Department of Anatomy, Center for Advanced Medical Education (BK21 project), Inha University School of Medicine, Incheon, Korea.
Department of Anatomy, Center for Advanced Medical Education (BK21 project), Inha University School of Medicine, Incheon, Korea.
Free Radic Biol Med. 2013 Dec;65:468-476. doi: 10.1016/j.freeradbiomed.2013.07.028. Epub 2013 Jul 24.
Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to exert robust neuroprotection in various neuropathological conditions, such as, cerebral ischemia and KA-induced seizure animal models. The neuroprotective effect of EP is attributable to the anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated convergence of anti-inflammatory and anti-oxidative functions of EP and present a novel molecular mechanism underlying anti-inflammatory effects of EP, which is conveyed by p300, a transcriptional co-activator for both Nuclear factor E2-related factor 2 (Nrf2) and p65. In BV2 cells, a microglia cell line, EP induced translocation of Nrf2 from the cytosol to the nucleus and enhanced the expression of hemeoxygenase 1 (HO-1) in a dose-dependent manner and 1h incubation with 10mM EP increased HO-1 to 4.9-fold. Nrf2 was found to translocate from the cytosol to the nucleus beginning 30 min after EP-treatment and binds to the antioxidant response element (ARE) located on HO-1 promoter. Interestingly, LPS-induced inducible NO synthase (iNOS) induction was substantially suppressed in EP-pre-treated BV2 cells and it was reverted by Nrf2 knockdown. We found that EP-induced Nrf2 accumulation in the nucleus recruits p300, a transcriptional co-activator of both Nrf2 and p65, inhibiting p65-p300 interaction. Competition between Nrf2 and p65 for p300 binding was confirmed by glutathione S-transferase (GST) pull down assay and reporter gene analysis. These results demonstrate that EP induced nuclear translocation of Nrf2 which binds to ARE along with p300 and hampers iNOS expression via depleting p300 from p65. This is a novel anti-inflammatory mechanism conveyed by EP, which enhances protective effect by converging anti-inflammatory and anti-oxidative effects and might be applicable to various Nrf2-activating agents, such as phytochemicals.
丙酮酸乙酯（EP）是丙酮酸的简单酯，已被证明在多种神经病理学条件下具有强大的神经保护作用，例如脑缺血和 KA 诱导的癫痫动物模型。EP 的神经保护作用归因于其抗炎、抗氧化和抗凋亡作用。在本研究中，我们研究了 EP 的抗炎和抗氧化功能的收敛，并提出了 EP 抗炎作用的新分子机制，该机制是通过转录共激活因子 p300 介导的，p300 是核因子 E2 相关因子 2（Nrf2）和 p65 的转录共激活因子。在 BV2 细胞（一种小胶质细胞系）中，EP 诱导 Nrf2 从细胞质易位到细胞核，并以剂量依赖的方式增强血红素加氧酶 1（HO-1）的表达，用 10mM EP 孵育 1 小时可将 HO-1 增加到 4.9 倍。EP 处理 30 分钟后，发现 Nrf2 从细胞质易位到细胞核，并与 HO-1 启动子上的抗氧化反应元件（ARE）结合。有趣的是，EP 预处理的 BV2 细胞中 LPS 诱导的诱导型一氧化氮合酶（iNOS）诱导显著受到抑制，并且通过 Nrf2 敲低可逆转。我们发现，EP 诱导的核内 Nrf2 积累招募了 p300，这是 Nrf2 和 p65 的转录共激活因子，抑制了 p65-p300 相互作用。通过谷胱甘肽 S-转移酶（GST）下拉测定和报告基因分析证实了 Nrf2 和 p65 与 p300 结合的竞争。这些结果表明，EP 诱导 Nrf2 向核内易位，与 p300 结合，并通过从 p65 中耗尽 p300 来抑制 iNOS 表达。这是 EP 传达的一种新的抗炎机制，通过收敛抗炎和抗氧化作用增强了保护作用，并且可能适用于各种 Nrf2 激活剂，如植物化学物质。
