Identification and validation of fermented milk-derived osteogenic peptides via molecular docking and osteoblastic cell model

Fermented milk has abundant bioactive peptides, and there is a growing interest in seeking osteogenic peptides of dairy origin. This study employed computer-assisted screening to identify potential osteogenic peptides from fermented milk by Lactobacillus helveticus and investigated the effects of these osteogenic peptides on the proliferation, differentiation, and mineralization of MC3T3-E1 cells, along with their underlying mechanisms. Three osteogenic peptides were identified: AF10 (ARHPHPHLSF), AY17 (AAGGPGAPADPGRPTGY), and NE19 (NIPPLTQTPVVVPPFLQPE). Treatment with 0.2 μM of AF10, AY17, and NE19 promoted the proliferation of MC3T3-E1 cells by increasing the proportion of cells in the S phase and G2/M phase. Further molecular docking revealed significant differences in the interaction of the three osteogenic peptides with the critical domain (Asn12-Lys13-Leu14-Thr15-Gln16-Leu17-Gly18) of the epidermal growth factor receptor (EGFR). The three peptides exhibited different degrees of promotion on the differentiation and mineralization of MC3T3-E1 cells, as well as significant differences in the expression of genes and proteins related to ERK1/2, RUNX2, and BMP2. The AF10 peptide was selected for further validation of the effect of the mitogen-activated protein kinase (MAPK) signaling pathway on the proliferation and expression of differentiation and mineralization-associated genes in MC3T3-E1 cells by combining MAPK pathway inhibitors. Molecular dynamics simulation revealed the stability of AF10 when binding to EGFR. These findings provide valuable insights into the screening of osteogenic peptides of various origins and the development of functional dairy products accordingly.