Electronic cloud-regulation strategy enabling rapid and sensitive near-infrared detection of ONOO⁻ for dynamic monitoring of ferroptosis-mediated drug-induced liver injury

Drug-induced liver injury (DILI) is caused by hepatotoxic effects resulting from drug metabolism. Extensive studies have demonstrated a close association between DILI progression and ferroptosis, a process characterized by redox imbalance triggered by lipid peroxidation. As a key product of redox imbalance and a critical biomarker of DILI, peroxynitrite (ONOO^-) plays a pivotal regulatory role in ferroptosis. To elucidate the precise mechanism of ONOO^- in ferroptosis-mediated DILI, there is an urgent need for ONOO^- fluorescent probes with high specificity, sensitivity and signal-to-noise ratio. This study innovatively modified the traditional pyridine hemicyanine fluorophore into a quinoline hemicyanine structure, achieving a red-shifted fluorescence emission wavelength through extended conjugation and enhanced electron delocalization. Systematic site screening identified diphenyl phosphate ester as the optimal reaction site, demonstrating exceptional specificity, sensitivity and 75 s response characteristics. By conjugating the optimized fluorophore with the reaction site via nucleophilic substitution, we successfully developed a novel ratiometric fluorescent probe, zt-4. Given the remarkable similarities in pathological mechanisms (including signaling pathways, regulatory nodes, and biomarkers) between osteoarthritis (OA) and DILI, we first validated the imaging performance of zt-4 in a complex inflammatory OA Model. Subsequently, we applied probe zt-4 to analyze ferroptosis-mediated DILI model at both cellular and murine levels. For the first time, our study revealed that the ferroptosis pathway dynamically regulates ONOO^- levels in DILI, elucidating its molecular mechanism. These findings provide new theoretical foundations and intervention strategies for DILI treatment.