Abstract: This study employed trypsin to hydrolyze tuna cooking juice to obtain hydrolysates with osteogenic activity, with the aim of elucidating the osteogenic potential and mechanism of an enzymatic hydrolysate of tuna cooking juice and establishing a theoretical foundation for developing novel fish-derived osteogenic peptides. The hydrolysates with different molecular masses were separated via ultrafiltration and their osteogenic activities were evaluated. Transcriptomic analysis was then used to investigate the underlying mechanism of action. Furthermore, nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) and molecular docking were adopted to identify oligopeptides that promote osteoblast proliferation. Evaluation of osteogenic activity revealed that the low-molecular-mass hydrolysates effectively increased the proliferation rate (135.21%) and alkaline phosphatase activity of MC3T3-E1 cells, leading to increased mineral deposition. Transcriptomic analysis clarified that the low-molecular-mass hydrolysates primarily promoted osteoblast proliferation by upregulating the transcription levels of the Ccnd1, Ccnd3, Ccn4, Tcf7l2, F2r, Pik3r2, Pdpk1, and Gnb2 genes in the Wnt/β-catenin and phosphatidylinositol-3-kinase/protein kinase B signaling pathways. A total of six potential peptides with high osteogenic activity were identified through nano LC-MS/MS, molecular docking, and in vitro cell validation experiments. Among these peptides, DG-10 (DGDAGA(Hyp)GPS) exhibited the highest osteogenic activity and excellent digestive stability. Its binding affinity for epidermal growth factor receptor (EGFR) (KD = 3.826 × 10-5 mol/L) was higher than that of the peptide VS-6 (VSFYD (Hyp)) (KD = 4.054 × 10-5 mol/L), and the binding sites (Thr15, Gln16, Leu17/14, Asn12, and Lys13) on EGFR were predicted to be potential functional residues.

Key words: tuna; osteogenic bioactivity; oligopeptide; cell experiment; molecular docking