Novel immunomodulatory peptides identified from Mytilus edulis: Molecular docking, immunomodulatory effects on...

This study sought to screen novel immunomodulatory peptides from Mytilus edulis hydrolysates (MEHs) after simulated gastrointestinal digestion (SGD) and to clarify their regulatory mechanisms. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 99 peptides with molecular weights ranging from 186.10 to 1197.59 Da. In silico screening combined with molecular docking analyses led to the selection of five novel candidates: FPR, FRE, KFP, LLPK, and PDRM. Among these, FPR and LLPK exhibited the most potent immunomodulatory activities, significantly enhancing RAW264.7 macrophage proliferation (up to 135% of control at 400 μg/mL, LLPK), phagocytosis (222.97% of control at 400 μg/mL, FPR), and nitric oxide secretion. Molecular dynamics simulations further verified the stable interactions of FPR and LLPK to TLR2 and the TLR4-MD2, supported by low root-mean-square deviation (RMSD), persistent hydrogen-bond networks, restrained root-mean-square fluctuation (RMSF), compact radius of gyration (Rg), and steady solvent-accessible surface area (SASA). Receptor blocking experiments showed that TLR2 inhibition significantly attenuated the effects of both peptides, while TLR4 inhibition had a negligible effect, confirming that FPR and LLPK primarily act through TLR2. Furthermore, FPR and LLPK significantly upregulated tumor necrosis factor-α (TNF-α) secretion (2.09- and 1.30-fold, respectively, p < 0.05), while interleukin-6 (IL-6) levels showed an increasing trend without reaching statistical significance. Overall, the results demonstrate that Mytilus edulis represents a promising source of immunomodulatory peptides with the potential to enhance immune function and regulate inflammatory responses.