Combining bioinformatics techniques to study the anti-inflammatory activity of two novel peptides from yak milk cheese

Background: Yak milk casein peptides exhibit promising anti-inflammatory activity, but there is a gap in the study of their anti-inflammatory mechanisms and specific molecular targets. Objective: This study aimed to elucidate the anti-inflammatory mechanisms of two novel yak casein-derived peptides, QEPVLGPVRGPFP (QP13) and VYPFPGPIPN (VN10), previously identified via bioinformatics screening. Methods: An LPS-induced RAW264.7 macrophage model was employed, and peptides were evaluated at concentrations of 187.5–1500 μg/mL, with indomethacin as a positive control. Molecular docking and 100 ns molecular dynamics simulations analyzed interactions with key inflammatory targets: inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Results: At 1500 μg/mL, VN10 and QP13 were non-toxic to macrophages and exerted maximal inhibition on nitric oxide (NO) production (90.44 ± 0.88 % and 87.58 ± 0.88 %, respectively), TNF-α (67.56 ± 3.94 % and 60.32 ± 1.72 %), and IL-6 (80.87 ± 0.52 % and 67.02 ± 1.38 %). Both peptides surpassed indomethacin in suppressing NO. Molecular docking revealed hydrogen bonding interactions with critical residues (Trp-366, Thr-77, Asn-61, Leu-62) of iNOS, TNF-α, and IL-6. Molecular dynamics simulations confirmed the stability of the peptide-target protein complexes. Conclusion: This study establishes a molecular mechanistic basis for the anti-inflammatory actions of yak milk-derived peptides QP13 and VN10, promoting the application of dairy-derived anti-inflammatory peptides in functional foods and human nutrition.