Rhus coriaria L. Fruit Extract Prevents UV-A-Induced Genotoxicity and Oxidative Injury in Human Microvascular Endothelial Cells

Abstract

Rhus coriaria L. (sumac) is a small plant widely diffused in the Mediterranean region. Its fruit are often consumed as a spice but are also present in traditional medicine of several countries. Recently, interest in this plant has increased and many scientific works reported its beneficial effects including antioxidant and anti-inflammatory properties. Plant extracts can be successfully used against ultraviolet rays, which are able to reach and damage the human skin; however, sumac extracts were never applied to this usage. Thus, in this study, we used a macerated ethanol extract of Rhus coriaria L. dried fruit (mERC) to demonstrate its preventive role against the damage induced by ultraviolet-A rays (UV-A) on microvascular endothelial cells (HMEC-1). In vitro effects of the extract pre-treatment and UV-A exposure were evaluated in detail. The antioxidant capacity was assessed by reactive oxygen species (ROS) formation and cellular antioxidant activity measurement. Genoprotective effects of mERC were investigated as well. Our findings indicate that the extract acts as a cell cycle inhibitor or apoptosis inducer, according to the level of damage. The present work provides new insights into the usage of Rhus coriaria extracts against skin injuries.

Keywords: HMEC-1; Rhus coriaria L.; UV-A; antioxidant; genotoxicity; microvascular endothelial cells; sumac.

Figure 1

ROS production, expressed as Fluorescence Units on protein content (μg), after mERC and UV-A treatment. HMEC-1 were treated for 1 h with 10 or 25 μg/mL of mERC (E10, E25) and exposed to 15 or 20 J/cm² UV-A (T15, T20). Results are expressed as mean ± SEM, n = 5. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. *** p < 0.001 vs. C20, ## p < 0.05 vs. T20.

Figure 2

Effect of Rhus coriaria L. extract and UV-A rays on total cellular antioxidant activity, measured as mM Trolox equivalents after normalization on cell’s viability. HMEC-1 were treated for 1 h with 10 or 25 μg/mL of mERC (E10, E25) and exposed to 20 J/cm² UV-A (T20). Results are expressed as mean ± SEM, n = 5. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ** p < 0.05 *** p < 0.001 vs. C20, # p < 0.01 vs. T20.

Figure 3

Effect of Rhus coriaria L. and UV-A rays on genotoxicity. Alkaline Comet assay evaluated DNA damage through (a) %DNA in Tail, (b) Tail length and (c) Tail moment (%DNA in Tail* Tail length). HMEC-1 cells were treated for 1 h with 25 μg/mL of mERC (E25) and exposed to 10, 15 or 20 J/cm² UV-A (T10, T15, T20). Results are expressed as mean of medians ± SEM, n = 6. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. * p < 0.05 *** p < 0.001 vs. C PBS, ### p < 0.001 vs. T20.

Figure 4

Comparison between Alkaline Comet assay performed after 0 and 24 h from UV-A exposure, expressed as Tail moment. HMEC-1 cells were treated for 1 h with the extract (25 μg/mL, E25) and exposed to 20 J/cm² UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis.* p < 0.05 *** p < 0.001 vs. C20, ## p < 0.01 ### p < 0.001 vs. T20 0h.

Figure 5

γ-H2AX formation after UV-A irradiation and mERC pre-treatment. DNA DSB quantification through immunofluorescence revealing high (>10) γ-H2AX foci presence. HMEC-1 cells were treated for 1 h with mERC (10 or 25 μg/mL, E10 and E25) and exposed to 10, 15, 20 J/cm² UV-A (T10, T15, T20). Results are expressed as mean ± SEM, n = 4. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. * p < 0.05 *** p < 0.001 vs. C PBS.

Figure 6

Effect of mERC and UV-A radiation on chromosome mis-segregation, measured as micronuclei percentage, detected by immunofluorescence. HMEC-1 cells were treated for 1 h with mERC (10 or 25 μg/mL, E10 and E25) and exposed to 10, 15, 20 J/cm² UV-A (T10, T15, T20). Results are expressed as mean ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ** p < 0.01 *** p < 0.001 vs. C PBS, ## p < 0.01 vs. T10.

Figure 7

Characterization of genotoxic damage induced by UV-A, and mERC extract’s role. Modified Comet assay measured direct DNA damage using (a) T4 PDG enzyme recognizing CPDs; indirect i.e., oxidative damage was identified by (b) ENDO III for oxidized pyrimidines or (c) FPG for oxidized purines. HMEC-1 cells were treated for 1 h with mERC (25 μg/mL, E25) and exposed to 20 J/cm² UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ### p < 0.001 vs. T20, °° p < 0.01 °°° p < 0.001 vs. T20+enzyme.

Figure 8

Cytotoxicity of UV-A rays and effects of Rhus coriaria pre-treatment, flow cytometry evaluation. Cellular population was grouped in (a) living cells and (b) apoptotic cells. HMEC-1 cells were treated for 1 h with 10 or 25 μg/mL of mERC (E10, E25) and exposed to 15, 20, 25 J/cm² UV-A (T15, T 20, T25). Results are expressed as mean ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. C PBS.

Figure 9

Flow cytometry cell cycle analysis after Rhus coriaria and UV-A treatment. Cellular population was grouped in five phases, comprising (a) G1 phase, (b) S phase, (c) SubG1 phase. HMEC-1 cells were treated for 1 h with 10 or 25 μg/mL of mERC (E10, E25) and exposed to 15, 20, 25 J/cm² UV-A (T15, T20, T25). Results are expressed as mean ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. C PBS.