The anti-melanogenic effects of 3-O-ethyl ascorbic acid via Nrf2-mediated α-MSH inhibition in UVA-irradiated keratinocytes and autophagy induction in melanocytes.

3-O-ethyl ascorbic acid (EAA) is an ether-derivative of ascorbic acid, known to inhibit tyrosinase activity, and is widely used in skincare formulations. Nevertheless, the molecular mechanisms underlying the EAA's effects are poorly understood. Here, the anti-melanogenic activity of EAA was demonstrated through Nrf2-mediated α-MSH inhibition in UVA-irradiated keratinocytes (HaCaT) and autophagy induction and inhibition of α-MSH-stimulated melanogenesis in melanocytes (B16F10). EAA pretreatment increased the HaCaT cell viability but suppressed ROS-mediated p53/POMC/α-MSH pathways in UVA-irradiated cells. Further, the conditioned medium from EAA-pretreated and UVA-irradiated HaCaT cells suppressed the MITF-CREB-tyrosinase pathways leading to the inhibition of melanin synthesis in B16F10 cells. EAA treatment increased nuclear Nrf2 translocation via the p38, PKC, and ROS pathways leading to HO-1, γ-GCLC, and NQO-1 antioxidant expression in HaCaT cells. However, Nrf2 silencing reduced the EAA-mediated anti-melanogenic activity, evidenced by impaired antioxidant gene expression and uncontrolled ROS (H0) generation following UVA irradiation. In B16F10 cells, EAA-induced autophagy was shown by enhanced LC3-II levels, AVO formation, Beclin-1 upregulation, and activation of p62/SQSTM1. Further, EAA-induced anti-melanogenic activity was substantially decreased in autophagy inhibitor (3-MA) pretreated or LC3 knockdown B16F10 cells. Notably, transmission electron microscopy data showed increased melanosome-engulfing autophagosomes in EAA-treated B16F10 cells. Moreover, EAA also down-regulated MC1R, TRP-1/-2, tyrosinase expressions, and melanin synthesis by suppressing the cAMP-CREB-mediated MITF expression in B16F10 cells stimulated with α-MSH. In vivo studies on the zebrafish model further confirmed that EAA inhibited tyrosinase expression/activity and endogenous pigmentation. In conclusion, 3-O-ethyl ascorbic acid is an effective skin-whitening agent and could be used as a topical agent for cosmetic purposes.