30 ANTI-POLLUTION
through the evaluation of Nrf2 and AhR pathways, NF-kB, and DJ-1. Finally, we evaluated the effect of S. chinensis on skin hydration, homogeneity, radiance and luminosity in Chengdu (China).
Materials and methods The urban dust chosen was SRM1649b from Sigma including PAHs and nitro-PAHs, polychlorobiphenyls, chlorinated pesticides, decabromodiphenyl ether, polychlorinated dibenzo-p-dioxins and dibenzofurans, inorganic constituents, heavy metals, and particulate matter (PM2.5 and PM10).
Transcriptomic analyses The transcriptomic approach was conducted on a 3D model of reconstructed human epidermis (RHEs) and consisted to measure by qRT-PCR the effects of S. chinensis extract on the expression of 93 genes involved in response and cellular protection mechanisms against pollution, namely the detoxification pathways, the inflammation and the antioxidant defence.
Effect of extract from
S.chinensis on the protection of human keratinocytes (NHEK) damages caused by pollution After the cytotoxicity evaluation for determination of cell viability using WST1 assay, Normal Human Epidermal Keratinocytes (NHEK) cells were cultured overnight at a 5000 cells/well of density in a 96 well plate, at 37°C, 5% CO2
. The cells
were treated then 24 hours with the S. chinensis extract 0.0.1% or control, then exposed during 6h to urban dust (80 μ
g.ml-1
) and S. chinensis extract. The cells
were fixed with formalin and the expression of AhR, Nrf2, DJ-1, and NF-kB were detected by immunofluorescence. After a step of permeabilisation/saturation, staining of the treated cells with antibodies (anti-AhR polyclonal, anti-Nrf2 polyclonal, anti-DJ-1 polyclonal and anti-NFkB polyclonal) was performed overnight at 4°
C.The secondary antibody AlexaFluor 488 tagged goat antibody was applied 1h at RT. Fluorescent labeling was imaged and quantified by automated microscopy (Array Scan Cellomics TM). The fluorescence was quantified by the bioapplication Compartmental Analysis.
Clinical study
The study was performed by comparison before and after hemi-face application of S. chinensis extract 1% vs placebo, twice a day. Thirty-one women were involved from 21 to 51 years old, with dilated pores or oily or dull skin or spot and with a wash-out of 15 days with placebo. Study was performed during 21 days where subjects have to be at least 4 hours a day in a polluted urban area. The different parameters analysed at D0, D7, D14, and D21 were: Clinical scorage
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Figure 1: Signalling pathway responses to global pollution. Air pollutants induce ROS generation and pro-inflammatory cytokines. Upon ligation by pollutants, the activated AhR translocates from the cytoplasm into the nucleus. This translocated AhR binds with ANRT, resulting in the activation of Cytochrome P450, family 1, member A1 (CYP1A1) transcription. ROS generated by CYP1A1 stimulates the production of TNF-α and IL-8. ROS also activates beneficial cellular responses, including Nuclear factor(erythroid-derived 2)-like 2 (Nrf2) activation. Under normal conditions, Nrf2 localizes in the cytoplasm where it interacts with the actin binding protein, Kelch-like ECH associating protein 1(Keap1), and is rapidly degraded by the ubiquitin-proteasome pathway. Signals from ROS target the Nrf2-Keap1 complex, dissociating Nrf2 from Keap1. The Parkinson’s- associated protein, DJ-1, is indispensable for Nrf2 stabilisation, by affecting Nrf2association with its inhibitor Keap1. Stabilised Nrf2 translocates to the nuclei, binds to the antioxidant response element and thereby regulates the expression of a large battery ofgenes involved in the cellular antioxidant protection, including NADPH quinine oxyreductase(NQO-1), heme oxygenase-1 (HO-1), glutathione (GSH)... ROS can also activate gene transcription via transcription factors, such as NF-kB that can interact directly with specific DNA motifs on promoters of target genes. The transcriptions of several MMP family members are strongly regulated by NF-kB. Increased activities of NF-kB lead to collagen breakdown, the downregulation of type I procollagen, and upregulations of MMPs resulting in premature skin ageing.
and self-assessment, Skin hydration by Corneometer CM825, Radiance and luminosity by glossymeter GL200 and spectrocolorimetry CM2600d.
Statistical analysis Results were expressed as mean value ± SEM and represent the mean of triplicate determinations obtained in four separate experiments. ANOVA followed by Bonferroni post-hoc test were performed by SPSS Software (version 16.00 for Windows, US) and statistical significance was considered at p < 0.05. Concerning the clinical study, descriptive statistics are expressed as mean (± standard-deviation). Evolutions between visits evaluated using analyses of variance in repeated measures.
Results Transcriptomic results Urban dust (80µ
g.mL-1) increases significantly the expression of CYP1A1 (p< 0.001), and CYP1B1 (p< 0.001), and ALDH3A1 (p<0.001). Other effects of urban
dust include over-expression of metalloproteinases MMP-1 (p< 0.01) and MMP-9 (p< 0.04) and an increase in GPX (p<0.006) and NAD(P)H deshydrogenase (p< 0.001). In presence of urban dust, S. chinensis extract (0.01%) activates over-expression of several genes involved in antioxidant response and in detoxification pathway, including FTL (+21%: p< 0.03), and GPX2 (+37%: p< 0.007). It also induces the over expression of SPRR1A (+20%: p<0.02) that is a gene coding for a cornifin-A, functioning as a cross-linked envelope precursor.
Effect of urban dust and co-treatment on NF-kB activation and cellular pathways in human keratinocytes
Urban dust significantly activated the cytoplasmic expression of NF-kB (+16%, p<0.001), when compared to control. Co-treatment of urban dust and S. chinensis extract decreased significantly it expression in cytoplasm (-8%, p< 0.001) and nucleus (-6%, p<0.001).
Urban dust induced an increase in Nrf2 November 2018
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