Hatori Yuta
Department of Medical Pharmacy, Faculty of Pharmacy, Yasuda Women's University.
Yakugaku Zasshi. 2019;139(12):1523-1530. doi: 10.1248/yakushi.19-00184.
Oxidative stress, including reactive oxygen species (ROS) generation and resulting glutathione oxidation, have been implicated in numerous aspects of cell physiology and human pathology such as cell senescence, cell differentiation, and inflammation. Significant effort has been made to establish methods of analyzing ROS levels and glutathione oxidation within a living cell. The recent development of redox-sensitive green fluorescent protein (GFP) variants enables a robust and accurate estimation of ROS level and glutathione oxidation at subcellular resolution. We created membrane-targeted versions of glutathione and hydrogen peroxide sensors by attaching palmitoylation signals to existing sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively), and demonstrated the nonuniform distribution of these oxidative elements within cytosol. In living cells, cytosolic glutathione is highly reduced, and hydrogen peroxide is barely detected. Nevertheless, near the cytoplasmic side of intracellular vesicular membranes, significant glutathione oxidation and hydrogen peroxide were successfully probed by our sensors, clearly showing the difference between various areas within cytosol. Currently, these sensors are being applied to an intestinal inflammation model which is constituted by co-culturing intestinal epithelial cells and macrophage-like inflammatory cells derived from THP-1. This review covers the current status of studies regarding the association of oxidative stress and intestinal inflammation, with a focus on the redox regulation of intracellular glutathione.
氧化应激，包括活性氧（ROS）的产生以及由此导致的谷胱甘肽氧化，与细胞生理学和人类病理学的诸多方面相关，如细胞衰老、细胞分化和炎症。人们已付出巨大努力来建立分析活细胞内ROS水平和谷胱甘肽氧化的方法。氧化还原敏感型绿色荧光蛋白（GFP）变体的最新进展使得能够在亚细胞分辨率下对ROS水平和谷胱甘肽氧化进行可靠且准确的评估。我们通过将棕榈酰化信号分别连接到现有的传感器（Grx1-roGFP2和roGFP2-Orp1）上，创建了膜靶向型谷胱甘肽和过氧化氢传感器，并证明了这些氧化成分在细胞质中的分布不均一。在活细胞中，胞质谷胱甘肽高度还原，几乎检测不到过氧化氢。然而，在细胞内囊泡膜的细胞质一侧附近，我们的传感器成功检测到了显著的谷胱甘肽氧化和过氧化氢，清楚地显示了细胞质内不同区域之间的差异。目前，这些传感器正应用于一种肠道炎症模型，该模型由共培养肠道上皮细胞和源自THP-1的巨噬细胞样炎症细胞构成。本综述涵盖了关于氧化应激与肠道炎症关联的研究现状，重点关注细胞内谷胱甘肽的氧化还原调节。
