β-Sitosterol ameliorates metabolic dysfunction-associated steatohepatitis by targeting the RAC1/mTOR/TFEB axis thus activating lipophagy-lysosomal pathway

Metabolic dysfunction-associated steatohepatitis (MASH), an inflammatory subtype of metabolic dysfunction-associated fatty liver disease (MAFLD), drives hepatic dysfunction and poses a significant health burden. Lipophagy dysfunction disrupts lipid droplet degradation and induces lysosomal damage, which is closely linked to MASH progression; thus, targeting lipophagy-lysosomal activation has emerged as a promising therapeutic strategy for the therapy of MASH. β-Sitosterol (β-SIT) derived from Polygonum hydropiper L. is structurally similar to cholesterol, and exhibits neuroprotective, antidiabetic and anti-obesity bioactivities. In this study, we explored the therapeutic potential of β-SIT for MASH. The mouse models of MASH were established by feeding a choline- deficient, L-amino acid-defined high-fat diet (CDAHFD) for 10 weeks, or high-fat diet (HFD) for 12 weeks. For in vitro experiments, AML-12 cells were treated with FFA mixture (OA:PA molar ratio = 2:1) to mimic lipid overload condition. MASH mice were administered β-SIT (10 or 20 mg·kg^-1 d^-1, i.g.) for 10 weeks. We showed that β-SIT treatment dose-dependently alleviated MASH by enhancing the lipophagy-lysosomal pathway in vivo and in vitro. In FFA-stimulated AML-12 cells, we demonstrated that β-SIT (20 μM) activated autophagic flux, promoted lysosomal biogenesis, and enhanced lysosome-lipid droplet interactions, as revealed by transmission electron microscopy, multi-SIM real-time fluorescence monitoring, and lipophagy-related marker detection. By integrated approaches including bioinformatics, molecular dynamics, CETSA and functional assays, we found that β-SIT inhibited mTOR pathway activation by directly targeting Ras-related C3 botulinum toxin substrate 1 (RAC1) in MASH mice. By conducting imaging/3D reconstruction, co-immunoprecipitation, immunofluorescence colocalization, lysosomal fractionation, and biochemical analyses in FFA-stimulated AML-12 cells, we confirmed that β-SIT modulated RAC1/mTOR interactions on lysosomes to restore lipophagy function. Critically, β-SIT promoted transcription factor EB (TFEB) nuclear translocation by modulating the RAC1-mTOR axis, thereby repairing lipophagy-lysosomal defects and attenuating MASH progression. Our results suggest that targeting the RAC1-mTOR-TFEB axis is a novel mechanism of β-SIT-driven lipophagy-lysosomal regulation, and highlight β-SIT as a potential candidate for the treatment of MASH.