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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.

Analyzing the Redox Status of Intracellular Glutathione and Its Application to an Intestinal Bowel Disease Model