Additive effect of combined pollutants to UV induced skin OxInflammation damage. Evaluating the protective topical application of a cosmeceutical mixture formulation

Abstract

Since the skin is one of the targets of the harmful effects of environmental insults, several studies have investigated the effects of outdoor stressors on cutaneous tissue. Ozone (O₃), particulate matter (PM), and ultraviolet radiation (UV) have all been shown to induce skin damage through disruption of tissue redox homeostasis, resulting in the so called "OxInflammation" condition. However, few studies have explored whether these stressors can act synergistically in cutaneous tissues. In the present work, we evaluated whether O₃, PM, and UV, which are the most common environmental skin insults, act synergistically in inducing skin damage, and whether this effect could be prevented through topical application of a cosmeceutical formulation mixture (CF Mix) containing 15% vitamin C (l-ascorbic acid), 1% vitamin E (α-tocopherol), and 0.5% ferulic acid. Human skin explants obtained from three different subjects were sequentially exposed to 200 mJ UV light, 0.25 ppm O₃ for 2 h, and 30 min of diesel engine exhaust (DEE), alone or in combination for 4 days (time point D1 and D4). We observed a clear additive effect of O₃ and DEE in combination with UV in increasing levels of several oxidative (4HNE, HO-1) and inflammatory (COX2, NF-κB) markers and loss of barrier-associated proteins, such as filaggrin and involucrin. Furthermore, daily topical pre-treatment with the CF Mix prevented upregulation of the inflammatory and oxidative markers and the loss of both involucrin and filaggrin. In conclusion, this study is the first to investigate the combined effects of three of the most harmful outdoor stressors on human skin and suggests that daily topical application may prevent pollution-induced skin damage.

Keywords: 4HNE; COX2; Inflammation; Pollution; Skin barrier.

Graphical abstract

Fig. 1

Combined exposure to UV light, O₃, and DEE does not alter tissue morphology but increases oxidative stress, which can be counteracted by topical application of a cosmeceutical formulation mixture. H&E staining of ex vivo human skin biopsies untreated or pre-treated with the cosmeceutical formulation mixture and then exposed to UV light alone or in combination with O₃ and DEE for 1 day (a) and 4 days (b).Levels of 4-HNE, a marker of lipid peroxidation, in ex vivo human skin biopsies exposed to UV light alone or in combination with O₃ and DEE and treated with the cosmeceuticals formulation mixture for 1 day (c) an 4 days (d). Green staining represents 4-HNE, and the blue staining (DAPI) for nuclei; original magnification 40x. Right panel represents the immunofluorescence (“c” and “d”) and immunoblotting (“e”) quantification performed by ImageJ. Data are expressed as arbitrary units (averages of three independent experiments), *p < 0.05 CF Mix vs pollutant, ^#p < 0.05 pollutants vs Ctrl by ANOVA) and β-actin was used as loading control (“e”). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Topical application of a cosmeceuticals formulation mixture inhibits outdoor stressor-induced inflammation. Levels of NFκB in ex vivo human skin biopsies exposed to different combinations of pollutants (UV, DEE and O₃) at day 1 (a) and day 4 (b) post-exposure was evaluated using immunofluorescence. Green staining represents NFκB, and the blue staining (DAPI) represents nuclei; Original magnification 40x. Quantification was performed using ImageJ (right panels). (c) Protein levels of COX2 were measured by immunoblotting. All Data are expressed as arbitrary units (averages of three different experiments), *p < 0.05 CF Mix vs pollutant, ^#p < 0.05 pollutants vs Ctrl by ANOVA and β-actin was used as loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Combined exposure to outdoor stressors decreases levels of skin barrier-associated proteins, which is mitigated by topical application of a cosmeceutical formulation mixture. Levels of skin-barrier associated proteins filaggrin (a, b) and involucrin (d, e) were assessed using immunofluorescence on ex vivo human skin biopsies exposed to different combinations of pollutants for 1 day (a, c) and 4 days (b, d) and pretreated with the cosmeceutical formulation mixture. Red staining represents filaggrin (a, b) or involucrin (c,d), and the blue staining (DAPI) represents nuclei; original magnificent 40X. Quantification (right panels) of filaggrin and involucrin levels was performed using ImageJ. Data are expressed as arbitrary units (averages of three different experiments), *p < 0.05 CF Mix vs pollutant, ^#p < 0.05 pollutants vs Ctrl by ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Exposure to combined stressors increases aryl hydrocarbon receptor (AhR) levels in the skin, which is inhibited by topical application of a cosmeceutical formulation mixture. Levels of AhR in ex vivo human skin biopsies exposed to different combination of pollutants (UV, DEE and O₃) at day 1. Green staining represents AhR, and the blue staining (DAPI) represents nuclei; Original magnification 40x. Fluorescence intensity levels were quantified using ImageJ (bottom panel). All Data are expressed as arbitrary units (averages of three different experiments), ^#p < 0.05 CF Mix vs Pollutant ^#p < 0.05 pollutants vs Ctrl by ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)