Identification and molecular mechanisms of novel wound healing potential peptides from eggshell membrane hydrolysates assisted by an integrated in silico and in vitro approach

The aim of this study was to demonstrate the wound-healing activity of novel peptides derived from the eggshell membranes (ESM) of fresh chicken eggs and to explore the underlying mechanisms using integrated in silico and in vitro approaches. The ESM hydrolysates prepared by papain (PapH) exhibited the highest antioxidant and wound-healing activity. Three novel peptides, KDFPCPM (P1), DKDGPFRLC (P2), and LVEKGDVAF (P3), were identified from PapH via peptidomics and subsequently synthesized. These synthetic peptides also displayed excellent antioxidant and wounding-healing effects, with P1 surpassed the other peptides. Molecular docking results indicated that P1 could form stable bonds with Keap1, a key antioxidant target identified through proteomics. Furthermore, in vitro experiments utilizing the Nrf2 inhibitor ML385 revealed that P1 from ESM promotes cell migration by activating the Keap1-mediated Nrf2/HO-1 signaling pathway, thereby enhancing the cellular antioxidant defense system. Collectively, these findings provided new insights for the high-value utilization of discarded ESM. • The papain hydrolysate of eggshell membranes performed the strongest antioxidative and wound healing activity. • Three novel peptides were identified by a combined peptidomics and bioinformatic analysis from papain hydrolysate. • The wound healing effect of ESM peptides was associated with their antioxidant activity. • The novel peptides KDFPCPM promoted cell migration through activating Keap1-mediated Nrf2/HO-1 signaling pathway.