Combined Effects of Tripeptide-3 and Hexapeptide-9 in Activating AMPKα to Inhibit D-Galactose-Induced Human Dermal Fibroblast Senescence

Dermatological senescence, characterized by functional deterioration and the accumulation of senescent fibroblasts, has prompted the investigation of safe and effective anti-ageing agents. The present study investigates the role of Tripeptide-3 (T-3) and Hexapeptide-9 (H-9) in countering D-galactose-induced senescence in human dermal fibroblasts (HDFs). Using a D-gal-induced senescence model, we found that non-cytotoxic concentrations of T-3 and H-9, both individually and in combination, led to a substantial reduction in senescence-associated β-galactosidase activity and concomitant downregulation of p21 expression. Notably, the combination exhibited superior anti-senescence effects. Mechanistically, both peptides activated the AMPKα pathway by increasing phosphorylation at Thr172, with their combination showing enhanced activation. It is of particular significance that the AMPKα inhibitor Compound C served to nullify these protective effects, thereby establishing AMPKα activation as a critical contributing pathway in the combined anti-senescence effect of T-3 and H-9. This study provides novel evidence that T-3 and H-9 inhibit fibroblast senescence via AMPKα activation, thus providing a mechanistic basis for the development of peptide-based anti-ageing skincare formulations.