Novel Peptide Inhibitor of Matrix Metalloproteinases-1 from Pufferfish Skin Collagen Hydrolysates and its Potential Photoprotective Activity Via the Mapk/Ap-1 Signaling Pathway

Abstract

With the maturation of aquaculture techniques, Takifugu bimaculatus has become the principal species for pufferfish farming in the southern coastal regions of Fujian Province. Compared to its wild counterparts, farmed T. bimaculatus possesses non-toxic flesh and collagen-rich skin, making it an excellent source of collagen peptides. In this study, we identified a novel pufferfish collagen-derived matrix metalloproteinase 1 (MMP-1) inhibitory peptide with anti-photoaging effects. We used multistage membrane and gel filtration chromatography to purify low-molecular-weight collagen peptides from T. bimaculatus skin (TBSCH-L). The amino acid sequences of TBSCH-L components were determined using nano-HPLC-MS/MS, and virtual molecular docking screening was employed to identify low-molecular-weight TBSCH peptides targeting MMP-1. Four anti-photoaging peptide sequences, GDRGFPGE, GPAGPRGA, FPGGPGAK, and RGFPGGDGAA, were identified by assessing the viability of UVB-induced L929 cells. GPAGPRGA (GP8) exhibited the highest MMP-1 inhibitory activity and cellular photoprotection. Surface plasmon resonance confirmed high-affinity binding between MMP-1 and GP8. Using UVB-exposed L929 cells, we elucidated the mechanism underlying GP8's anti-photoaging effects. GP8 showed potent antioxidant capabilities, significantly reducing intracellular reactive oxygen species (ROS) levels and enhancing superoxide dismutase (SOD) activity at concentrations of 100−200 μM. At 200 μM, GP8 significantly decreased malondialdehyde (MDA) content. GP8 also accelerated the migration of L929 cells, demonstrating its wound-healing potential. It markedly reduced intracellular β-galactosidase levels and downregulated the phosphorylation levels of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 proteins, as well as c-Jun protein expression within the MAPK/AP-1 signaling pathway, thereby lowering MMP expression in L929 cells. The protective effects of TBAPP on zebrafish exposed to UVB radiation indicated that 25−100 μM GP8 effectively repaired UVB-damaged zebrafish caudal fins, achieving a recovery rate of up to 31.2%. GP8 effectively cleared UVB-induced ROS in zebrafish, decreased lipid peroxidation, and alleviated cellular apoptosis. In conclusion, GP8 holds promise as a novel natural marine anti-photoaging ingredient for applications in the cosmetic and functional food industries.

Keywords: Takifugu bimaculatus, ultraviolet B, collagen hydrolysate, skin photoaging, matrix metalloproteinase 1 inhibitory peptide