Liver-targeted alkaline phosphatase-activatable fluorescent probe for imaging liver fibrosis and screening anti-fibrotic natural products.

Liver fibrosis is a progressive consequence of chronic liver injury and remains difficult to diagnose dynamically and to treat effectively. Current fibrosis evaluation mainly relies on histology or nonspecific biochemical indices, and small-molecule fluorescent tools that can sensitively track fibrosis-associated enzymatic activity in vivo while supporting therapeutic discovery remain limited. Here, we developed a liver-targeted, alkaline phosphatase (ALP)-activatable fluorescent probe, ALP-YL, by integrating a phosphate trigger for enzymatic recognition, an intramolecular charge-transfer (ICT) fluorophore for signal output, and a cholic acid moiety for hepatic enrichment, and evaluated it by spectroscopic analysis, cell imaging, and animal imaging together with probe-guided screening of botanical extracts. ALP-YL showed high selectivity for ALP, a detection limit of 0.052 U/L, good cytocompatibility, and rapid fluorescence activation in vitro. In hepatocyte injury models and TGF-β-activated LX-2 cells, the probe sensitively reported endogenous ALP fluctuations associated with fibrogenic activation. In CCl4-induced fibrotic mice, ALP-YL preferentially accumulated in the liver and generated in vivo and ex vivo fluorescence signals that agreed with histopathology, serum biochemistry, and fibrosis-related protein expression. Using the probe as a functional fluorescence readout, we further identified a glycosylated resveratrol trimer (B2) from peony seeds that displayed stronger anti-fibrotic activity than silibinin in activated LX-2 cells and modulated the TGF-β/Smad and SLC7A11-GPX4 pathways. This work establishes ALP-YL as a biosensing platform for fibrosis-relevant imaging and fluorescence-guided anti-fibrotic lead discovery.