SKIN PROTECTION 79
It is important to protect this pool of skin stem cells against oxidative stress to restore a better cell-turnover and to fight skin ageing.
recognised to contribute to stem cell ageing.21
Data in Figure 1 show that the new
strombine derivative amino-acid can protect epidermal stem cells from oxidative stress in a dose-dependent way, respectively +26.3%* at a dose of 0.00001%, +34.2%* at a dose of 0.0001% and 42.4%* at a dose of 0.001% Protocol: Human keratinocytes were obtained from a 51 year old woman donor. Cells were cultivated at 37°C in a humid atmosphere and 5% CO2
. The culture was
then enriched into stem cells using Goodell method.22
After enrichment, cells were
incubated for 24h without (control) or with an increasing concentration of the amino- acid (0.00001, 0.0001 and 0.001% v/v). Then UVB irradiation was applied (30 mJ/cm²) or not (control). Finally, cells were incubated again with the amino-acid for 13 days. At the end of the incubation, cell viability assay was performed (dosage of intracellular phosphatase activity). All conditions were tested in triplicate (n=3).
Ex-vivo skin regeneration assay The skin thins progressively over adult life at a rate that accelerates with age. Epidermal thickness decreases about 6.4% per decade and even faster in women. This is due to a lower keratinocyte cells number as well as a slower cell turnover.23
The phenomenon is
even increased in the case of oxidative stress as the high level of ROS will induce premature cell senescence resulting in a growth arrest of cells.24
With external
assaults and build-up of oxidative stress, there is an accumulation of senescent cells in skin, with an irreversible arrest of proliferation and an incapacity to divide.25 Ki-67 is used as a marker of cell proliferation.26, 27 Data obtained from the ex vivo assay show that the new strombine derivative
Control 0.001
Figure 2: Ki67 specific immunostaining in skin biopsies.
amino-acid, at a low dosage (0.001%), can stimulate epidermal cell renewal (+37,1%*), even under oxidative stress, thus promoting cell renewal and epidermis turnover. Protocol: Skin biopsies were obtained
from woman donor of 44 years old. They were incubated over 24 hours at 37°C in a humid atmosphere and 5% CO2
, without
(control) or with an increasing concentration of the amino-acid (0.00001, 0.0001 and 0.001% v/v, diluted at 10% in DMSO and added to the culture medium). At the end of the incubation period, explants were fixated in paraffin and sliced twice using microtome. Cell division was assessed using a specific immunostaining of Ki67 protein. Positive cells we counted on 5 different microscopic fields randomly chosen on the 2 different cuts.
In vitro DNA repair assay Activation of PARP is one of the early DNA damages responses among other DNA sensing and repair molecules. PARP constitute a family of cell signalling enzymes that have emerged as critical regulatory components of the immediate cellular response to DNA damage. They play a key role in maintaining genome integrity through modulation of multiple cellular responses (including base excision
5000 4000 3000 2000 1000 0
4788* 4406 3665 4176
18000 14000 10000 6000 2000 0
Control 0.00001 0.0001 Collagen-1 0.001 16759* 12785 10902 11751 +42.4* +7.5
Control No UV
0.001
Control UV
Figure 3: Levels of PARP Activity in keratinocytes. (percentage versus control / p<0.05)
repair, necrosis and apoptosis) in the face of oxidative stress.28 Data in Figure 3 show that the new
strombine derivative amino-acid (at 0.01%) can stimulate DNA repair mechanism increasing PARP activity (+42.4%*), only in case of oxidative stress. Protocol: keratinocytes were cultured at 37°C in a humid atmosphere and 5% CO2
,
when confluences was reached, on batch was exposed to UVB irradiation (200 mJ/cm²), then incubated without (control) or with an increasing concentration of amino-acid (0.00001, 0.0001 and 0.001% v/v, pre-diluted at 10% in DMSO and added to the culture medium). At the end of the incubation period, PARP activity was quantified. Second batch was performed the same way without UVB irradiation. All conditions were tested in triplicate (n=3).
Control 0.00001 0.0001 Fibronectin Figure 4 & 5: Levels of Pro-collagen-I and Fibronectin in culture medium (ng/ml/ p<0.05). February 2020 0.001
In vitro and ex vivo anti-ageing efficacy Dermal epidermal junction (DEJ) is an acellular region of the skin that separates the epidermis and the dermis. It makes the link between basal layer of the epidermis
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0.001
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