Orthogonal NIR/Ratiometric Probe for Simultaneous Imaging of NO and ATP in Acute Lung Injury: Elucidating Metabolic Dysregulation and Therapeutic Screening

Acute lung injury (ALI) involves severe mitochondrial dysfunction, where the dynamic interplay between nitric oxide (NO) and adenosine triphosphate (ATP) remains elusive due to the lack of tools for their simultaneous visualization. Here, we developed ANRC, an orthogonal near-infrared (NIR) and mitochondria-targeted fluorescent probe that enabled ratiometric imaging of NO and turn-on detection of ATP in live cells and <i>in vivo</i>. ANRC exhibited high selectivity, with a linear response to ATP in the 0.5-8 mM range and to NO in the 0-40 μM range, and detection limits of 1.3 μM for ATP and 0.26 μM for NO. In a lipopolysaccharide (LPS)-induced ALI model, ANRC visualized a metabolic imbalance characterized by elevated NO and depleted ATP. Transcriptomic analysis revealed that NO disrupted mitochondrial respiration and the tricarboxylic acid (TCA) cycle, impairing ATP synthesis. Using ANRC as a screening tool, we identified dexmedetomidine (DEX) as a therapeutic candidate that restored NO/ATP homeostasis and alleviated lung injury. This work presents a powerful dual-analyte imaging platform for studying mitochondrial metabolism and oxidative stress in ALI, offering new opportunities for diagnosis and drug development.