Division of Pharmacology and Experimental Therapeutics, Department of Biomedical Sciences & Biotechnologies and National Institute of Neuroscience, School of Medicine, University of Brescia, Italy.
Division of Pharmacology, Department of Neuroscience and National Institute of Neuroscience, School of Medicine, Federico II University of Naples, Italy.
Neurobiol Dis. 2013 Jan;49:177-89. doi: 10.1016/j.nbd.2012.08.018. Epub 2012 Aug 30.
Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter.
With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol.
We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD).
The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation. Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20μg/kg and 200μg/kg) or resveratrol (6800μg/kg) individually. However, the administration of lowest doses of MS-275 (2μg/kg) and resveratrol (68μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-xL promoter and decreased at the Bim promoter.
Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window.
核因子-κB（NF-κB）p50/RelA 是一种具有双重作用的关键分子，在缺血性中风的进展中发挥作用。在有害性缺血中，但不在预处理损伤中，p50/RelA 的神经毒性激活表现为 RelA 特异性赖氨酸 310（K310）乙酰化和其他赖氨酸残基去乙酰化。RelA 乙酰化的紊乱与 Bim 启动子的激活有关。
通过纠正脑缺血中 RelA 改变的乙酰化，我们结合了组蛋白去乙酰化酶（HDAC）1-3 的药理学抑制，这些酶已知可降低全局 RelA 乙酰化，以及 Sirtuin 1 的激活，Sirtuin 1 具有 RelA K310 残基的特异性去乙酰化酶活性。为了实现这一目标，我们测试了临床使用的 HDAC 1-3 抑制剂恩替诺司他（MS-275）和 Sirtuin 1 激活剂白藜芦醇。
我们使用短暂性大脑中动脉闭塞（MCAO）的小鼠模型和暴露于氧葡萄糖剥夺（OGD）的原代皮质神经元。
MS-275 和白藜芦醇的联合使用通过恢复正常的 RelA 乙酰化，在暴露于 OGD 的神经元中产生协同的神经保护作用。这种作用与 MS-275 增加总 RelA 乙酰化的能力以及白藜芦醇通过激活 AMP 激活的蛋白激酶-Sirtuin 1 途径降低 RelA K310 乙酰化的能力相关。协同治疗再现了预处理缺血特有的 RelA 乙酰化状态。暴露于联合药物的神经元完全恢复了最佳的组蛋白 H3 乙酰化。单独用 MS-275（20μg/kg 和 200μg/kg）或白藜芦醇（6800μg/kg）处理 MCAO 小鼠可再现神经保护作用。然而，最低剂量的 MS-275（2μg/kg）和白藜芦醇（68μg/kg）联合使用可减少梗死体积和神经功能缺损。重要的是，即使在中风发作后 7 小时进行治疗，也有效。用治疗小鼠的皮质进行染色质免疫沉淀分析显示，RelA 结合和组蛋白乙酰化在 Bcl-xL 启动子处增加，在 Bim 启动子处减少。
我们的研究表明，表观遗传治疗塑造 RelA 和组蛋白的乙酰化可能是一种有前途的策略，可以通过延长治疗窗口来限制缺血后损伤。
