Study of α-Amyrin targeting Sirt1/PGC-1α/PPARα axis to regulate fatty acid metabolism against liver fibrosis

Background: Hepatic fibrosis represents a major global health burden with no approved first-line therapeutic agents. The urgent need for innovative targeted small molecule interventions has prompted investigation of natural compounds with anti-fibrotic potential. While α-amyrin demonstrates established anti-inflammatory properties, its therapeutic efficacy against hepatic fibrosis remains unexplored.

Purpose: The present study was designed to assess the anti-fibrotic efficacy of α-amyrin and determine its molecular mechanisms of action.

Study design: Firstly, the efficacy of α-Amyrin was evaluated through the liver fibrosis model. Further, the mechanism of α-Amyrin was analyzed using multi-omics and Molecular Biology experiments, and was verified using the target knockdown model. Finally, the effective components of α-Amyrin were analyzed through bioinformatics analysis and experimental verification.

Methods: In this study, we use an in vitro model of fibrosis in human hepatic stellate LX-2 cells induced with TGF-β1 and an in vivo mouse model with CCl₄ to evaluate the anti-fibrosis potential of α-Amyrin using multiple approaches. Employing multi-omics and Molecular Biology techniques to investigate the mechanism of α-Amyrin counteracting hepatic fibrosis. The interaction targets of α-Amyrin were examined through molecular docking, molecular dynamics simulations, and surface plasmon resonance. The anti-hepatic fibrosis mechanism of α-Amyrin was further validated using both in vitro and in vivo PPARα knockout or inhibition models.

Results: In vivo, α-Amyrin significantly inhibited the formation of hepatic pseudolobules and suppressed hepatic inflammation compared with the model control group. In vitro, α-Amyrin significantly down-regulated the expression levels of well-established markers of liver fibrosis. The results of transcriptomics and metabolomics provided clues to the mechanisms of the PPARα pathway and fatty acid metabolism. Further experimental validation of these mechanisms was conducted. In vitro and in vivo, α-Amyrin significantly promoted fatty acid oxidation levels which is consistent with up-regulation of PPARα pathway involved in this process, including SIRT1, PPARα, PGC-1α, CPT1A, ACOX1, and its downstream targets. Moreover, α-Amyrin markedly enhanced PPARα nuclear translocation and facilitated the Sirt1-PGC-1α interaction. Molecular docking, dynamics simulations, and surface plasmon resonance confirmed direct binding of α-Amyrin to PPARα. Critically, the anti-hepatic fibrosis effect of α-Amyrin was significantly compromised in both the in vivo PPARα Inhibitor model and the in vitro PPARα knockout model.

Conclusions: The possible mechanism of α-Amyrin's anti-fibrosis effect is to target the PPARα axis to promote the reprogramming of fatty acid oxidation metabolism, which in turn exerts an anti-inflammatory effect and reverses the inflammation-fibrosis pathological process. The above results are expected to provide an innovative strategy for the development of small molecule drugs targeting liver fibrosis.