Revisiting The Impact Of H2o2 On Intracellular Nrf2 Levels Usıng A Newly Developed Oxygen-Independent Nrf2 Bıosensor

Reactive oxygen species (ROS) are central to cellular communication, and Nrf2 serves as a crucial transcription factor in maintaining redox balance. Despite extensive research, the interplay between Nrf2 and hydrogen peroxide (H2O2) remains contentious. This study aimed to explore how varying intracellular H2O2 concentrations impact Nrf2 activity in brain endothelial cells under physiological oxygen conditions (5 kPa O2), as opposed to conventional hyperoxic conditions. We developed the Pericellular Oxygen-Insensitive Nrf2 Total LEvel Reporter (POINTER), a novel biosensor for oxygen-independent Nrf2 monitoring in human cerebral microvascular endothelial cells (hCMEC/D3). By utilizing external H2O2, chemogenetic production of H2O2 via modified D-amino acid oxidase (mDAAO), and Auranofin-induced oxidative stress, we evaluated the effects of H2O2 on Nrf2. Our findings demonstrated significantly lower Nrf2 levels under physioxia compared to room air, with Auranofin and chemogenetically produced H2O2 showing distinct effects on Nrf2 modulation. POINTER revealed that while Auranofin significantly increased Nrf2 levels under physiological oxygen, exogenous and chemogenetically produced H2O2 did not sustain this effect. These results underscore the nuanced regulation of Nrf2 by both antioxidant inhibition and H2O2 elevation under physioxic conditions.