Expression (fold/control)
86 NATURALS
reporter gene. Emitted bioluminescence was quantified using a luminometer set for 560 nm readings.
Gene expression qPCR was performed by extracting mRNA from human skin keratinocytes that was subsequently copied into cDNA using a reverse transcriptase. A specific AQP3 primer was used. AQP3 mRNA expression was quantified using the Delta Ct method. The Brasenia extract was tested at 50 µg/ml. TGFb (10 ng/ml) was used as a positive control. Transcriptomic analysis was performed based on the RNA Seq method. Total mRNA was isolated and enriched from human skin keratinocytes and copied as cDNA by a reverse transcriptase enzyme. High-throughput sequencing of cDNA fragments was performed and results were aligned with a reference genome sequence. The Brasenia extract was tested at concentrations between 25 and 100 µg/ml.
Identification of skin barrier and water transporter components In vitro reconstructed skin substitutes were produced from human keratinocytes and fibroblasts. Skin substitutes were grown at the air/liquid interface for 14 days and incubated, or not (control) with the Brasenia extract (50 µg/ml) for five days. Skin substitutes were then embedded in
paraffin wax blocks. Five micrometre sections were reacted with anti-loricrin, -involucrin, -filaggrin or -AQP3 specific antibodies and revealed by immunofluorescence. Fluorescence intensity (green) was measured by pixel intensity using an appropriate software. Cell nuclei (blue) were counterstained with DAPI.
Results and discussion The skin barrier function is part of the epidermis and consist in a highly orchestrated scenario controlling the synthesis, cell migration and appropriate positioning of structural and functional components. The transcription factor EGR3 was recently
identified as a master regulator of the skin barrier function especially in modulating the expression of the late differentiation components loricrin, involucrin and filaggrin.4 The gene expression of EGR3 is stimulated in the presence of the Brasenia extract in a concentration-dependent fashion (Figure 1). Parallel to the EGR3 expression, incubation of keratinocytes with the Brasenia extract led to an up-regulation of the synthesis of loricrin (+80%), involucrin (+47%) and filaggrin (+31%) (Figure 2). Loricrin is the most abundant component of the stratum corneum (SC), and along with involucrin and filaggrin are mainly expressed in the stratum granulosum (SG) of the epidermis. Interestingly, the SG is also the site of EGR3 expression. Filaggrin is synthesized as a precursor
– profilaggrin – that is processed during its migration path between the SG and the SC. In the terminal stages of keratinocyte differentiation, filaggrin is finally degraded into individual amino acids added to the pool of
PERSONAL CARE March 2024 skin natural moisturizing factors (NMF).5 Our
results are in line with a stimulating action of the Brasenia extract on the synthesis of the skin barrier actors loricrin, involucrin and filaggrin through the EGR3-regulated axis. Aquaporin-3 (AQP3) is also an active player
in skin hydration. AQP3 integrates into the cell membrane and form pores allowing selective permeation of water and glycerol. This supports the role of AQP3 in promoting skin hydration and maintaining/repairing the water permeability barrier. Furthermore, importing glycerol (also a
NMF) into the cell could lead to the formation of signaling molecules (phosphatidylglycerol) or for energy generation. AQP3 is also involved in keratinocyte proliferation, early differentiation and viability.6
AQP3 is
produced in the stratum basale (SB) layer of the epidermis where it fills up the whole cytoplasm. As keratinocytes migrate upward into
the stratum spinosum (SS), AQP3 becomes glycosylated and strongly localized at the cell periphery forming transmembrane pores (Figure 3A). The Brasenia extract displays a strong stimulating action of the AQP3 protein signal (+86%) (Figure 3B). This effect is paralleled by an increase in the corresponding gene expression (Figure 3C).
5 4 3 2 1
0 25 50 100 Barrier & Beyond (µg/ml)
Figure 1: Increased expression of the transcription factor EGR3 in the presence of the Brasenia extract
The action of the Brasenia extract on APQ3
synthesis is likely to be mediated through a pathway different from that controlled by EGR3. The latter is itself expressed in the SG layer where AQP3 disappears to become undetectable in the SC (Figure 3).7 The epidermal barrier is not only important
for the skin water management but is also involved in protecting internal organs against environmental insults. On a daily basis, the skin is exposed to various stresses that elicit pro-oxidative reactions within cells. The probe DH-RHO 123 is able to permeate the cell membrane and once into the cytoplasm will react with ROS leading to a green fluorescent moiety providing a mean to monitor stress- induced oxidative reactions. We sought to investigate the protective
intracellular action of the Brasenia extract when cells are exposed to radiation or chemical insults. The presence of the Brasenia extract
successfully prevented the pro-oxidative surge elicited by exposure of WS1 human skin fibroblasts to UVB (-81%) and Fe3+
(-71%)
(Figure 4). More studies will be necessary to unravel the mechanism(s) of action but the Brasenia extract appears to exert an antioxidant action either directly and/ or through the induction of downstream protective pathways. Parallel to its antioxidant action, the
Brasenia extract may also act through anti- inflammatory pathways. When incubated with activated macrophages, the Brasenia extract inhibits the production of the pro- inflammatory cytokines IL-1b, IL-6, IL-8 and TNFa (Figure 5). Furthermore, it also prevents the activation of the transcription factor NF-kB (Figure 5). Interestingly, NF-kB is activated in response to stressors such as ROS, heavy metals and UVB and plays an important role in the induction of senescence-associated secretory phenotype.8 The action of the Brasenia extract in
protecting against the UVB is especially interesting as this stress is also known to disrupt the barrier function linked to an increase in TEWL.9
Fe3+ can penetrate cells via
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