Quantitative Fluorescence Imaging of the Intracellular Redox State by Real-Time Spatial and Temporal Simultaneous Analysis of O2•- Levels and Keap1 Translocation

Dysregulated redox homeostasis under pathological conditions can eventually culminate in oxidative stress and associated disease damage. Spatial and temporal regulation of intracellular redox states involves two crucial parameters for elucidating oxidative stress-related molecular mechanisms. However, the lack of methods for real-time analysis of redox states is a considerable hurdle for the in-depth interpretation of pathogenic mechanisms. Herein, given the over-produced reactive oxygen species (ROS) and the translocation of redox-sensitive proteins as the potential biomarkers of oxidative stress, we developed a novel ROS-macromolecular protein synergistic imaging strategy that relied on a small-molecule fluorescent CPR-SK probe. The CPRSK specifically activated the caffeic acid moieties or targeting peptides (EWWW) toward the biomarkers, including superoxide (O-2(center dot-)) fluctuations and Keap1 translocation, achieving simultaneous real-time imaging of dual molecular events during oxidative stress. Importantly, in situ, CPR-SK exhibited both gentle elevation of O-2(center dot-) and subsequent migration of Keapl from the cytoplasm to the nucleus, which were key indicators for determining slight injuries induced by hepatic ischemia-reperfusion. The results clearly demonstrated that this spatiotemporal imaging method was a reliable tool for analyzing dynamic intracellular changes of the redox state and elucidating the molecular mechanisms of oxidative stress-related diseases.

Automated summary

Dysregulated redox homeostasis under pathological conditions can lead to oxidative stress and associated disease damage. However, the lack of real-time methods for analyzing redox states is a barrier to understanding pathogenic mechanisms. We developed a novel imaging strategy that allows real-time imaging of dual molecular events during oxidative stress, providing insights into dynamic intracellular changes of the redox state and elucidating the molecular mechanisms of oxidative stress-related diseases.