Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages
Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages
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DOI：
10.1016/j.jnutbio.2010.03.008
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发表时间：
2011-05-01
影响因子：
5.6
通讯作者：
Masella, Roberta
中科院分区：
文献类型：
--
作者：
Vari, Rosaria;D'Archivio, Massimo;Masella, Roberta
原儿茶酸（PCA）是花青素的主要代谢产物，其日摄入量远高于其他多酚类物质。五氯苯甲醚具有生物效应，例如，它诱导抗氧化剂/解毒酶基因表达。本研究旨在确定PCA诱导J774 A.1巨噬细胞谷胱甘肽（GSH）过氧化物酶（GPx）和GSH还原酶（GR）过度表达的分子机制。新的证据表明PCA通过诱导C-JUN NH（2）-末端激酶（JNK）介导的核因子红细胞2（NF-E2）-相关因子2（Nrf 2）磷酸化而增加GPx和GR表达。从用PCA（25 μ M）处理不同时间点的细胞中提取RNA和蛋白质。定量实时聚合酶链反应和免疫印迹分析显示，这两种酶的mRNA（>60%）和蛋白质（>50%）迅速增加。在此之前，Nrf 2在mRNA和蛋白质方面上调，并且通过增加的Nrf 2磷酸化和核转位（+60%）评估其显著活化。通过使用特异性激酶抑制剂和检测活化形式，我们发现JNK是负责Nrf 2活化的主要上游激酶。令人信服的证据提供了PCA诱导的Nrf 2激活和酶表达增加之间的因果关系。通过沉默Nrf 2和使用JNK抑制剂，酶增强被抵消。最后，与ChIP测定，我们证明了PCA激活的Nrf 2特异性结合酶基因启动子中的ARE序列。我们的研究首次证明PCA通过JNK介导的Nrf 2激活发挥重要作用的机制提高巨噬细胞内源性抗氧化潜力。这一知识可能有助于新的饮食为基础的方法，旨在抵消氧化损伤，通过加强内源性防御。(C)2011 Elsevier Inc. All rights reserved.
Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH(2)-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 mu M) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences. (C) 2011 Elsevier Inc. All rights reserved.