Isolation, identification, and computational analysis of antioxidant peptides from deer blood and its therapeutic effects on alcoholic liver injury in mice

Alcoholic liver disease (ALD) is a prevalent liver disorder caused by excessive alcohol consumption, and oxidative stress plays a key role in its pathogenesis. Antioxidant peptides are promising candidates for ALD therapy, and in this study, we investigated the protective effects of deer blood peptides (DBP) against ALD. We established an efficient method of extracting DBP through single-factor optimization and response surface methodology, identifying papain as the most effective protease and determining optimal hydrolysis conditions. DBP consists primarily of peptides with molecular weights <3 kDa, which exhibit strong scavenging capacity against multiple free radicals and excellent biocompatibility. In H2O2-induced HepG2 cells, DBP pretreatment increased the cell survival rate, increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and reduced malondialdehyde (MDA) levels. In an alcohol-induced liver injury mouse model, DBP reduced the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). It improved the hepatic antioxidant status and alleviated fatty liver lesions. Through peptidomics and bioinformatics analysis, 13 potential antioxidant peptides were identified. Molecular docking revealed that these peptides spontaneously bind to 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). We synthesized QGPLEPGPAGNRGCGRK (QK17) and PPSPGAMPRPP (PP11), both of which have excellent radical scavenging activity and cellular protective effects. Moreover, molecular docking revealed that PP11 binds to Keap1 (−10.2 kcal/mol), potentially regulating the Keap1-Nrf2 pathway. These findings indicate that DBP may serve as a promising therapeutic agent for ALD, assisting in its prevention while promoting the utilization of deer blood resources. • Isolation of small-molecule peptides (DBP) with high antioxidant activity from deer blood. • DBP shows potent antioxidant activity in H₂O₂-induced HepG2 cells and alcoholic liver injury mice. • LC-MS/MS and bioinformatics screening identified 13 potential antioxidant peptides from DBP. • Molecular docking suggests peptides may act via the Keap1-Nrf2 and NF-κB signaling pathways.