasts and inhibit the occurrence and development of osteoporosis after ovarian loss in mice. |
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