Near-Infrared Fluorogenic Probe Enables Real-Time Imaging of HOCl to Reveal MAR1-Mediated Suppression of Pyroptosis in Limb Ischemia-Reperfusion Injury

The interplay between oxidative stress and programmed cell death plays a pivotal role in the development of several inflammation-related diseases. As one of the important reactive oxygen species (ROS) in living organisms, the specific function of hypochlorous acid (HOCl) in pyroptosis, as well as lower limb ischemia-reperfusion injury (LL-IRI), has not been fully explored. Herein, we fabricated an activatable near-infrared fluorogenic (NIRF) probe, BFP-HOCl, which enabled the highly selective and sensitive detection of HOCl. In the presence of HOCl, the fluorescence of BFP-HOCl was remarkably enhanced at 652 nm and exhibited a good linear response relationship. Using BFP-HOCl, imaging of exogenous and endogenous HOCl in cells was successfully achieved. Intriguingly, BFP-HOCl was able to track the elevated HOCl levels during LPS+ATP-induced pyroptosis and its fluorescence enhancement could be suppressed by MAR1 dose-dependently. In a mouse LL-IRI model, a combination of transcriptomic analysis, immunohistochemistry, qPCR, and <i>in vivo</i> imaging revealed a critical role for HOCl as an oxidative activator upstream of the ROS-NLRP3-GSDMD signaling axis. MAR1 effectively attenuated the degree of tissue injury by downregulating the level of HOCl and inhibiting the expression of pyroptosis-associated proteins (NLRP3, ASC, Caspase-1, and GSDMD). This work not only established that HOCl could be used as a potential marker of pyroptosis and LL-IRI but also further confirmed the functional status of pyroptosis in LL-IRI and the intervention potential of MAR1, which provided a new strategy for the diagnosis and intervention of LL-IRI.