Counteracting Skin Aging In Vitro by Phytochemicals

Abstract

The skin is the most extensive organ in the human body. Photo exposure to ultraviolet (UV) rays causes several damages to skin cells, including premature skin aging, the onset of possible DNA mutations, and the risk of developing cancers, including melanoma. Protecting skin from the damaging effects of sun exposure through the application of creams and filters is important to prevent irreversible damages. Several natural extracts and biomolecules with antioxidant activity are widely used in the production of dietary supplements or topical products, for the prevention and treatment of skin affections. Within this context, we pre-treated human skin fibroblasts (HFF1), skin-isolated stem cells (SSCs) and keratinocytes (HaCaT) with two creams containing a specific solar protection factor (SPF) for 72 h and then exposed the cells to UV light. Gene expression analysis was performed for the key cell cycle regulators (p16, p19, p21, p53 and TERT). Cell senescence was assessed by colorimetric assays of beta-galactosidase and antioxidant potential, revealing the ability of treated cells to counteract free radical production as a result of oxidative stress. Finally, possible mutations in DNA induced by photo exposure were studied. The results obtained demonstrated that the tested products elicit positive effects on all skin cell populations, preserving them from photo exposure damages and premature senescence, being also able to increase the DNA repairing mechanisms and inducing a youngest phenotype.

Keywords: antioxidants; bioactive molecules; cell senescence; fibroblasts; gene expression; repairing mechanisms; skin aging; stem cells.

FIGURE 1

MTT Viability assay of HaCaT, HFF1 and SSCs after 72 h of pre‐treatment with creams and exposure to UV. Cell viability was expressed as % cell viability referred to untreated control cells. Data are expressed as mean ± SD (n = 6) referring to the control (****p ≤ 0.001).

FIGURE 2

Proliferation of HaCaT, HFF1 and SSCs after 72 h of pre‐treatment with creams and exposure to UV. Cell proliferation is expressed in OD units as compared to control untreated cells. Data are expressed as mean ± SD (n = 6) referring to the control (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.001).

FIGURE 3

Expression of cell‐cycle regulator genes after 72 h of pre‐treatment with creams and exposure to UV. The expression of p16 (panel A), p19 (panel B) p21 (panel C), p53 (panel D), TERT (panel E) and HSP70 (panel F) was evaluated in HaCaT, HFF1 and SSCs after exposure to UV. The mRNA levels for each gene were normalised to Glyceraldehyde‐3‐Phosphate‐Dehydrogenase (GAPDH) and expressed as fold of change (2−ΔΔCt) of the mRNA levels observed in untreated control cells (Ctrl) defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to the control (*p ≤ 0.05), (**p ≤ 0.01), (***p ≤ 0.001), (****p ≤ 0.0001).

FIGURE 4

Total antioxidant capacity of HaCaT, HFF1 and SSCs after 72 h of pre‐treatment with creams and exposure to UV. Data are expressed as mean ± SD (n = 6, in triplicate with two experiments per group) (****p ≤ 0.001).

FIGURE 5

AP sites formation in HaCaT, HFF1 and SSCs after 72 h of pre‐treatment with creams and exposure to UV. Data are expressed as mean ± SD (n = 6, in triplicate with two experiments per group) (**p ≤ 0.01), (****p ≤ 0.0001).

FIGURE 6

Comet assay in HaCaT, HFF1 and SSCs after 72 h of pre‐treatment with creams and exposure to UV. Data are expressed as mean ± SD (n = 6, in triplicate with two experiments per group) (****p ≤ 0.0001). The figures are representative of different independent experiments. Fields with the highest yield of positively stained cells are shown. Scale bars: 40 μm.

FIGURE 7

Senescence‐associated β‐galactosidase activity. β‐galactosidase was evaluated in HaCaT (panel A), HFF1 (panel B) and SSCs (panel C) after 72 h of pre‐treatment with creams and exposure to UV. All cells were compared to control untreated HSPCs (Ctrl). Scale bar = 100 μm.