Purification and identification of novel osteogenic peptides from sea cucumber intestine hydrolysates and their pro-osteogenesis effects on MC3T3-E1 cells

The present study aimed to purify and identify osteogenic peptides from sea cucumber intestine hydrolysates (Mw<3kDa) and further evaluate their pro-osteogenesis properties on MC3T3-E1 cells. Two novel osteogenic peptides, RPQYPQYPS and LSFSPY, were purified and identified from the alcalase hydrolysate of sea cucumber intestine by gel filtration chromatography, ion exchange column chromatography, RP-HPLC, which were further sequenced by LC-MS/MS and synthesized with solid-phase synthesis method. Peptides RPQYPQYPS and LSFSPY significantly increased the proliferation of MC3T3-E1 cells by 38.7% and 32.8%. Alizarin red staining also confirmed that peptides RPQYPQYPS and LSFSPY significantly promoted MC3T3-E1 cell mineralization in a dose-dependent manner. Further, molecular docking results showed that RPQYPQYPS and LSFSPY had a higher affinity with integrin α5β1. Western blot results confirmed that RPQYPQYPS and LSFSPY significantly upregulated the expression of integrin downstream signals PI3K, ERK and Wnt10b. These findings provided evidence for the osteogenic peptides from sea cucumber intestines as a functional food ingredient.