A Previously Unreported Octapeptide from Ganoderma lucidum Attenuates Oxidative Stress and Inflammation in LPS-Stimulated Macrophages via Modulation of NF-κB and COX-2 Signaling

Abstract Ganoderma lucidum is a source of bioactive peptides with reported immunomodulatory properties. In this study, an octapeptide, TNFSNCTL, was identified from G. lucidum hydrolyzates using de novo sequencing coupled with LC–MS/MS and subsequently synthesized via solid-phase peptide synthesis. Querying both the NCBI nonredundant (NR) database and G. lucidum protein sequences returned no exact contiguous alignment, suggesting that TNFSNCTL has not been described before. Its antioxidant and anti-inflammatory properties were then characterized in cell-free systems and in RAW264.7 macrophages challenged with lipopolysaccharide (LPS). Treatment lowered the intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO) while remaining minimally toxic to fibroblasts, keratinocytes, and macrophages across the concentrations tested. The peptide also downregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and dampened NF-κB p65 phosphorylation, leaving the total NF-κB pool unchanged. As this exploratory study evaluated only low- and high-dose conditions, future studies incorporating intermediate doses will further refine the dose–response relationship and strengthen mechanistic understanding. Proteomic profiling revealed differential regulation of proteins associated with phosphorylation processes, redox homeostasis, and interferon-responsive pathways, including upregulation of Ppp1cc and downregulation of Lrpprc. Molecular docking suggested a potential interaction between TNFSNCTL and COX-2. These findings indicate that TNFSNCTL modulates oxidative stress and inflammatory signaling in macrophages, supporting its potential as a promising candidate for functional food or nutraceutical applications.