Immunomodulatory peptides from thick-shelled mussel (Mytilus coruscus): Isolation, identification, molecular docking and immunomodulatory effects on RAW264.7 cells

Low molecular weight (MW) peptide fraction (<1 kDa, MCP) from protein hydrolysate of thick-shelled mussel (Mytilus coruscus) exhibited significant proliferative and phagocytic effects on RAW264.7 macrophages. However, the immunomodulatory peptides in MCP have not been identified. Therefore, 60 specific immunomodulatory peptides with MWs ranged from 428.30 Da to 935.46 Da were purified from MCP by series chromatography methods and identified by LC-MS/MS. Among 60 identified peptides, LH-6 (LVVLGH) could significantly activate RAW264.7 cells to increase proliferation, promote phagocytosis and secrete NO and cytokines, including IL-6, IL-1β and TNF-α. Moreover, Western blot, immunofluorescence, and molecular docking experiments proved that LH-6 (LVVLGH) exerted in vitro immune regulation in RAW264.7 cells by binding to TLR4 receptor to activate NF-κB signal pathway by promoting nuclear translocation of NF-κB p65 and phosphorylation of IκBα and activate MAPK signal pathway by promoting the phosphorylation of JNK, ERK and P38. In addition, LH-6 (LVVLGH) actively bound to TLR4 receptors by forming hydrogen bonds, hydrophobic bonds, π-π stacking, salt bridge and π-cation interaction. Therefore, this study provides evidence for the in vitro immune regulation of LH-6 (LVVLGH) and supports the possibility acting as a potential nutraceutical supplement to strengthen the immune system.