Narayanan Srinivasan V, Dave Kunjan R, Saul Isa, Perez-Pinzon Miguel A
From the Cerebral Vascular Disease Research Laboratories (S.V.N., K.R.D., I.S., M.A.P.-P.), Neuroscience Program (S.V.N., K.R.D., M.A.P.-P.), and Department of Neurology (S.V.N., K.R.D., I.S., M.A.P.-P.), University of Miami Miller School of Medicine, FL; and University of Miami Miller School of Medicine MD/PhD Program (S.V.N.).
Stroke. 2015 Jun;46(6):1626-32. doi: 10.1161/STROKEAHA.115.008921. Epub 2015 Apr 23.
Nuclear erythroid 2 related factor 2 (Nrf2) is an astrocyte-enriched transcription factor that has previously been shown to upregulate cellular antioxidant systems in response to ischemia. Although resveratrol preconditioning (RPC) has emerged as a potential neuroprotective therapy, the involvement of Nrf2 in RPC-induced neuroprotection and mitochondrial reactive oxygen species production after cerebral ischemia remains unclear. The goal of our study was to study the contribution of Nrf2 to RPC and its effects on mitochondrial function.
We used rodent astrocyte cultures and an in vivo stroke model with RPC. An Nrf2 DNA binding ELISA and protein analysis via Western blotting of downstream Nrf2 targets were performed to determine RPC-induced activation of Nrf2 in rat and mouse astrocytes. After RPC, mitochondrial function was determined by measuring reactive oxygen species production and mitochondrial respiration in both wild-type and Nrf2-/- mice. Infarct volume was measured to determine neuroprotection, whereas protein levels were measured by immunoblotting.
We report that Nrf2 is activated by RPC in rodent astrocyte cultures, and that loss of Nrf2 reduced RPC-mediated neuroprotection in a mouse model of focal cerebral ischemia. In addition, we observed that wild-type and Nrf2-/- cortical mitochondria exhibited increased uncoupling and reactive oxygen species production after RPC treatments. Finally, Nrf2-/- astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular antioxidants after RPC treatment.
Nrf2 contributes to RPC-induced neuroprotection through maintaining mitochondrial coupling and antioxidant protein expression.
核红细胞2相关因子2（Nrf2）是一种在星形胶质细胞中高度富集的转录因子，先前已证明其可响应缺血而上调细胞抗氧化系统。尽管白藜芦醇预处理（RPC）已成为一种潜在的神经保护疗法，但Nrf2在RPC诱导的脑缺血后神经保护及线粒体活性氧生成中的作用仍不清楚。我们研究的目的是探讨Nrf2对RPC的贡献及其对线粒体功能的影响。
我们使用了啮齿动物星形胶质细胞培养物和采用RPC处理的体内中风模型。通过Nrf2 DNA结合酶联免疫吸附测定以及对Nrf2下游靶点进行蛋白质免疫印迹分析，来确定RPC诱导的大鼠和小鼠星形胶质细胞中Nrf2的激活情况。RPC处理后，通过测量野生型和Nrf2基因敲除小鼠的活性氧生成和线粒体呼吸来确定线粒体功能。测量梗死体积以确定神经保护作用，通过免疫印迹法检测蛋白质水平。
我们发现，在啮齿动物星形胶质细胞培养物中，RPC可激活Nrf2，并且在局灶性脑缺血小鼠模型中，Nrf2缺失会降低RPC介导的神经保护作用。此外，我们观察到，RPC处理后，野生型和Nrf2基因敲除小鼠的皮质线粒体均表现出解偶联增加和活性氧生成增加。最后，Nrf2基因敲除的星形胶质细胞中线粒体抗氧化剂表达降低，且在RPC处理后无法上调细胞抗氧化剂水平。
Nrf2通过维持线粒体偶联和抗氧化蛋白表达，对RPC诱导的神经保护发挥作用。
