The in vitro and in vivo skin‐whitening activity of Ectoine through enhanced autophagy in melanocytes and keratinocytes and zebrafish model

Ectoine, a natural bacterial osmolyte, suppressed UVA irradiated-α-melanocyte stimulating hormone (MSH) stimulated melanogenesis through antioxidant Nrf2 pathways in human keratinocytes; however, the underlying skin whitening mechanisms were not elucidated. The depigmenting efficiency of Ectoine (0-400 μM) through antimelanogenesis and melanin degradation by autophagy promotion was investigated in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo zebrafish model. MTT assay, Western blotting, GFP-LC3 puncta, AVO formation, melanin assay, immunofluorescence staining, TEM techniques, siLC3 transfection, and zebrafish model were utilized. Ectoine-induced autophagy in B16F10 and HaCaT cells was shown by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta, autolysosome AVOs formation, ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. The immunoprecipitation data revealed that Ectoine increased the association between LC3-II and p62 proteins in B16F10 and HaCaT cells. Importantly, antioxidant NAC pretreatment antagonized the Ectoine-induced ATG4B diminution in B16F10 and HaCaT cells. Ectoine inhibited melanogenesis by suppressing melanosome gp100, tyrosinase, TRP-1/-2, and/or melanin formation via autophagy in α-MSH-stimulated B16F10 and melanin-feeding HaCaT cells. TEM findings displayed that Ectoine increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 and melanin-feeding HaCaT cells. Ectoine-inhibited melanogenesis in α-MSH-stimulated B16F10 cells and melanin-feeding HaCaT cells was reversed by pretreatment with the autophagy inhibitor 3-MA or LC3 silencing. In vivo study demonstrated that Ectoine (5 mM) suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. The in vitro and in vivo study demonstrated that Ectoine inhibits melanogenesis and enhances melanin degradation by triggering autophagy. Ectoine could be utilized as a whitening ingredient in cosmetic formulations.