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

Acta Pharmacologica Sinica volume 47, pages 946–963 (2026)Cite this article

Abstract

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.

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Fig. 1: β-SIT ameliorates FFA-induced lipid deposition in vitro.
Fig. 2: β-SIT ameliorates CDAHFD and HFD diet-induced MASH in mice.
Fig. 3: β-SIT-activated autophagic flux and lipophagy in FFA-treated hepatocytes.
Fig. 4: β-SIT ameliorates steatosis in MASH mice by activating lipophagy.
Fig. 5: β-SIT regulates lipophagy by modulating RAC1 expression.
Fig. 6: Improvement of MASH by β-SIT is reversed by overexpression of hepatic RAC1.
Fig. 7: β-SIT regulates lysosomal function by inhibiting co-localization of RAC1 and mTOR on lysosomes.
Fig. 8: β-SIT promotes TFEB nuclear translocation by regulating the RAC1-mTOR pathway.

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Acknowledgements

This work received partial financial support from the National Natural Science Foundation of China (grant number: 82073851).

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  1. Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, 116044, China

    Yang Wang, Yi Sun, Chang-yuan Wang, Min Cao, Ru Hao, Shan Wang & Hui-jun Sun

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YW, YS and CYW performed the experiment; YW drafted the article; MC and RH collected and interpreted the data; SW performed the analysis with constructive discussions; HJS designed the study, obtained funding, supervised the study, drafted and critically revised the paper. All the authors have read and approved the final paper. All data were generated in-house, and no paper mill was used.

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Correspondence to Hui-jun Sun.

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Wang, Y., Sun, Y., Wang, Cy. et al. β-Sitosterol ameliorates metabolic dysfunction-associated steatohepatitis by targeting the RAC1/mTOR/TFEB axis thus activating lipophagy-lysosomal pathway. Acta Pharmacol Sin 47, 946–963 (2026). https://doi.org/10.1038/s41401-025-01702-8

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