A Visible/NIR-II Dual-Channel Fluorescent Probe for Investigating the Roles of NLRP3 Inflammasome and Ferroptosis during Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion (I/R) injury is a major cause of postoperative liver dysfunction and liver failure, leading to irreversible hepatocellular damage. The NLRP3 inflammasome and ferroptosis are recognized as key contributors to hepatic I/R injury, but their synergistic role in the underlying pathological mechanism remains unclear. Since dysregulated lipid droplets (LDs) metabolism is closely linked to oxidative stress and ferroptosis, real-time visualizing LDs dynamics offers a promising approach to study the interplay between NLRP3 inflammasome and ferroptosis during hepatic I/R injury. Herein, we designed and synthesized a dual-channel fluorescent probe, PX-P, based on a donor-π-acceptor-donor (D-π-A-D) structure, enabling specific targeting of LDs, a large Stokes shift (>120 nm), excellent photostability, and zero-crosstalk between two emission channels (Δλ_em = 363 nm). Using PX-P, we monitored LDs polarity changes in hepatocytes and mice during I/R injury, revealing distinct differences between normal and cancer cells, dynamic alterations in LDs polarity during inflammation and ferroptosis, and interactions between LDs and lysosomes, nucleolus, and nucleus in the processes of lipophagy and lipid homeostasis disruption. Visible/NIR-II dual-channel imaging revealed that LDs accumulation increased during ischemia and early stages of reperfusion, then gradually decreased with prolonged reperfusion. Notably, simultaneous inhibition of the NLRP3 inflammasome and ferroptosis pathways effectively alleviated hepatocellular injury. Collectively, PX-P provides a versatile cross-scale imaging platform for tracking LDs dynamics from cells to living organisms, offering insights into the pathogenic mechanism of hepatic I/R injury and potential therapeutic strategies for early intervention.