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

Abstract

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 CPR-SK specifically activated the caffeic acid moieties or targeting peptides (EWWW) toward the biomarkers, including superoxide (O 2 •−) 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•− and subsequent migration of Keap1 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.

Mitochondria-Targetable Near-Infrared Fluorescent Probe for Visualization of Hydrogen Peroxide in Lung Injury, Liver Injury, and Tumor Models

“…The clinical liver injury diagnostic methods are generally not suitable for the early assessment of liver injury. Since the pathogenesis of HIRI is closely related to oxidative stress disorder, − for example, previous evidence suggests that H 2 O 2 can activate the G-protein to destroy the junctional complex and enhance ischemia-reperfusion injury . In addition, distinct from other highly ROS (e.g., O 2 •– , • OH, ONOO – ) that are partially involved in ischemic damage, the stable H 2 O 2 is diffusible across the cell membrane, making it the main ROS that exacerbates ischemia-reperfusion injury.…”