Unveiling aucubin-mediated inhibition of PANoptosis in lower limb ischemia-reperfusion injury with a near-infrared H2O2 fluorogenic probe

Although reactive oxygen species (ROS)-mediated PANoptosis plays a pivotal role in the pathological dysfunction of lower limb ischemia/reperfusion injury (LL-IRI), its exact mechanism remains unclear. Real-time detection of H 2 O 2 will help to reveal the complicated relationship between ROS, PANoptosis and LL-IRI. In this work, we designed and synthesized a novel near-infrared fluorogenic (NIRF) probe, BFP-H 2 O 2 , which exhibited a pronounced fluorescence-enhanced response to H 2 O 2 at 650 nm. BFP-H 2 O 2 was successfully applied to a TAK1i/LPS-induced PANoptosis cell model, revealing up-regulation of H 2 O 2 during PANoptosis. To the best of our knowledge, there were no suitable chemical tools available for high-throughput screening of anti-PANoptosis compounds from Plantagodepressa Willd (PW). Leveraging BFP-H 2 O 2 , aucubin (AU) from PW was identified for the first time as the most promising anti-PANoptosis active ingredient. Additionally, BFP-H 2 O 2 enabled visual imaging of the dynamic changes of H 2 O 2 in the LL-IRI mice model. Particularly importantly, it was confirmed that AU could effectively inhibit H 2 O 2 -mediated oxidative stress and attenuate PANoptosis in gastrocnemius muscle tissues during LL-IRI. This work not only afforded a reliable chemical tool for screening anti-PANoptosis drugs, but also further elucidated the mechanism of PANoptosis and LL-IRI and advanced the development of therapeutic drugs for PANoptosis-related diseases. • A novel NIR fluorogenic probe BFP-H 2 O 2 enabled real-time imaging of H 2 O 2 during PANoptosis. • Aucubin was screened via BFP-H 2 O 2 as a potent natural inhibitor of PANoptosis in LL-IRI. • Aucubin alleviated LL-IRI via reducing oxidative stress and inhibiting PANoptosis.