Immunomodulatory mechanism of the crocodile-derived peptides AKLDLEEVIK and DFLDLPSIER mediated by macrophage metabolic reprogramming

Crocodile has long been utilized as a traditional high-protein food resource and is recognized as a rich source of health-promoting bioactive peptides. Previously, we identified two immunomodulatory peptides, AKLDLEEVIK (AK-10) and DFLDLPSIER (DR-10), from crocodile head protein hydrolysates, which enhanced basal immunity function and suppressed inflammatory responses under distinct immune microenvironments. However, the underlying mechanisms remain unclear. This study investigated the mechanism by which adaptive metabolic reprogramming mediates the effects of AK-10 and DR-10 on macrophage phenotype and function in resting and LPS-induced inflammatory microenvironments. The results showed that AK-10 and DR-10 triggered metabolic reprogramming adapted to the immune microenvironment, with loss-of-function experiments confirming that these metabolic shifts are required for macrophage phenotypic polarization and functional shifts. Under resting conditions, both peptides enhanced glycolytic flux and upregulated the mRNA expression of HK2, PKM2, and LDHA, providing metabolic support for M1 polarization, which in turn promoted the expression of CD86, iNOS, and pro-inflammatory cytokines, thereby enhancing basal immune surveillance. Under inflammatory stress, they suppressed aberrant glycolysis and restored oxidative phosphorylation, accompanied by upregulation of PGC-1α, CPT1a, and CS mRNA expression. This metabolic transition drove macrophage polarization from M1 toward the anti-inflammatory M2 phenotype (elevated CD206, Arg-1, IL-10, TGF-β), thereby exerting anti-inflammatory effects. Moreover, AK-10 and DR-10 regulated intracellular ROS levels and enhanced antioxidant defense capacity, thereby maintaining redox homeostasis. Collectively, these findings reveal the metabolic mechanisms by which AK-10 and DR-10 orchestrate macrophage immune responses across distinct immune microenvironments, suggesting a potential basis for their application as functional food ingredients.