Rijun Zhang

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.

Oxidative stress and inflammatory responses often occur concomitantly, and they are key causative factors in various human and animal diseases. Evidence suggests that mulberry leaf protein (MLP) may have potential antioxidant and anti-inflammatory properties, but there are significant challenges in enhancing their bioactivities. In this study, MLP was enzymatically hydrolyzed using papain, protamex, alkaline protease, trypsin, and neutral protease, followed by comprehensive evaluation of the antioxidant capacity, anti-inflammatory properties, and cytotoxicity of the hydrolysates. Our findings revealed that some enzymes significantly enhanced the peptide production and antioxidant activity of MLP (p < 0.01), and its activity was positively correlated with the degree of hydrolysis. Among the five hydrolysates, neutral protease hydrolysate (NeuH) exhibited the best antioxidant properties, with free radical scavenging rates of 71.58 ± 0.42% (ABTS), 26.38 ± 0.15% (OH), and 73.91 ± 0.37% (DPPH) at a concentration of 0.1 mg/mL. In addition, NeuH significantly suppressed IL-6 secretion (p < 0.01) and downregulated mRNA expression of IL-6, iNOS, and COX-2 inflammatory markers. This study not only establishes a correlation between enzymatic parameters and MLP biological functions but also demonstrates the potential of optimized MLP hydrolysates, particularly NeuH, as valuable natural antioxidant and anti-inflammatory ingredients for functional foods or nutraceuticals aimed at mitigating oxidative stress and inflammation-related disorders.

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.

Mulberry-derived postbiotics (MDP) have demonstrated promising bioactive properties, including antioxidant and anti-inflammatory effects; however, their specific role in modulating gut inflammation and microbiota composition remains underexplored. Given the growing interest in functional food ingredients for gut health and managing inflammatory disorders, this study aims to evaluate the effects of MDP in alleviating intestinal inflammation and altering the gut microbiota in an LPS-induced mouse model of systemic inflammation. MDP administration significantly mitigated LPS-induced pathological changes in the intestine, liver, spleen, and kidneys, thereby improving systemic health and immune function. Histological analysis revealed reduced inflammation and tissue damage in the intestinal epithelium, supporting the potential of MDP to improve gut barrier integrity. An antioxidant assay revealed that MDP decreased the malonaldehyde (MDA) levels and increased the enzymatic activities of CAT, SOD, and GSH in response to LPS administration, indicating enhanced cellular antioxidant defenses. Inflammatory cytokine analysis showed that MDP downregulated proinflammatory markers such as TNF-α, IL-1β, IL-6, MYD88, Nrf2 COX-2, and HO1, while upregulating TLR4, resulting in potential anti-inflammatory effects by modulating the TLR4-NF-κb pathway. Moreover, MDP promoted beneficial alterations in gut microbiota composition by increasing the abundance of Firmicutes and Bacteroidetes, which are linked to gut health and inflammation regulation. The changes in gut microbiota composition suggest a potential mechanism by which MDP may help restore gut homeostasis and reduce systemic inflammation. These findings suggest that MDP may serve as promising functional food ingredients that support immune health, reduce inflammation, and promote gut microbiota balance, offering potential applications in fortified foods and nutraceuticals aimed at mitigating inflammatory and metabolic disorders.

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.

Microbially synthesized postbiotics have unique properties and advantages; however, systematic studies on the efficient production and biological functions of postbiotics from Bacillus subtilis are limited, which greatly restricts their applications. In this study, we obtained a novel crude exopolysaccharide (EPS) postbiotic from Bacillus subtilis H4. We systematically optimized the EPS production strategy using single-factor analysis, Plackett–Burman design, the path of steepest ascent method, and response surface methodology. The optimized EPS yield was significantly improved, with a maximum yield of 15.01 g/L under the addition of 4.12% soy peptone, 8.99% sucrose, and 0.06% MnSO₄. We found that EPS is a neutral, heterogeneous polysaccharide with a pyranose ring, with a molecular weight of 44,304.913 kDa and a melting point of 218 °C. It consists of glucose, galactose, arabinose, glucosamine, and mannose at a molar ratio of 58.85:19.81:14.75:10.89:6.58. EPS exhibits strong antioxidant capacities, scavenging ABTS and DPPH radicals with IC50 values of 1 and 6 mg/mL, respectively. Moreover, it shows notable anti-inflammatory properties, dramatically inhibiting the lipopolysaccharide (LPS)-induced elevation of nitric oxide (NO) levels and over-activation of the TLR4-NF-κB signaling pathway. These findings highlight the potential of EPS as a multifunctional bioactive compound, offering great promise for its application in the food, clinical, and feed industries.

The hybrid FowlTα1 peptide represents a promising biomolecule synthesized from two naturally occurring peptides, namely Fowlicidins (Fowl) and Thymosin α1 (Tα1). This particular peptide exhibits remarkable anti-inflammatory and antimicrobial properties and demonstrates the capacity to effectively interact with lipopolysaccharide (LPS), while simultaneously inducing minimal cytotoxicity and hemolytic repercussions. Despite its potential, the high cost of this peptide has limited its use. To overcome this limitation, the present study developed a cost-effective and biocompatible method for expressing the FowlTα1 peptide in Pichia pastoris (P. pastoris). We obtained a transgenic strain of the hybrid FowlTα1 peptide with a predicted molecular weight of 3.1 kDa. The FowlTα1 peptide was purified followed by reverse-phase high-performance liquid chromatography (RP-HPLC), yielding 7.2 mg with a purity of 98.2%. Furthermore, physiochemical and structural analysis revealed an amphipathic helical configuration that enhances bioactivity. Moreover, in LPS-stimulated HD11 macrophages, the hybrid FowlTα1 peptide significantly reduced the release of nitric oxide (NO), TNF-α, IL-6, and IL-1β in a dose-dependent manner (p < 0.05) and displayed robust antimicrobial activity against Escherichia coli (E. coli) compared to conventional antibiotic. Overall, the results of this study highlighted the production method and potential of the FowlTα1 peptide as a novel therapeutic agent for antimicrobial, anti-inflammatory, and anti-endotoxin applications.

Background: Natural food-derived bioactive compounds have garnered increasing attention for their potential to modulate immune responses and promote gut health. In particular, compounds like mulberry-derived postbiotics (MDP) may offer novel therapeutic strategies to address inflammation, a key driver of many metabolic disorders.Methodology: This study examines the protective effects of MDP against inflammation in LPS-induced mice, using transcriptomic and microbiome analyses to explore underlying mechanisms.Results: MDP pretreatment alleviates LPSinduced villous atrophy and intestinal barrier damage, promoting recovery of intestinal morphology. Transcriptomic profiling revealed significant changes in gene expression, with 983 upregulated and 1220 downregulated genes in the NC vs LPS comparison, and 380 upregulated and 204 downregulated genes in the LPS vs LPS+MDP comparison. Enrichment analysis using GO and KEGG pathways revealed significant associations with transcriptional regulatory activity, and the NOD-like receptor signaling pathway among the differentially expressed genes. Protein-protein interaction analysis identified key genes involved in inflammation and immune regulation, with hub genes like IL6, CXCL10, and MYD88 in the LPS group and CD74, CIITA, and H2-AB1 in the MDP-treated group.Conclusion: Microbiome analysis suggested MDP may also influence gut microbiota composition, supporting systemic immune regulation. These findings highlight MDP’s potential as a food additive for immune modulation and gut health.

With the increasing recognition of the role of immunomodulation and oxidative stress in various diseases, designing peptides with both immunomodulatory and antioxidant activities has emerged as a promising therapeutic strategy. In this study, a hybridization design was applied as a powerful method to obtain multifunctional peptides. A total of 40 peptides with potential immunomodulatory and antioxidant activities were designed and screened. First, molecular docking was employed to screen peptides with a high binding affinity to MD2, a key receptor protein in the NFκB immune pathway. For the in vitro high-throughput screening, we constructed a reporter gene-based stable cell line, IPEC-J2-Lucia ARE cells, which was subsequently used to screen peptides with antioxidant activity. Furthermore, the biocompatibility, immunomodulatory, and antioxidant activities of these peptides were assessed. Among the candidates, the hybrid peptide VA exhibited the strongest immune-enhancing activity through the activation of the NF-κB pathway and significant antioxidant activity via the Nrf2-ARE pathway. Additionally, VA demonstrated protective effects against H₂O₂-induced oxidative damage in HepG2 cells. This study not only demonstrates the potential of peptide hybridization, but also develops a screening platform for multifunctional peptides. It provides a new tool for the treatment of autoimmune diseases and oxidative stress-related diseases.