Green Tea Extract Protects Human Skin Fibroblasts From Reactive Oxygen Species Induced Necrosis

October 2011 | Volume 10 | Issue 10 | Original Article | 1096 | Copyright © 2011

Jonathan I. Silverberg MD PhD MPH,a,b,c Jared Jagdeo MD MS,a Mital Patel MD,a Daniel Siegel MD MS,a Neil Brody MD PhDa

aDepartment of Dermatology and bCenter for Allergy and Asthma Research, State University of New York, Downstate Medical Center, Brooklyn, NY cDepartment of Dermatology, St. Luke's-Roosevelt Hospital Center, New York, NY

Abstract

Oxidative damage by reactive oxygen species (ROS) plays a major role in skin aging, carcinogenesis and inflammation. Little is known about the protective effects of green tea extract (GTE) on toxic ROS-induced skin death. We use an in vitro model of normal human skin fibroblasts (AG13145) to study the effects of green tea extract (GTE) on hydrogen peroxide (H2O2) induced necrosis. Cell morphology, numbers, apoptosis, necrosis, and ROS were assessed by epifluorescence microscopy and flow cytometry. This study demonstrates that GTE protected from H2O2-induced necrosis in a dose-dependent manner, with highest dose GTE (100 ng/mL) resulting in the most protection from necrosis, as assessed by improved cell morphology, increased cell numbers, and decreased necrosis. The protective effects of GTE on H2O2-induced necrosis appear to be mediated directly by decreasing intracellular ROS. The present study suggests that pretreatment with high doses of GTE could protect from toxic ROS-induced injury of skin in the clinical setting. However, additional studies are necessary to determine the clinical utility of GTE for decreasing skin cell ROS, necrosis and inflammation.

J Drugs Dermatol. 2011;10(10):1096-1101.

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INTRODUCTION

Reactive oxygen species (ROS) contribute to skin aging, carcinogenesis and inflammation.1,2 Increased apoptosis and necrosis, and decreased mitochondrial function result from ROS mediated cellular damage.1-10 Therefore, the study of antioxidants to counteract ROS and associated damage in skin is of great interest.

Green tea contains a number of bioactive ingredients, including epigallocatechin gallate, caffeine and vitamins. These agents have anti-inflammatory properties on skin when administered both topically and orally. Topical application of select green tea polyphenols to human and mouse skin prior to ultraviolet (UV) irradiation can decrease erythema, myeloperoxidase activity, and infiltration of leukocytes.11-13 Oral administration of green tea extract to mice mitigated the effects of psoralen—UVA treatments, including skin erythema, edema, hyperplasia and hyperkeratosis.14

Necrotic cell death in vivo results in the spillage of intracellular contents leading to local inflammation.15 The direct anti-necrosis, and by extension anti-inflammatory, effects of green tea on human skin fibroblasts are unknown. In the present study, we use an in vitro model to study the protective effects of green tea extract (GTE) against high dose hydrogen peroxide (H2O2) -induced necrotic cell death.

METHODS

Cell Culture

Human skin fibroblasts (AG13145, Coriell Institute for Medical Research, Camden, NJ) were grown in Dulbecco's minimum essential medium plus nonessential amino acids (DMEM) plus L-glutamine (Gibco, Grand Island, NY), Penicillin-Streptomycin- Neomycin (Gibco) and 15% fetal calf serum (Gibco); media were changed every 2-3 days. All experiments were carried out on young fibroblasts (passage 7 to 10). Cells were grown in a 95:5% air:CO2 humidified incubator at 37° C.

Experimental Protocol

It has been previously shown that H2O2 induces ROS-mediated apoptosis at low levels and necrosis at high levels.16,17 The concentration of H2O2 [1.2 mM] was used based upon results from our laboratory that showed high levels of exogenously generated intracellular ROS in fibroblasts. Fibroblasts were plated in 35 mm tissue culture dishes (Cellstar, Greiner Bio-one, Frickenhausen, Germany) at a density of 1x105 cells in 2mL of DMEM plus 15% fetal calf serum (Gibco). GTE treatment was performed 24 hours after plating and visual confirmation that all cells were adherent. Plated cells were washed twice with DMEM (Gibco), incubated for four hours with various concentrations of GTE (1, 10, and 100 ng/mL) in a 95:5% air:CO2 humidified incubator at 37° C. After incubation,

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