Anti-oxidative and anti-apoptosis effects of egg white peptide, Trp-Asn-Trp-Ala-Asp, against H2O2-induced oxidative stress in human embryonic kidney 293 cells

Abstract

Peptides derived from egg white protein are well-known for their abundant species and biological properties. The aim of this study was to investigate the anti-oxidative and anti-apoptosis effects of (Trp-Asn-Trp-Ala-Asp) WNWAD, a pentapeptide derived from egg white ovomucin pepsin hydrolysates, against the oxidative stress induced by H₂O₂ in HEK-293 cells. Oxygen radical absorbance capacity (ORAC) results showed that WNWAD possessed extraordinary oxygen radical absorption capacity (with an ORAC value = 2.91 μM TE μM⁻¹) in vitro. Then, at the cellular level, MTS assay results displayed that WNWAD dose-dependently inhibited H₂O₂-induced cellular oxidative stress in HEK-293 cells and fully recovered the oxidative damage induced by 400 μM H₂O₂ at a concentration of 1 μM. The 2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe assay further proved that WNWAD inhibited cellular oxidative stress by reducing intracellular ROS accumulation in intact HEK-293 cells. In addition, scanning electron microscopy (SEM) images showed that the morphology of HEK-293 cells under H₂O₂ treatment displayed a cell apoptosis phenotype, while WNWAD pretreatment prevented the development of this phenotype. Furthermore, Western blot results indicated that WNWAD up-regulated the level of the anti-apoptotic protein Bcl-2 and down-regulated the levels of apoptosis executor proteins, cleaved caspase-3 and cleaved PRAP, in H₂O₂-induced oxidative stress in HEK-293 cells. All the above results suggested that WNWAD protected HEK-293 cells against H₂O₂-induced oxidative stress by inhibiting intracellular ROS accumulation and blocking the ROS activated mitochondria-mediated cell apoptosis pathway.