Identification and molecular mechanisms of novel antioxidant peptides from two sources of eggshell membrane hydrolysates showing cytoprotection against oxidative stress: A combined in silico and in vitro study

Abstract

Eggshell membranes (ESM) from fresh and hatched chicken eggs are important agricultural byproducts. In this study, we investigated the antioxidant and cytoprotective activity of hydrolysates from fresh and hatched ESM, identified the antioxidant peptides and explored their potential molecular mechanism using a combined in silico and in vitro approach. The results showed that the hydrolysates fractions (MW < 3 kDa) of both ESM exhibited excellent antioxidant effects and could protect H₂O₂-induced RAW264.7 cells by reducing ROS and MDA levels involving the modulation of the Keap1-Nrf2 pathway. Six novel peptides identified by integrated approaches of peptidomics and in silico bioinformatic analysis were synthesized, exhibiting significantly higher ORAC values (629.41-1823.77 µmol TE/mmol) than GSH (397.21 µmol TE/mmol). Among these, KPLCPP, MDGWPR, and LWNPR possessed stronger ABTS scavenging and cytoprotective activities than GSH. All the six peptides could dock onto the Keap1-Kelch domain. Moreover, KPLCPP and LWNPR could regulate the Keap1-Nrf2 pathway and induced the overexpression of antioxidant enzymes including HO-1, SOD and GSH-Px. With the molecular docking and western blot analysis, the underlying molecular mechanism of the ESM antioxidant peptides might be related to the activation of Keap1-Nrf2 pathway by occupying the Nrf2-binding site on Keap1. This study provides a theoretical basis for the application of fresh and hatched ESM antioxidant peptides in functional foods, as well as insights for the identification and the mechanisms research of more food-derived antioxidant peptides.

Keywords: Bioactive peptides; Bioinformatics; Egg byproducts; Identification; Keap1-Nrf2 signaling pathway; Molecular docking; Peptidomics.