Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin

Abstract

Solar ultraviolet (UV) radiation, particularly its UVB (290-320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti-tumor-promoting properties. In this study, we determined the effect of pomegranate-derived products--POMx juice, POMx extract and pomegranate oil (POMo)--against UVB-mediated damage using reconstituted human skin (EpiDerm(TM) FT-200). EpiDerm was treated with POMx juice (1-2 microl/0.1 ml/well), POMx extract (5-10 microg/0.1 ml/well) and POMo (1-2 microl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm(2)) irradiation and was harvested 12 h post-UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate-derived products resulted in inhibition of UVB-induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8-dihydro-2'-deoxyguanosine (8-OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate-derived products resulted in inhibition of UVB-induced (i) collagenase (MMP-1), (ii) gelatinase (MMP-2, MMP-9), (iii) stromelysin (MMP-3), (iv) marilysin (MMP-7), (v) elastase (MMP-12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate-derived products inhibited UVB-induced MMP-2 and MMP-9 activities. Pomegranate-derived products also caused a decrease in UVB-induced protein expression of c-Fos and phosphorylation of c-Jun. Collectively, these results suggest that all three pomegranate-derived products may be useful against UVB-induced damage to human skin.

Figure 1. Effect of POMx juice, POMx extract and POMo on UVB-mediated formation of CPD in three dimensional human reconstituted skin (EpiDerm™ FT-200)

EpiDerm™ FT-200 was topically treated with or without POMx juice (2μl), POMx extract (10 μg) or POMo (2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post-UVB, skin were harvested and immunostaining was performed to detect UVB-induced DNA damage in the form of (A) CPD and (B) 8-OHdG positive cells as described in “Materials and Methods”. Representative sections of immunostaining are shown from three independent experiments with similar results.

Figure 2. Effect of POMx juice, POMx extract and POMo on UVB-mediated protein oxidation in three dimensional human reconstituted skin (EpiDerm™ FT-200)

EpiDerm™ FT-200 was topically treated with or without POMx juice (1-2μl), POMx extract (5-10 μg) or POMo (1-2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post UVB, EpiDerm™ FT-200 was harvested and cell lysates prepared and western blot analysis performed after derivitization of transferred protein PVDF membrane with DNPH. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The values above the figures represent relative density of the bands normalized to β-actin. The immunoblots shown here are representative of three independent experiments with similar results.

Figure 3. Effect of POMx juice, POMx extract and POMo on UVB-mediated increase in protein expression of PCNA in three dimensional human reconstituted skin (EpiDerm™ FT-200)

(A) EpiDerm™ FT-200 was topically treated with or without POMx juice (1-2μl), POMx extract (5-10 μg) or POMo (1-2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). [A] Twelve h post-UVB, EpiDerm was harvested and cell lysates prepared for western blot analysis. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The values above the figures represent relative density of the bands normalized to β-actin. The immunoblots shown here are representative of three independent experiments with similar results. (B) EpiDerm™ FT-200 was topically treated with or without POMx juice (2μl), POMx extract (10 μg) or POMo (2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post-UVB, EpiDerm was harvested and immunostaining was performed to detect UVB-induced PCNA positive cells as described in “Materials and Methods”. Representative sections of immunostaining are shown from five independent experiments with similar results.

Figure 4. Effect of POMx juice, POMx extract and POMo on UVB-mediated increase in tropoelastin in three dimensional human reconstituted skin (EpiDerm™ FT-200)

(A) EpiDerm™ FT-200 was topically treated with or without POMx juice (1-2μl), POMx extract (5-10 μg) or POMo (1-2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). [A] Twelve h post-UVB, EpiDerm was harvested and cell lysates prepared for western blot analysis. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The values above the figures represent relative density of the bands normalized to β-actin. The immunoblots shown here are representative of three independent experiments with similar results. (B) EpiDerm™ FT-200 was topically treated with or without POMx juice (2μl), POMx extract (10 μg) or POMo (2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post-UVB, EpiDerm was harvested and immunostaining was performed to detect UVB-induced PCNA positive cells as described in “Materials and Methods”. Representative sections of immunostaining are shown from five independent experiments with similar results.

Figure 5. Effect of POMx juice, POMx extract and POMo on UVB-mediated increase in MMPs protein and gelatinase activity in human reconstituted skin (EpiDerm™FT-200)

(A) EpiDerm™ FT-200 was topically treated with or without POMx juice (1-2μl), POMx extract (5-10 μg) or POMo (1-2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post-UVB, EpiDerm was harvested and cell lysates prepared for western blot analysis. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The values above the figures represent relative density of the bands normalized to β-actin. The immunoblots shown here are representative of three independent experiments with similar results. (B) EpiDerm™ FT-200 was topically treated as described above. Twelve h post-UVB, culture media was collected and gelatin zymography performed as described in “Materials and Methods”. The gels shown here are representative of three independent experiments with similar results.

Figure 5. Effect of POMx juice, POMx extract and POMo on UVB-mediated increase in MMPs protein and gelatinase activity in human reconstituted skin (EpiDerm™FT-200)

(A) EpiDerm™ FT-200 was topically treated with or without POMx juice (1-2μl), POMx extract (5-10 μg) or POMo (1-2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post-UVB, EpiDerm was harvested and cell lysates prepared for western blot analysis. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The values above the figures represent relative density of the bands normalized to β-actin. The immunoblots shown here are representative of three independent experiments with similar results. (B) EpiDerm™ FT-200 was topically treated as described above. Twelve h post-UVB, culture media was collected and gelatin zymography performed as described in “Materials and Methods”. The gels shown here are representative of three independent experiments with similar results.

Figure 6. Effect of POMx juice, POMx extract and POMo on UVB-mediated phosphorylation of c-jun and expression of c-fos protein in human reconstituted skin (EpiDerm™ FT-200

EpiDerm™ FT-200 was topically treated with or without POMx juice (1-2μl), POMx extract (5-10 μg) or POMo (1-2μl) diluted in EFT media (100 μl/tissue/well) containing 0.1% DMSO for 1 h after which the agents were removed and EpiDerm™ FT-200 washed with PBS and exposed to UVB (60mJ/cm²). Twelve h post-UVB, EpiDerm was harvested and cell lysates prepared for western blot analysis. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The values above the figures represent relative density of the bands normalized to β-actin. The immunoblots shown here are representative of three independent experiments with similar results.