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When the Dust Settles: Keratinocyte Differentiation is the Anti-pollution Solution

June 29, 2017 | Contact Author | By: Anne-Françoise Clay, Valère Busuttil, Isabelle Imbert, Christophe Capallere, Laurine Bergeron, Catherine Serre, Christelle Plaza, Gilles Oberto, Karine Cucumel, Jean-Marie Botto and Nouha Domloge, Ashland Specialty Ingredients, Vincience, Nice Sophia-Antipolis, France
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Pollution

Keywords: testing | in vitro | ex vivo | in vivo | skin care | ingredients | study | biomarkers | moisturization

Abstract: This article describes in vitro, ex vivo and in vivo models to examine the effects of PM in skin, with particular interest in a botanical extract to mitigate the described effects. Urban pollution including particulate matter (PM) can affect the skin barrier by inducing numerous cell stresses.

Daily, the human body encounters many stresses to which the skin acts as the first external barrier. Environmental pollution is one stressor and is present in many forms, including volatile organic compounds, cigarette smoke, ozone as well as particulate matter (PM). This persistent presence of urban pollution has become increasingly problematic in large cities worldwide. It affects not only the quality and beauty of skin, but also barrier function; these deleterious effects have been studied and demonstrated.

In human skin, the major pollutants to which detrimental effects can be linked are PM-containing compounds, e.g., heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs), ranging in size from the nano- to the micrometer. As this article explains, to study these effects and develop preventative solutions, in vitro and ex vivo models were designed using PM to mimic urban pollution exposure in human skin biopsies, three-dimensional (3D) reconstructed human epidermises (RHEs) and cultured normal human keratinocytes (NHK).

Beyond the effect of PM on epidermal differentiation and barrier function, possible epigenetic impacts were investigated as well as their consequences in vivo on skin protein carbonylations. A botanical ingredient was then developed and tested for its ability to shield against PM.

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Daily, the human body encounters many stresses to which the skin acts as the first external barrier. Environmental pollution is one stressor and is present in many forms, including volatile organic compounds, cigarette smoke, ozone as well as particulate matter (PM). This persistent presence of urban pollution has become increasingly problematic in large cities worldwide. It affects not only the quality and beauty of skin, but also barrier function; these deleterious effects have been studied and demonstrated.1

In human skin, the major pollutants to which detrimental effects can be linked are PM-containing compounds, e.g., heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs), ranging in size from the nano- to the micrometer. As this article explains, to study these effects and develop preventative solutions, in vitro and ex vivo models were designed using PM to mimic urban pollution exposure in human skin biopsies, three-dimensional (3D) reconstructed human epidermises (RHEs) and cultured normal human keratinocytes (NHK).

Beyond the effect of PM on epidermal differentiation and barrier function, possible epigenetic impacts were investigated as well as their consequences in vivo on skin protein carbonylations. A botanical ingredient was then developed and tested for its ability to shield against PM.

Materials and Methods

In vitro, ex vivo: The in vitro and ex vivo tests described employed both fine suspended particles of < 10 µm (PM10) and small particles of < 2.5 µm (PM2.5)—smaller than cutaneous pores. After exposure to these stressors, the integrity of the skin’s barrier function was evaluated using specific biomarkers or probes, claudin-1, hyaluronic acid, epidermal lipids and Lucifer Yellow skin penetration in skin biopsies, RHEs and cultured NHKs.

Test ingredient: A sustainable botanical ingredient derived from Schinus molle (Brazilian peppertree) extracta, which is rich in bioflavonoids such as quercitrin and miquelianin, was then developed and evaluated for its activities—more particularly, for its potential to protect the quality of the skin exposed to PM.

Clinical study: In addition, twenty volunteers participated in a double-blind, eight-week study of the effects of PM on epigenetics and the potential for the extract to mitigate them. The arms of the subjects were exposed to cigarette smoke, to simulate air pollutant exposure, for a determinate amount of time. Skin samples were then collected by tape-stripping and skin protein carbonylations were analyzed. The effects of the extract on carbonylations also were assessed.

Results: Keratinocytes

As noted, air pollution is associated with the presence of both fine suspended particles (PM10) and small particles (PM2.5), i.e., smaller than cutaneous pores, which can penetrate the skin and directly induce damage. These PM were applied separately to NHK in culture pre-treated or not with the botanical ingredient to evaluate cell stress induced in vitro, as measured by lactate dehydrogenase (LDH) release (see Figure 1a-b; SEM = standard error of the mean). The in vitro results indicate the NHK treated with the botanical extract were better protected from stress induced by both PM10 and PM2.5 exposure, compared with untreated keratinocytes.

Results: Differentiation Markers

After in vitro treatment of keratinocytes with the botanical ingredient, the expression levels of genes including long-non-coding RNAs (lncRNAs) specifically involved in skin renewal and barrier functions were assessed by quantitative real-time PCR (qPCR) in keratinocytes.2, 3 Of particular interest were differentiation antagonizing non-protein coding RNA (DANCR) and tissue differentiation-inducing non-protein-coding RNA (TINCR) (see Figure 2a-b).

Results showed DANCR decreased with epidermal differentiation whereas TINCR increased with epidermal differentiation. This profile is similar to that observed in normal epidermal differentiation. Thus, we conclude the botanical ingredient positively modulated tissue differentiation in vitro, leading to an improvement in the skin barrier function.

Caspase 14 was assessed since this enzyme is required for filaggrin degradation into natural moisturizing factors.

Results: Barrier Biomarkers

To evaluate the effects of the botanical ingredient as a pollution shield, key markers were again evaluated via qPCR, including E-cadherin, involucrin and transglutaminase-1, which are known to play a role in skin cohesion and mechanical resistance to stress. Caspase 14 also was assessed since this enzyme is required for filaggrin degradation into natural moisturizing factors (NMF) and is associated with healthy-looking skin (see Figure 2c).

The mRNA levels of involucrin, transglutaminase-1, caspase 14 and E-cadherin, markers of skin barrier function, were significantly increased in vitro in keratinocytes treated with the botanical ingredient at 0.1% for 48 hr, compared with untreated cells.

Results: Tight Junction

To compare the effects of the botanical ingredient vs. placebo on the skin barrier, the expression of claudin-1, an important protein involved in the integrity of tight junctions, was assessed in human skin biopsies (see Figure 3a). The ex vivo results showed a significant increase of claudin-1 expression (+128.2%) in skin biopsies treated with the botanical ingredient, compared with the placebo.

Results: Moisturization

To keep skin moisturized and protect against environmental stress, levels of hyaluronic acid (HA), a hallmark of youthful-looking skin, also were important to evaluate (see Figure 3b). Ex vivo results showed a significant increase (+58.9%) in HA expression in skin biopsies treated with the botanical ingredient, compared with placebo.

Results: Lipid Expression

To evaluate and visualize the effects of the botanical ingredient on skin lipid content, Nile red staining was used in vitro on NHK and ex vivo in skin biopsies. Nile red binds to and fluoresces intensely in the presence of skin’s hydrophobic epidermal lipids including cholesterol sulfate, phospholipids, sphingomyelin and glucosylceramides—key components for barrier function.

In vitro results showed a significant increase in lipid expression (+102%) in keratinocytes treated with the botanical ingredient at 0.1% for 48 hr, compared with untreated cells (see Figure 4a). Ex vivo results also showed a significant increase in lipid expression (+41%) in biopsies treated with the botanical in the same manner, compared with placebo (see Figure 4b).

Results: Barrier Reinforcement

Changes to skin barrier functioning after SDS stress deterioration were next assessed on RHEs treated with either placebo or the botanical ingredient at 1% for 48 hr, and were then submitted to 0.15% SDS stress for a period of 3 hr (see Figure 5). A significant decrease in passive dye diffusion was observed (-16%) in the RHE pre-treated with the botanical ingredient, compared with RHE treated only with placebo.

The botanical ingredient positively modulated tissue differentiation in vitro, leading to improved skin barrier function.

Results: Clinical Efficacy

Finally, to test the ability of the botanical extract to shield against atmospheric pollution, protein carbonylations were evaluated in vivo in 20 Asian volunteers between the ages of 36 and 65 years.

The volunteers applied a cream containing 1% of the botanical extract or its placebo to each of their forearms. After eight weeks, the subjects’ forearms were exposed to cigarette smoke to mimic air pollution; samples were then collected and protein carbonylation levels were analyzed. A significant decrease in protein carbonylations was observed in forearms treated with the botanical extract, compared with the placebo side, providing positive indication of the extract’s protective activity (see Figure 6).

Conclusion

Protecting skin against urban pollution, especially PM, is an important factor to reducing skin damage and the subsequent appearance of the visible signs of aging. In vitro, the application of a botanical ingredient on primary keratinocytes is shown here to protect against stress induced by exposure to PM. Beneficial modulations of two epigenetic lncRNA biomarkers involved in tissue differentiation also were observed in vitro with application of the botanical ingredient.

Furthermore, skin barrier protection markers as well as epidermal lipid content were enhanced in vitro or ex vivo following application of the botanical ingredient. Taken together, these in vitro tests demonstrate the benefit of this ingredient for preserving the skin’s barrier function ex vivo.

Finally, positive in vivo effects of the botanical ingredient against protein carbonylations verified its ability to guard against air pollution damage to skin, and by doing so, to maintain skin health and subsequent signs of aging.

References

  1. TL Pan et al, The impact of urban particulate pollution on skin barrier function and the subsequent drug absorption, J Dermatol Sci 78(1) 51-60 (2015)

  2. M Ketz et al, Suppression of progenitor differentiation requires the long noncoding RNA ANCR, Genes Dev 26(4) 338-43 (2012)

  3. M Ketz, et al, Control of somatic tissue differentiation by the long non-coding RNA TINCR, Nature 493(7431) 231-5 (2013)

 

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Figure 1. LDH activity measurement

Figure 1

Figure 1. LDH activity measurement in NHK treated or not with 0.1% extract twice daily for 48 hr prior to exposure or not to: PM10 at 250 µg/mL or 500 µg/mL once for 24 hr (a); or PM2.5 at 100 µg/mL for 24 hr (b)

Figure 2. qPCR evaluation

Figure 2

Figure 2. qPCR evaluation of DANCR (a) TINCR (b) and involucrin, transglutaminase-1, caspase 14 and E-cadherin mRNA levels (c) in keratinocytes after 48 hr treatment or not twice daily with 0.1% botanical

Figure 3. Claudin-1 and HA immunofluorescent staining

Figure 3

Figure 3. Claudin-1 (a) and HA immunofluorescent staining (b) in skin biopsies after twice daily, 48-hr treatment with placebo or 1% botanical; 20× objective; green = claudin-1 or HA staining; blue = nucleus staining using DAPI

Figure 4. Quantification of Nile red staining

Figure 4

Figure 4. Quantification of Nile red staining after 48 hr treatment twice daily with 1% botanical ingredient in NHK (a) or skin biopsies (b)

Figure 5. Quantification of Lucifer Yellow penetration

Figure 5

Figure 5. Quantification of Lucifer Yellow penetration via the skin barrier after treatment and SDS stress

 

Figure 6. Variation of SCCP

Figure 6

Figure 6. Variation of stratum corneum carbonylated protein (SCCP); * = significant per student t-test; n = 20 +/- SEM

Footnote

a Elixiance (INCI: Propanediol (and) Water (aqua) (and) Bioflavonoids)

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