Korean J Physiol Pharmacol.  2016 May;20(3):269-277. 10.4196/kjpp.2016.20.3.269.

Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts

• Baek B
• Lee SH
• Kim K
• Lim HW
• Lim CJ

• KMID: 2285599
• DOI: http://doi.org/10.4196/kjpp.2016.20.3.269

Abstract

Ellagic acid (EA), an antioxidant polyphenolic constituent of plant origin, has been reported to possess diverse pharmacological properties, including anti-inflammatory, anti-tumor and immunomodulatory activities. This work aimed to clarify the skin anti-photoaging properties of EA in human dermal fibroblasts. The skin anti-photoaging activity was evaluated by analyzing the reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity levels as well as cell viability in dermal fibroblasts under UV-B irradiation. When fibroblasts were exposed to EA prior to UV-B irradiation, EA suppressed UV-B-induced ROS and proMMP-2 elevation. However, EA restored total GSH and SOD activity levels diminished in fibroblasts under UV-B irradiation. EA had an up-regulating activity on the UV-B-reduced Nrf2 levels in fibroblasts. EA, at the concentrations used, was unable to interfere with cell viabilities in both non-irradiated and irradiated fibroblasts. In human dermal fibroblasts, EA plays a defensive role against UV-B-induced oxidative stress possibly through an Nrf2-dependent pathway, indicating that this compound has potential skin antiphotoaging properties.

Keyword

Ellagic acid; Glutathione; Superoxide dismutase; UV-B radiation

MeSH Terms

Cell Survival
Ellagic Acid*
Fibroblasts*
Glutathione
Humans*
Matrix Metalloproteinase 2
Oxidative Stress*
Plants
Reactive Oxygen Species
Skin
Superoxide Dismutase
Ellagic Acid
Glutathione
Matrix Metalloproteinase 2
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Fig. 1 The chemical structure of ellagic acid (EA).

• 

  Fig. 2 Attenuating effect of EA on the reactive oxygen species (ROS) elevation in human dermal fibroblasts under UV-B irradiation.Fibroblasts were subjected to fresh media with the varying concentrations (0, 5, 12 or 30 µmol/L) of EA for 30 min before irradiation. The intracellular ROS levels were determined using both DCFH-DA in a microplate fluorometer (A) and dihydrorhodamine 123 via confocal microscopic analysis (B). In A, ROS levels are represented as DCF fluorescence arbitrary units expressed as the percentage of control. In the lower panel of B, the ROS-associated fluorescent signals were quantified using Adobe Photoshop software. Data are presented as % of control versus the non-irradiated control (Mean±SD, n=3). *p<0.05; **p<0.01; ***p<0.001 versus the non-treated control (UV-B irradiation alone).

• 

  Fig. 3 Non-toxic effects of EA on the cellular viability in human dermal fibroblasts without (A) or with (B) UV-B irradiation.Fibroblasts were subjected to fresh media with the varying concentrations (0, 5, 12 or 30 µmol/L) of EA for 30 min before irradiation. The viable cell numbers were determined using MTT assay. Data are presented as % of control versus the non-treated (A) or non-irradiated (B) control (Mean±SD, n=3).

• 

  Fig. 4 Attenuating effect of EA on the gelatinolytic activity of promatrix metalloproteinase-2 (proMMP-2) in the conditioned media from human dermal fibroblasts under UV-B irradiation.Fibroblasts were subjected to fresh media with the varying concentrations (0, 5, 12 or 30 µmol/L) of EA for 30 min before irradiation. In A, the proMMP-2 gelatinolytic activity in conditioned medium was detected using gelatin zymography. The relative band strength was determined with densitometry using ImageJ software. In B, the equal loading of conditioned media was shown by the silver staining. Data are presented as % of control versus the non-irradiated control (Mean±SD, n=3). *p<0.05 versus the non-treated control (UV-B irradiation alone).

• 

  Fig. 5 Attenuating effect of EA on promatrix metalloproteinase-2 (proMMP-2) protein levels in human dermal fibroblasts under UV-B irradiation.Fibroblasts were subjected to fresh media with the varying concentrations (0, 5, 12 or 30 µmol/L) of EA for 30 min before the irradiation. The proMMP-2 proteins were determined using western blotting analysis with anti-MMP-2 antibodies. GAPDH was used as a protein loading control. The relative band strength was determined with densitometry using ImageJ software. Data are presented as % of control versus the non-irradiated control (Mean±SD, n=3). *p<0.05; ***p<0.001 versus the non-treated control (UV-B irradiation alone).

• 

  Fig. 6 Enhancing effects of EA on the total GSH (A) and SOD (B) activity levels in human dermal fibroblasts under UV-B irradiation.Fibroblasts were subjected to fresh media with the varying concentrations (0, 5, 12 or 30 µmol/L) of EA for 30 min before the irradiation. In A, total GSH content, expressed as µg/mg protein, was determined with an enzymatic recycling assay using GR. In B, total SOD activity was measured using a spectrophotometric assay and presented as % of control versus the non-irradiated control (Mean±SD, n=3). ##p<0.01 versus non-irradiated control; **p<0.01; ***p<0.001 versus non-treated control (UV-B irradiation alone).

• 

  Fig. 7 Enhancing effect of EA on the Nrf2 levels in human dermal fibroblasts under UV-B irradiation.Fibroblasts were subjected to fresh media with the varying concentrations (0, 5, 12 or 30 µmol/L) of EA for 30 min before the irradiation. The Nrf2 proteins were determined using western blotting analysis with anti-Nrf2 antibodies. GAPDH was used as a protein loading control. The relative band strength was determined with densitometry using ImageJ software. Data are presented as % of control versus the non-irradiated control (Mean±SD, n=3). ##p<0.01 versus non-irradiated control; **p<0.01 versus non-treated control (UV-B irradiation alone).

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