Figure 1. Bixin upregulates NRF2 signaling and antioxidant defenses in epidermal keratinocytes.

(A) For Oxidative Stress RT² Profiler^™ PCR Expression Array analysis, HEKs were exposed to bixin (20 μM, 24 h) followed by gene expression analysis; upper panel: scatter blot depiction of bixin-induced gene expression (versus untreated); cut-off lines: threefold up- or down-regulation; the insert shows the chemical structure of bixin; bottom panel: numerical expression changes [n=3, mean ± SD; (p<0.05)]. (B) Bixin (0-20 μM, 0-24 h) increased the protein levels of NRF2 and its target genes as assessed by immunoblot analysis; left panel: dose-response, right panel: time course. (C) HaCaT keratinocytes cotransfected with NQO1-ARE firefly luciferase and Renilla luciferase reporters were treated with bixin (0-40 μM) for 16 h. Dual luciferase activities were measured; data are expressed as means ± SD (*p<0.05, ctrl. vs. bixin treated groups). (D) HaCaT keratinocytes were treated with bixin (20 μM; 0-48 h exposure time), and cell lysates were subjected to immunoblot analysis. (E) HaCaT keratinocytes were treated with bixin (0-40 μM, 24 h), and total cellular glutathione was determined [n=3; means ± SD (*p<0.05, ctrl. vs. bixin groups]. (F) HaCaT keratinocytes were exposed to bixin (20 μM; 1 and 24 h exposure time) followed by dye sensitization (generating ¹O₂) and subsequent loading with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA)]. Intracellular oxidative stress was then assessed by flow cytometric determination of DCF fluorescence intensity [means ± SD, n=3; means without a common letter differ (p < 0.05)]. (G) HaCaT keratinocytes were exposed to various anti-oxidants [1 h pretreatment: trolox (1 mM), tiron (500 μM), N-acetyl-L-cysteine (NAC; 10 mM)] followed by addition of bixin (40 μM; 4 h) and NRF2/KEAP1 immunoblot analysis.