College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea.
J Nutr. 2010 Jul;140(7):1211-9. doi: 10.3945/jn.110.121277. Epub 2010 May 12.
Cytoprotective effects of chemopreventive agents may be attributed to the induction of antioxidant enzymes. Among these, the induction of glutamate-cysteine ligase (GCL) protects cells from oxidative injury by increasing glutathione (GSH) content. Nuclear factor erythroid-2-related factor 2 (Nrf2) transcriptionally regulates the expression of genes encoding for GCL and other cysteine-metabolizing enzymes. Despite extensive studies on the components in garlic, little information is available on organosulfur by-products made from garlic. In this study, we investigated whether ajoene, a chemically stable garlic by-product, has the ability to activate Nrf2 and induce GCL, and, if so, what is the role of activating Nrf2 in cytoprotection against oxidative stress. Immunoblottings and reporter gene assays were performed in HepG2 cells. Ajoene treatment activated Nrf2, as indicated by increased phosphorylation and nuclear accumulation of Nrf2, decreased interaction with Kelch-like ECH-associated protein-1, and decreased Nrf2 ubiquitination. Consistently, treatment of ajoene increased antioxidant response element reporter gene activity and the mRNA and protein levels of GCL subunits. Ajoene activated protein kinase C-delta (PKCdelta). Inhibition of PKCdelta activation by rottlerin abrogated its ability to activate Nrf2 and induce GCL, suggesting that ajoene promotes the Nrf2-dependent antioxidant defense system via PKCdelta activation. Consequently, ajoene prevented cell death, GSH depletion, and hydrogen peroxide production elicited by tert-butylhydroperoxide. The important role of Nrf2 in cytoprotection was verified by the reversal of ajoene's ability to protect hepatocytes in Nrf2-knockout mice. Our results demonstrate that ajoene increases PKCdelta-dependent Nrf2 activation, GCL induction, and the cellular GSH concentration, which may contribute to protecting cells from oxidative stress.
化学预防剂的细胞保护作用可能归因于抗氧化酶的诱导。其中,谷氨酸半胱氨酸连接酶 (GCL) 的诱导通过增加谷胱甘肽 (GSH) 含量来保护细胞免受氧化损伤。核因子红细胞 2 相关因子 2 (Nrf2) 转录调节编码 GCL 和其他半胱氨酸代谢酶的基因的表达。尽管对大蒜中的成分进行了广泛的研究,但关于大蒜产生的有机硫副产物的信息很少。在这项研究中,我们研究了化学稳定的大蒜副产物阿霍烯是否具有激活 Nrf2 和诱导 GCL 的能力,如果有,激活 Nrf2 在对抗氧化应激的细胞保护中的作用是什么。在 HepG2 细胞中进行免疫印迹和报告基因分析。阿霍烯处理激活了 Nrf2,如 Nrf2 的磷酸化和核积累增加、与 Kelch-like ECH-associated protein-1 的相互作用减少以及 Nrf2 泛素化减少所表明的。一致地,阿霍烯处理增加了抗氧化反应元件报告基因活性以及 GCL 亚基的 mRNA 和蛋白水平。阿霍烯激活蛋白激酶 C-δ (PKCdelta)。用罗特林抑制 PKCdelta 激活可消除其激活 Nrf2 和诱导 GCL 的能力,表明阿霍烯通过激活 PKCdelta 促进 Nrf2 依赖性抗氧化防御系统。因此,阿霍烯可防止叔丁基过氧化物氢诱发的细胞死亡、GSH 耗竭和过氧化氢产生。在 Nrf2 敲除小鼠中逆转阿霍烯保护肝细胞的能力验证了 Nrf2 在细胞保护中的重要作用。我们的结果表明,阿霍烯增加了 PKCdelta 依赖性 Nrf2 激活、GCL 诱导和细胞内 GSH 浓度,这可能有助于保护细胞免受氧化应激。