Immunomodulatory effects, digestive stability, and action mechanism of casein peptide SPAQILQW in activating macrophages via N-terminal serine

The peptide SPAQILQW, with potential immunomodulatory activity, in casein hydrolysates was obtained in a previous study, but the stability of its activity and key active sites remain to be explored. This study demonstrates that SPAQILQW could promote the proliferation and phagocytic activity of macrophages, increase the expression of key inflammatory mediators and chemokines in macrophages, and significantly modulate immune activity in vitro. The results of an in vitro gastrointestinal protease-mimicking hydrolysis assay and a Caco-2 cell monolayer membrane model demonstrated that SPAQILQW could overcome hydrolysis by digestive Enzymes in the gastrointestinal tract and that 10.15% ± 0.08% of it could enter the bloodstream through the intestinal brush border membrane to exert its active effects, including activating macrophages by promoting the expression of chemokines (CXCL1), TNF-α, and NO. Furthermore, SPAQILQW interacted with TLR2 and TLR4 in RAW264.7 cells. The C₆ = O₇ site with a highest occupied molecular orbital contribution of 34.59453% was the active site in SPAQILQW, and the serine where the C₆ = O₇ was located was the active amino acid of SPAQILQW. Additionally, the immunomodulatory activities were activated by SPAQILQW in macrophages via the MAPK signaling pathway. This study offers insight into the digestive stability and action mechanism of immunomodulatory peptides with an N-terminal serine.