Dual-Site Chemosensor for Visualizing ^•OH-GSH Redox and Tracking Ferroptosis-Inducing Pathways In Vivo

Oxidative stress, characterized by an imbalance between oxidative and antioxidant processes, results in excessive accumulation of intracellular reactive oxygen species. Among these responses, the regulation of intracellular hydroxyl radicals (^•OH) and glutathione (GSH) is vital for physiological processes. Real-time in situ monitoring these two opposing bioactive species and their redox interactions is essential for understanding physiological balance and imbalance. In this study, we developed a dual-site fluorescence chemosensor OG-3, which can independently image both exogenous and endogenous ^•OH and GSH in separate channels both within cells and in vivo, eliminating issues of spatiotemporal inhomogeneous distribution and cross-interference. With its imaging capabilities of monitoring ^•OH-GSH redox, OG-3 elucidated two different pathways for ferroptosis induction: (i) inhibition of system x_c^- to block cystine uptake (extrinsic pathway) and (ii) GPX4 inactivation, leading to the loss of antioxidant defense (intrinsic pathway). Moreover, we assessed the antiferroptotic function and effects of ferroptosis inhibitors by monitoring ^•OH and GSH fluctuations during ferroptosis. This method provides a reliable platform for identifying potential ferroptosis inhibitors, contributing to our understanding of relevant metabolic and physiological mechanisms. It shows potential for elucidating the regulation of ferroptosis mechanisms and investigating further strategies for therapeutic applications.