Unveiling the Crucial Roles of O(2)(•–) and ATP in Hepatic Ischemia–Reperfusion Injury Using Dual-Color/Reversible Fluorescence Imaging

[Image: see text] Hepatic ischemia–reperfusion injury (HIRI) is mainly responsible for morbidity or death due to graft rejection after liver transplantation. During HIRI, superoxide anion (O(2)(•)(–)) and adenosine-5′-triphosphate (ATP) have been identified as pivotal biomarkers associated with oxidative stress and energy metabolism, respectively. However, how the temporal and spatial fluctuations of O(2)(•)(–) and ATP coordinate changes in HIRI and particularly how they synergistically regulate each other in the pathological mechanism of HIRI remains unclear. Herein, we rationally designed and successfully synthesized a dual-color and dual-reversible molecular fluorescent probe (UDP) for dynamic and simultaneous visualization of O(2)(•)(–) and ATP in real-time, and uncovered their interrelationship and synergy in HIRI. UDP featured excellent sensitivity, selectivity, and reversibility in response to O(2)(•)(–) and ATP, which rendered UDP suitable for detecting O(2)(•)(–) and ATP and generating independent responses in the blue and red fluorescence channels without spectral crosstalk. Notably, in situ imaging with UDP revealed for the first time synchronous O(2)(•)(–) bursts and ATP depletion in hepatocytes and mouse livers during the process of HIRI. Surprisingly, a slight increase in ATP was observed during reperfusion. More importantly, intracellular O(2)(•)(–)—succinate dehydrogenase (SDH)—mitochondrial (Mito) reduced nicotinamide adenine dinucleotide (NADH)—Mito ATP—intracellular ATP cascade signaling pathway in the HIRI process was unveiled which illustrated the correlation between O(2)(•)(–) and ATP for the first time. This research confirms the potential of UDP for the dynamic monitoring of HIRI and provides a clear illustration of HIRI pathogenesis.