Simultaneous Dual-Channel Tracking Lipid Droplets Dynamic Behaviors and Revealing the Protective Role in Nonalcoholic Fatty Liver in mice

Lipophagy, a selective autophagic process, facilitates lipolysis and mitigates hepatocyte steatosis, thereby emerging as a significant research focus for the prevention and treatment of nonalcoholic fatty liver disease (NAFLD). The development of methodologies for real-time monitoring of lipophagy and its dynamic alterations is essential for elucidating its role in lipid metabolism disorders. This study introduces three fluorescent probes with dual-channel responsiveness to pH and viscosity, among which probe D-VP was identified as the most promising candidate. This is attributed to its straightforward one-step synthesis, substantial Stokes shift (199 nm), and minimal signal crosstalk between the two channels (Δλ = 130 nm). D-VP demonstrates excellent lipid droplet targeting capability, allowing for sensitive tracking of dynamic processes such as lipid droplet formation, accumulation, and degradation. Changes in dual-channel fluorescence signals can indicate the start and progression of lipophagy, marked by decreased fluorescence in the viscosity channel and increased fluorescence in the pH channel. Imaging with this probe showed higher viscosity and lower pH in the liver tissues of mice with NAFLD, suggesting its potential for NAFLD diagnostic imaging. Lipophagy induction led to noticeable changes in liver fluorescence signals, and the fluorescence intensity ratio between channels could assess NAFLD treatment efficacy. High-dose, short-duration autophagy induction proved more effective than low-dose, long-duration treatment. Western blot analysis confirmed that lipophagy-mediated NAFLD reversal primarily occurs through AMPK pathway activation. This probe holds promise as a diagnostic tool for developing precision therapies targeting lipophagy activation in NAFLD.