Artemether relieves liver fibrosis by triggering ferroptosis in hepatic stellate cells via DHHC12-mediated S-palmitoylation of the BECN1 protein

Abstract

Liver fibrosis, a pivotal stage in chronic liver disease progression, is driven by hepatic stellate cell (HSC) activation. Ferroptosis is a novel form of programmed cell death, which offers therapeutic potential for liver fibrosis. Although artemether (ART) exhibits antifibrotic properties, its mechanisms in liver fibrosis remain unclear. This study aimed to determine the therapeutic effects of ART on liver fibrosis and explore the role of S-palmitoylation in HSC ferroptosis.

Methods

A mouse model of liver fibrosis was constructed by carbon tetrachloride (CCl₄) injection. Transforming growth factor-β (TGF-β) was used for stimulating HSC activation in vitro. Histopathological and serological assays were performed to analyze the therapy effects of ART. Liquid Chromatography/Mass Spectrometry (LC/MS) and acyl-biotinyl exchange (ABE) were used to determine the role of S-palmitoylation in ART-induced HSC ferroptosis. Western blot and Co-Immunoprecipitation (Co-IP) were performed to examine the effects of autophagy in ART-induced HSC ferroptosis through regulating BECN1 S-palmitoylation.

Results

ART ameliorated liver fibrosis by inducing HSC ferroptosis, and the ferroptosis inhibitor ferrostatin-1 (Fer-1) impaired the inhibitory effect of ART. Interestingly, the levels of S-palmitoylation were elevated by upregulating the palmitoyltransferase DHHC12 during ART-induced HSC ferroptosis. DHHC12 knockdown reduced S-palmitoylation levels and impaired ART-mediated HSC ferroptosis. RNA-seq analysis indicated that autophagy activation was essential for ART to induce HSC ferroptosis. 3-methyladenine (3-MA) suppressed autophagy and ART-induced HSC ferroptosis. Importantly, BECN1 S-palmitoylation by DHHC12 drove ART to activate autophagy. DHHC12 bound to the cysteine 21 residue of BECN1, thereby stabilizing the BECN1 protein and facilitating autophagy activation. Mutation of the cysteine 21 residue decreased BECN1 protein stability, autophagy activation and ferroptosis in ART-treated HSCs. In a mouse model of hepatic fibrosis, HSC-specific inhibition of BECN1 S-palmitoylation reversed ART-induced HSC ferroptosis and the improvement of fibrotic liver.

Conclusions

ART alleviates liver fibrosis by inducing HSC ferroptosis via DHHC12-mediated BECN1 protein S-palmitoylation.