Elucidation of the Relationship between Hydrogen Peroxide and Pyroptosis by a Superior near-Infrared Fluorescent Probe

Pyroptosis is an inflammatory programmed cell death process closely related to reactive oxygen species (ROS), in which hydrogen peroxide (H 2 O 2 ) is considered to play a significant role. However, the specific stage at which H 2 O 2 changes and whether it serves as the predominant ROS during pyroptosis remain unclear, hindering a deeper understanding of the underlying mechanism. In this study, a near-infrared fluorescence probe ( DXM-CHO-B ) with a high fluorescence quantum yield and large Stokes shift was obtained by molecular modification. DXM-CHO-B exhibited excellent specificity and high sensitivity to H 2 O 2, featuring a wide linear range (50 nM–92 μM), a low detection limit (39 nM), and the ability to perform real-time H 2 O 2 tracking in both cell and zebrafish models. DXM-CHO-B also maintained a high selectivity for H 2 O 2 in cells, and the accuracy of the high selectivity was verified. Based on the excellent detection performance of DXM-CHO-B, the pyroptosis process of cells was monitored. The experimental results revealed that H 2 O 2 was overexpressed during pyroptosis, and its amount undergoes significant changes in the early stage of pyroptosis. This conclusion was further supported by scanning electron microscopy imaging, lactate dehydrogenase release testing, and propidium iodide staining staining assays of pyroptosis cells at different time points. More importantly, further research has shown that the majority of ROS produced during pyroptosis were H 2 O 2 . These findings established H 2 O 2 as the core mediator of pyroptosis, providing insights into its molecular mechanism and potential therapeutic strategies for enhancing tumor immunotherapy.