Dual-Phase Immunomodulation by the Bovine β-Casein Peptide KEMPFPK: Insights into Potential TLR Interaction and Gut Microbiota-Mediated Effects

This study investigates the immunomodulatory effects and underlying mechanisms of KEMPFPK, a peptide derived from bovine β-casein, using integrated in vitro, in silico, and in vivo approaches. In RAW264.7 macrophages, KEMPFPK enhanced proliferation, phagocytosis, and migration and selectively upregulated the chemokine MCP-1. Under LPS-induced inflammation, KEMPFPK suppressed pro-inflammatory cytokines (IL-1β, TNF-α) and NO production while promoting the anti-inflammatory cytokine IL-10. These effects were mediated through the inhibition of NF-κB and MAPK signaling pathways. Molecular docking predicted high-affinity binding of KEMPFPK to Toll-like receptors (TLR2 and TLR4), suggesting a potential mechanism for its immunomodulatory activity. In cyclophosphamide (CTX)-induced immunosuppressed mice, KEMPFPK administration restored immune organ indices, rebalanced serum cytokine levels, and modulated humoral immunity. Importantly, KEMPFPK was associated with a significantly reshaped gut microbiota profile, characterized by the promotion of beneficial genera (e.g., Ligilactobacillus, Adlercreutzia) and the suppression of opportunistic pathogens (e.g., Escherichia–Shigella). These findings establish KEMPFPK as a dual-phase immunomodulator and suggest that its effects may involve direct immune cell regulation coupled with indirect microbiota remodeling. This study provides a scientific foundation for the application of KEMPFPK in immunomodulatory functional foods.