Screening and Identification of TRPV1-Targeting Peptides from Apis mellifera Venom with Inflammatory Modulatory Potential

Bee venom from Apis mellifera has been traditionally used to treat inflammatory disorders, largely due to its bioactive peptide components. However, comprehensive characterization of these peptides and their interaction with transient receptor potential vanilloid 1 (TRPV1), a key receptor in nociception and inflammation, remains limited. In this study, we systematically profiled the A. mellifera venom peptidome and identified TRPV1-targeting peptides with inflammatory modulatory potential. De novo sequencing-based peptidomics generated an in-house database of 472 peptides (average local confidence, ALC ≥ 50%), including 103 high-confidence peptides (ALC ≥ 80%). Ten novel peptides were identified as candidate TRPV1-targeting peptides through pull-down screening. Among them, three peptides (P1: GNGGGGLGSGGSLGLGHE, P2: MLLAVARSVP, and P3: NQEVEEERLKY) were synthesized and tested for anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages. All three peptides significantly suppressed TNF-α secretion, with P1 achieving 62.5% inhibition at 10 μM, and modulated IL-6 and IL-1β in a dose-dependent manner. Proteomic profiling and pathway enrichment analyses indicated that these peptides exert anti-inflammatory effects through NF-κB suppression and the modulation of redox and metabolic pathways. This study provides the first evidence that A. mellifera venom contains peptides identified through TRPV1-based affinity screening, including three novel TRPV1-targeting peptides with inflammatory modulatory potential.