Limonin mitigates hepatic senescence and fibrosis in MASH mice by targeting STAT3 to inhibit Galectin-3/mTORC1 signaling.

BACKGROUND

Despite existing reports on the hepatoprotective effects of limonin (Lim), its specific impact on hepatic fibrosis and cellular senescence in metabolic dysfunction-associated steatohepatitis (MASH) remains unclear. The precise molecular mechanisms and direct targets underpinning its pharmacological activity are also poorly defined.

PURPOSE: This research aimed to investigate the therapeutic potential of Lim against MASH-related hepatic fibrosis and senescence, and to delineate the underlying molecular pathways.

METHODS

A murine MASH model was generated by feeding a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). To elucidate the mechanism of action of Lim, an integrated strategy was adopted, comprising AAV8-mediated gene manipulation, RNA sequencing (RNA-seq), and pharmacological interventions with agonists or inhibitors. The direct protein targets of Lim were identified using human proteome microarrays and validated through molecular docking, pull-down assays, and site-directed mutagenesis.

RESULTS

Our findings indicate that Lim effectively alleviates hepatic fibrosis and senescence in MASH mice, while suppressing galectin-3 expression and mechanistic target of rapamycin complex 1 (mTORC1) activation. Notably, targeted knockdown of galectin-3 in the liver hindered aging-related changes in MASH mice, with mTORC1 functioning as a downstream effector. Further results revealed that mTORC1 acts as a key mediator of Lim's protective effects against hepatic fibrosis and senescence in MASH mice. Mechanistically, Lim binds to the Src homology 2 (SH2) domain of signal transducer and activator of transcription 3 (STAT3), inhibiting its activity and leading to reduced galectin-3 expression and mTORC1 activation. Moreover, the application of STAT3 inhibitor has been shown to alleviate hepatic fibrosis and senescence in MASH mice, further corroborating the suppression of galectin-3 and mTORC1 activity.

CONCLUSION

In conclusion, our study provides compelling evidence for the efficacy of Lim in ameliorating hepatic senescence and fibrosis in MASH mice, elucidating the involvement of the STAT3/galectin-3/mTORC1 signaling in these processes.