Myconoside and Calceolarioside E Restrain UV-Induced Skin Photoaging by Activating NRF2-Mediated Defense Mechanisms

Abstract

Chronic and excessive ultraviolet (UVA/UVB) irradiation exposure is known as a major contributor to premature skin aging, which leads to excessive reactive oxygen species generation, disturbed extracellular matrix homeostasis, DNA damage, and chronic inflammation. Sunscreen products are the major preventive option against UVR-induced photodamage, mostly counteracting the acute skin effects and only mildly counteracting accelerated aging. Therefore, novel anti-photoaging and photopreventive compounds are a subject of increased scientific interest. Our previous investigations revealed that the endemic plant Haberlea rhodopensis Friv. (HRE) activates the antioxidant defense through an NRF2-mediated mechanism in neutrophiles. In the present study, we aimed to investigate the photoprotective potential of HRE and two of its specialized compounds-the phenylethanoid glycosides myconoside (MYC) and calceolarioside E (CAL)-in UVA/UVB-stimulated human keratinocytes in an in vitro model of photoaging. The obtained data demonstrated that the application of HRE, MYC, and CAL significantly reduced intracellular ROS formation in UVR-exposed HaCaT cells. The NRF2/PGC-1α and TGF-1β/Smad/Wnt signaling pathways were pointed out as having a critical role in the observed CAL- and MYC-induced photoprotective effect. Collectively, CAL is worth further evaluation as a potent natural NRF2 activator and a promising photoprotective agent that leads to the prevention of UVA/UVB-induced premature skin aging.

Keywords: NRF2; calceolarioside; keratinocytes; myconoside; photoaging; ultraviolet radiation.

Figure 1

Phototoxicity irradiation doses of UVA/UVB (A), UVA (B), and UVB (C) on cell viability in human keratinocytes. For each irradiation mode, the half-maximal inhibitory concentration (IC₅₀) was calculated. Error bars indicate the mean ± SEM for cell viability expressed as a percentage from the native dark control.

Figure 2

Photoprotective effects of Haberlea rhodopensis extract (HRE; (A)), myconoside (MYC; (B)), and calceolarioside E (CAL; (C)) in UVA/UVB-exposed human keratinocytes. Error bars indicate the mean ± SEM for cell viability expressed as a percentage from the dark control. Statistical significance between the groups was determined via one-way ANOVA, followed by Tukey’s post hoc test; * p < 0.05 and ** p < 0.01 compared to the UVA/UVB group.

Figure 3

Myconoside (MYC) and calceolarioside E (CAL) reduce UVA/UVB-induced ROS production in human keratinocytes. The fluorescence images of the experimental groups stained with DCF-DA reagent after UVA/UVB irradiation were observed at 20x magnification (scale bar = 50 μm) with an FITC filter (A). Quantification of the normalized fluorescence intensity of intracellular ROS generation in HaCaT cells (B). The quantification of the stained oxygen radicals was measured as an average pixel intensity using ImageJ software version 1.53t and was represented as the normalized pixel intensity against the UVA/UVB group. Statistical significance between the groups was determined via one-way ANOVA, followed by Tukey’s post hoc test; ** p < 0.01 compared to the UVA/UVB group.

Figure 4

Gene expression profile modulation associated with UVA/UVB-induced photoaging by H. rhodopensis extract (HRE), calceolarioside E (CAL), and myconoside (MYC). Clustergram and heatmap of the relative gene expression analysis from the RT-qPCR. The results are expressed as the mean ± SEM compared to the UVA/UVB-exposed model group from three independent experiments. * p < 0.05 and ** p < 0.01 compared to the photoaging model group.

Figure 5

Calceolarioside E (CAL) activates NRF2 signaling in UVA/UVB-photoaged HaCaT cells. Western blot of STAT1 (A) and NRF2 (B) 24 h after UVA/UVB irradiation and 1 hr pre-treatment with HRE (1, 5, and 10 μg/mL), MYC, or CAL (1, 5, and 10 μM) and representative bands from the Western blot analysis (C). The results are presented as the mean ± SEM from three independent experiments. ** p < 0.01 compared to the UVA/UVB group.

Figure 6

Molecular-based modeling of the anti-photoaging activity of myconoside (MYC) and calceolarioside E (CAL) mediated through NRF2/PGC-1α and TGF-β/Smad/Wnt signaling in UVA/UVB-exposed HaCaT cells.