Exposure to solar ultraviolet (UV) radiation is a causative factor in skin photocarcinogenesis and photoaging, and an urgent need exists for improved strategies for skin photoprotection. The redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defense against environmental electrophilic insult, has recently emerged as an important determinant of cutaneous damage from solar UV, and the concept of pharmacological activation of Nrf2 has attracted considerable attention as a novel approach to skin photoprotection. In this study, we examined feasibility of using tanshinones, a novel class of phenanthrenequinone-based cytoprotective Nrf2 inducers derived from the medicinal plant Salvia miltiorrhiza, for protection of cultured human skin cells and reconstructed human skin against solar simulated UV. Using a dual luciferase reporter assay in human Hs27 dermal fibroblasts pronounced transcriptional activation of Nrf2 by four major tanshinones [tanshinone I (T-I), dihydrotanshinone (DHT), tanshinone IIA (T-II-A) and cryptotanshinone (CT)] was detected. In fibroblasts, the more potent tanshinones T-I and DHT caused a significant increase in Nrf2 protein half-life via blockage of ubiquitination, ultimately resulting in upregulated expression of cytoprotective Nrf2 target genes (GCLC, NQO1) with the elevation of cellular glutathione levels. Similar tanshinone-induced changes were also observed in HaCaT keratinocytes. T-I and DHT pretreatment caused significant suppression of skin cell death induced by solar simulated UV and riboflavin-sensitized UVA. Moreover, feasibility of tanshinone-based cutaneous photoprotection was tested employing a human skin reconstruct exposed to solar simulated UV (80 mJ/cm(2) UVB; 1.53 J/cm(2) UVA). The occurrence of markers of epidermal solar insult (cleaved procaspase 3, pycnotic nuclei, eosinophilic cytoplasm, acellular cavities) was significantly attenuated in DHT-treated reconstructs that displayed increased immunohistochemical staining for Nrf2 and γ-GCS together with the elevation of total glutathione levels. Taken together, our data suggest the feasibility of achieving tanshinone-based cutaneous Nrf2-activation and photoprotection.
Keywords: CHX, cycloheximide; CT, cryptotanshinone; DHT, dihydrotanshinone; DMEM, Dulbecco's modified Eagle's medium; Dihydrotanshinone; H&E, hematoxylin and eosin; HMOX1, heme oxygenase-1; IHC, immunohistochemistry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2; Nrf2, nuclear factor-E2-related factor 2; ROS, reactive oxygen species; SF, sulforaphane; SLL, solar simulated UV light; Skin photoprotection; Solar simulated ultraviolet light; T-I, tanshinone I; T-II-A, tanshinone IIA; Tanshinone I; UVA, ultraviolet; UVB, ultraviolet B; γ-GCS, gamma-glutamate-cysteine ligase.