Original Contribution
Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-κB signaling in human epidermal keratinocytes

https://doi.org/10.1016/j.freeradbiomed.2005.12.032Get rights and content

Abstract

Solar ultraviolet (UV) radiation-induced oxidative stress has been implicated in various skin diseases. Here, we report the photoprotective effect of grape seed proanthocyanidins (GSPs) on UV-induced oxidative stress and activation of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways using normal human epidermal keratinocytes (NHEK). Treatment of NHEK with GSPs inhibited UVB-induced hydrogen peroxide (H2O2), lipid peroxidation, protein oxidation, and DNA damage in NHEK and scavenged hydroxyl radicals and superoxide anions in a cell-free system. GSPs also inhibited UVB-induced depletion of antioxidant defense components, such as glutathione peroxidase, catalase, superoxide dismutase, and glutathione. As UV-induced oxidative stress mediates activation of MAPK and NF-κB signaling pathways, we determined the effects of GSPs on these pathways. Treatment of NHEK with GSPs inhibited UVB-induced phosphorylation of ERK1/2, JNK, and p38 proteins of the MAPK family at the various time points studied. As UV-induced H2O2 plays a major role in activation of MAPK proteins, NHEK were treated with H2O2 with or without GSPs and other known antioxidants, viz. (−)-epigallocatechin-3-gallate, silymarin, ascorbic acid, and N-acetylcysteine. It was observed that H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was decreased by these antioxidants. Under identical conditions, GSPs also inhibited UVB-induced activation of NF-κB/p65, which was mediated through inhibition of degradation and activation of IκBα and IKKα, respectively. Together, these results suggest that GSPs could be useful in the attenuation of UV-radiation-induced oxidative stress-mediated skin diseases in human skin.

Section snippets

Chemicals and antibodies

GSPs were kindly provided by the Kikkoman Corp. (Japan), as a generous gift for our research work. Dihydrorhodamine 123 (DHR) was purchased from Molecular Probes (Eugene, OR, USA). OxyBlot Protein Oxidation Detection Kit was purchased from the Intergen Co. (Purchase, NY, USA). The phosphorylated ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), and p38 (Thr180/Tyr182) and nonphosphorylated ERK1/2, JNK, and p38 antibodies and the anti-β-actin antibody were purchased from Cell Signaling Technology,

GSPs inhibit UVB-induced intracellular release of H2O2 in NHEK

Based on our prior studies with UVB and NHEK [14], NHEK were exposed to a UVB dose of 30 mJ/cm2, which resulted in more than 12-fold induction of intracellular release of H2O2 (Fig. 1A), which was determined as a marker of UVB-induced oxidative stress. Pretreatment of NHEK cells with GSPs (10–50 μg/ml) before UVB (30 mJ/cm2) exposure resulted in a dose-dependent inhibition of UVB-induced intracellular release of H2O2 (30–89%; *p < 0.01, ¶p < 0.001) when measured in terms of relative

Discussion

The UVB (290–320 nm) component of solar ultraviolet radiation acts as a tumor initiator, a tumor promoter, and a complete carcinogen [20]. It has been recognized that UVB-induced oxidative stress in an in vitro or in vivo system contributes to several adverse biological effects on the skin [1], [2], [21]. There is considerable evidence that the tumor-promoting effects of UVB radiation are mediated through UVB-induced oxidative stress-mediated activation of signal transduction pathways that

Acknowledgments

This work was supported by funds from USPHS Grant CA104428, the Merit Review Award from the Veterans Administration, and the UAB Skin Diseases Research Center (AR050948-01).

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