Oxidative stress plays a crucial role in the development and progression of various diseases. Antioxidant peptides have attracted great attention in agricultural, food, and clinical fields due to their low toxicity, high efficacy, and easy absorption, but the development of antioxidant peptides and their in-depth molecular mechanisms are still lacking. The previous study established a platform for the high-throughput design and screening of multifunctional peptides and successfully identified a novel hybrid peptide, VLP-Aβ (VA), which exhibits both antioxidant and immunomodulatory properties. This study aimed to evaluate the antioxidant activity of VA and investigate the underlying molecular mechanisms. The antioxidant effects of VA were evaluated using both in vitro (H₂O₂-induced oxidative damage in HepG2 cells) and in vivo (CCl₄-induced liver damage in mice) models. VA exhibited significant antioxidant activity both in vitro and in vivo, significantly improving the cell viability and increasing the levels of antioxidant enzymes (SOD, CAT, GSH-Px) to alleviate oxidative stress. These findings indicated that the antioxidant effect of VA is dependent on NRF2, as evidenced by NRF2 knockdown experiments. Further investigation revealed that VA alleviates oxidative stress by modulating the KEAP1-NRF2-ARE signaling pathway. These findings provide insights into the properties of the antioxidant peptide VA, expand the understanding of its molecular mechanisms, and suggest new opportunities for developing VA as a novel functional agent in the agricultural, food, and clinical industries.

Lipopolysaccharide (LPS), an essential structural molecule in the outer membrane of Gram-negative bacteria, is recognized as a principal trigger of inflammatory responses and oxidative stress. Thus, the control and clearance of LPS is essential to inhibit LPS-induced excessive inflammation, oxidative stress, and liver injury. In recent years, some native bioactive peptides, such as human β-defensin 126 (DEFB126) and thymopentin (TP5), have been reported to have inhibitory effects against LPS-induced inflammation and oxidative stress. However, the cytotoxicity, weak stability, and poor biological activity have hindered their practical application and clinical development. The development of novel hybrid peptides is a promising approach for overcoming these problems. In this study, we designed a novel hybrid peptide [DTP, DEFB126 (1-39)-TP5] that combines the active center of DEFB126 and full-length thymopentin (TP5). Compared to the parental peptides, DTP has a longer half-life, lower cytotoxicity, and greater anti-inflammatory and antioxidant activity. The anti-inflammatory and antioxidant effects of DTP were demonstrated in a murine LPS-induced sepsis model, which showed that DTP successfully inhibited the indicators associated with LPS-induced liver injury; decreased the contents of TNF-α, IL-6, and IL-1β; increased the level of glutathione (GSH); and improved the activities of catalase (CAT) and superoxide dismutase (SOD). Furthermore, our study revealed that the anti-inflammatory and antioxidant activities of DTP were associated with LPS neutralization, blockade of LPS binding to the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex, reduction in reactive oxygen species content, and inhibition of the activation of the nuclear factor kappa-B (NF-кB) signaling pathway. These results elucidate the structural and functional properties of the peptide DTP, reveal its underlying molecular mechanisms, and shed light on its potential as a multifunctional agent for applications in agriculture, food technology, and clinical therapeutics.

Immunosuppression dramatically increases tissue and organ susceptibility to infection, injury, and even cancer. This poses a serious threat to human and animal health. In a previous study, we established a platform for high-throughput design and screening of multifunctional peptides. Using this platform, we successfully identified a novel hybrid peptide, VLP-Aβ (VA), which exhibits both immunomodulatory and antioxidant properties. This study aimed to evaluate the immunomodulatory activity of VA and investigate the underlying molecular mechanisms. In the cyclophosphamide (CTX)-induced immunodeficient mouse model, VA significantly alleviated CTX-induced weight loss. It also restored thymus and spleen indices, and increased serum immunoglobulins (IgA, IgM, IgG) and cytokines (TNF-α, IL-6, IL-1β) levels. VA also improved splenic lymphocyte proliferation, CD4⁺/CD8⁺ T cell ratios, and NK cell cytotoxicity. At the cellular level, western blot analysis showed that VA activated the TLR4-NF-κB pathway in RAW264.7 macrophages. Mechanistically, inhibition of the MD2 protein by L6H21 abolished VA’s immunomodulatory effects. This confirms MD2 as a critical mediator. Molecular docking and dynamics simulations revealed that VA binds stably to the hydrophobic pocket of MD2. These findings suggest that VA exerts immunomodulatory effects by stimulating MD2 and activating the TLR4-NF-κB pathway, which provides new ideas, techniques, and approaches for the development of novel peptide immunomodulators.