Abstract
The tripeptide Leu-Pro-Lys (LPK), derived from the Sipunculus nudus protein, was synthesized and studied to investigate its potential protective effect on bone formation. The effect and mechanism of LPK were analyzed through network pharmacology, bioinformatics, and experimental pharmacology. The study found that LPK at concentrations of 25 μg/mL and 50 μg/mL significantly increased ALP activity and mineralization in C3H10 cells. LPK also increased the expression of COL1A1 and promoted bone formation in zebrafish larvae. Network pharmacology predicted 148 interaction targets between LPK and bone development, and analysis of the protein-protein interaction network identified 13 hub genes, including ESR1, MAPK8, and EGFR, involved in bone development. Through KEGG enrichment pathways analysis, it was determined that LPK promotes bone development by regulating endocrine resistance, the relaxin signaling pathway, and the estrogen signaling pathway. Molecular docking results showed direct interactions between LPK and ESR1, MAPK8, and MAPK14. Additional verification experiments using western blot assay revealed that LPK significantly upregulated the expression of genes related to bone formation, including COL1A1, OPG, RUNX2, ESR1, phosphorylated MAPK14, and phosphorylated MAPK8 in C3H10 cells. These results suggest that LPK promotes bone formation by activating the estrogen/MAPK signaling pathway.