King's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UK.
King's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UK.
Redox Biol. 2020 Oct;37:101708. doi: 10.1016/j.redox.2020.101708. Epub 2020 Sep 8.
Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O levels. Using an O-sensitive phosphorescent nanoparticle probe, we measured an intracellular O level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O. Induction of HO-1 and GCLM by SFN (2.5 μM) was significantly attenuated in cells adapted to 5 kPa O, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O and subjected to hypoxia (1 kPa O, 1 h) and reoxygenation. In cells adapted to 18 kPa O, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 μM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O levels.
缺血性中风与再灌注期间活性氧物种生成的激增有关。静脉溶栓和血管内血栓切除术的治疗窗口狭窄限制了患者的治疗选择。因此，了解调节神经血管氧化还原防御的机制对于改善临床转化至关重要。我们之前在缺血性中风的啮齿动物模型中的研究表明，用萝卜硫素（SFN）预处理激活 Nrf2 防御酶可提供针对神经血管和神经功能缺损的保护。我们在这里进一步研究了 SFN 在适应长期（5 天）高氧（18kPa）和常氧（5kPa）O 水平的小鼠脑微血管内皮细胞（bEnd.3）中的介导保护作用。使用氧敏感的磷光纳米颗粒探针，我们在培养于 5kPa O 下的 bEnd 3 细胞中测量到细胞内 O 水平为 3.4±0.1kPa。SFN（2.5μM）诱导的 HO-1 和 GCLM 在适应 5kPa O 的细胞中显著减弱，尽管 Nrf2 核积累。为了模拟缺血性中风，bEnd.3 细胞适应于 18 或 5kPa O，并进行缺氧（1kPa O，1h）和再氧合。在适应于 18kPa O 的细胞中，再氧合诱导的自由基生成被 PEG-SOD 消除，并用 SFN（2.5μM）预处理显著减弱。沉默 Nrf2 转录消除了 HO-1 和 NQO1 的诱导，并导致再氧合诱导的自由基生成显著增加。值得注意的是，使用发光探针 L-012 和荧光探针 MitoSOX™Red 和 FeRhoNox™-1 检测到的再氧合诱导的氧化应激在培养于 5kPa O 下的细胞中减少，表明脑微血管细胞的氧化还原表型发生改变适应于生理常氧。由于氧化还原和其他细胞内信号通路受到 O 的严重影响，因此针对 Keap1-Nrf2 防御途径的抗氧化治疗的开发靶向缺血再灌注损伤的中风、冠心病和肾病，需要在明确的 O 水平下进行体外研究。
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