Department of Dermatology, 1st Affiliated Hospital, Zhejiang University School of Medicine, 79# Qing Chun Road, Hangzhou 310003, China.
J Dermatol Sci. 2010 Apr;58(1):19-27. doi: 10.1016/j.jdermsci.2010.02.002. Epub 2010 Feb 16.
Genistein, as an active compound of dietary antioxidants, has shown considerable promise as an effective agent against aging process. However, the effect of genistein on skin photoaging and the associated mechanism remain unclear.
To delineate the effect of genistein on UVB-induced senescence in human dermal fibroblasts (HDFs) with emphasis on the mechanism of oxidative pathway regulated by p66Shc involved in the events.
HDFs were induced to premature senescence by repetitive subcytotoxic doses of UVB irradiation. Cellular apoptosis and DNA cell cycle were analyzed using flow cytometry. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by ELISA. Mutation levels of two large deletions of mitochondrial DNA, 4977bp and 3895bp deletion, were determined by quantitative PCR. Western blot was applied to detect the expression and activation of p66Shc (the 66-kilodalton isoform of the growth factor adapter Shc) and FKHRL1 (a forkhead protein that is intimately linked with intracellular oxidation).
Strong activity of senescence-associated beta-galactosidase (SA-beta-gal), high percent of cell apoptosis as well as cell cycle arrest in G0/G1 phase, and increased intracellular oxidative stress were observed in HDFs irradiated by UVB. Genistein exerted dramatically protective effects on HDFs in a dose-dependent manner. Elevated copy numbers of large deletions in mitochondrial DNA were also inhibited by genistein. Down-regulation of total and phosphorylated p66Shc on Ser36, as well as FKHRL1 and its phosphorylation on Thr32, were observed after genistein treatment.
The results indicate that genistein protects UVB-induced senescence-like characteristics in HDFs via maintenance of antioxidant enzyme activities and modulation of mitochondrial oxidative stress through down-regulation of a p66Shc-dependent signaling pathway, which may provide potential prevention against skin aging and even photoaging.
染料木黄酮作为膳食抗氧化剂的一种活性化合物，在对抗衰老过程方面显示出相当大的潜力。然而，染料木黄酮对皮肤光老化的影响及其相关机制尚不清楚。
阐述染料木黄酮对 UVB 诱导的人真皮成纤维细胞（HDFs）衰老的影响，并重点探讨涉及 p66Shc 依赖性信号通路的氧化途径的调控机制。
采用重复亚细胞毒性剂量的 UVB 照射诱导 HDFs 提前衰老。采用流式细胞术分析细胞凋亡和 DNA 细胞周期。通过 ELISA 检测超氧化物歧化酶（SOD）和丙二醛（MDA）的细胞内水平。通过定量 PCR 检测线粒体 DNA 两个大片段缺失（4977bp 和 3895bp 缺失）的突变水平。采用 Western blot 检测 p66Shc（生长因子衔接子 Shc 的 66kDa 同工型）和 FKHRL1（与细胞内氧化密切相关的叉头蛋白）的表达和激活。
UVB 照射的 HDFs 中，衰老相关的β-半乳糖苷酶（SA-β-gal）活性增强，细胞凋亡率高，细胞周期停滞在 G0/G1 期，细胞内氧化应激增加。染料木黄酮呈剂量依赖性地对 HDFs 发挥显著的保护作用。线粒体 DNA 大片段缺失的拷贝数也被染料木黄酮抑制。染料木黄酮处理后，总 p66Shc 和 Ser36 磷酸化、FKHRL1 及其 Thr32 磷酸化水平均下调。
研究结果表明，染料木黄酮通过维持抗氧化酶活性和调节线粒体氧化应激，下调 p66Shc 依赖性信号通路，从而保护 UVB 诱导的 HDFs 出现衰老样特征，这可能为皮肤衰老甚至光老化的预防提供潜在途径。
