BACKGROUND: Hyperpigmentation is primarily caused by ultraviolet (UV) radiation, which triggers oxidative stress and melanin accumulation in the skin. Moderate UVB exposure activates autophagy, which facilitates cell repair, while prolonged UVB exposure disrupts this process, worsening skin damage and pigmentation. Nelumbo nucifera extract (NnE) drew our attention in our preliminary screening experiments for its potential to regulate keratinocyte autophagy. Nevertheless, the molecular mechanisms underlying the extract of NnE's therapeutic effects and its bioactive constituents remain incompletely characterized.
PURPOSE: The aim of this study was to evaluate the effects of NnE on UVB-induced hyperpigmentation and to explore the mechanisms underlying its autophagy-promoting activity of bioactive ingredients.
METHODS: B16 murine melanoma cells and human epidermal keratinocytes (HEKa) exposed to UVB radiation were used to model autophagy inhibition and melanosome accumulation. 3D pigmented skin organoid model and UVB-exposed guinea pigs were used to assess the effects of NnE on pigmentation. Various techniques, including western blot, immunofluorescence, and transcriptome sequencing, were employed to analyze autophagy regulation and melanosome clearance. Furthermore, we used molecular docking and in vitro enzyme activity detection methods to search for the active substances of NnE acting on the target.
RESULTS: In HEKa cells, NnE effectively reversed UVB irradiation-induced inhibition of autophagy and promoted melanosome clearance. NnE also reduced the melanin content in HEKa cells. In MelaKutis® and UVB-exposed guinea pigs, topical application of NnE effectively alleviated pigmentation. Mechanistically, NnE suppressed the interaction between AMPK and YAP, thereby promoting nuclear translocation of YAP and upregulation of key autophagy-related genes, including ATG5, and ultimately enhancing autophagic activity. Finally, through molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability assay (DARTS), in vitro enzyme activity detection, and RNA interference, we demonstrated that quercetin-3-O-glucuronide, the major active constituent of NnE, exerts anti-photoaging effects by inhibiting NOX4 and subsequently regulating the AMPK/YAP/ATG5 signaling axis.
CONCLUSIONS: This study demonstrates that the anti-pigmentation effect of NnE and its bioactive compound, quercetin 3-O-glucuronide, is mediated through the YAP-ATG5 signaling axis and Q3Q-driven NOX4 inhibition, which establishes the mechanistic basis for their potential application in preventing UVB-induced skin pigmentation and provides a novel perspective for the development of anti-photoaging interventions.
