Novel immunomodulatory peptides identified from Mytilus edulis: Molecular docking, immunomodulatory effects on RAW264.7 cells and molecular dynamics simulation studies.

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.