Cyclo(Val-Pro) and cyclo(Phe-Phe) alleviate MASLD via distinct CCR2- and ADCY7-associated pathways.

Metabolic dysfunction-associated steatotic liver disease (MASLD), hallmarked by hepatic metabolic disorders and chronic inflammation, is a growing global public health threat with limited approved pharmacological options. Natural cyclodipeptides exhibit favorable metabolic stability and bioactivity profiles, making them ideal molecules for drug discovery in metabolic diseases; however, their roles and mechanisms in MASLD remain poorly defined. Here, we investigated the protective effects of cyclo(Val-Pro) and cyclo(Phe-Phe), two bioactive cyclodipeptides previously identified in hydrolyzed chicken meat extract (HCE), against MASLD. Administration of both cyclodipeptides effectively ameliorated metabolic disturbance and hepatic inflammation in high-fat diet-induced MASLD mice, and mitigated lipopolysaccharide-triggered acute inflammation in a complementary in vivo model. Liver transcriptomic analysis revealed that the two cyclodipeptides significantly modulated the hepatic chemokine signaling pathway in MASLD mice. Mechanistically, both cyclodipeptides reduced hepatic CD11b- and F4/80-associated signals and modulated macrophage-associated pro-inflammatory and anti-inflammatory marker expression in vivo and in vitro. Further validation using molecular docking, target inhibitor intervention, drug affinity responsive target stability, cellular thermal shift assay, site-directed mutagenesis, and gene knockout approaches supported that cyclo(Val-Pro) regulated the expression of macrophage CC chemokine receptor type 2 (CCR2), while cyclo(Phe-Phe) modulated adenylyl cyclase 7 (ADCY7), an upstream chemokine signaling regulator, to block downstream inflammatory cascades. Collectively, our findings support further investigation of these two cyclodipeptides and suggest that CCR2- and ADCY7-associated pathways may contribute to their effects in MASLD.