H3K9 acetylation-dependent CHAC1 transcription in dihydroartemisinin-induced hepatic stellate cell ferroptosis.

Background: The activation of hepatic stellate cells (HSCs) plays a crucial role in the progression of liver fibrosis, and eliminating activated HSCs is regarded as an effective strategy for combating fibrosis. Ferroptosis has emerged as a potential mechanism for HSC depletion. Dihydroartemisinin (DHA), a derivative of artemisinin, has shown anti-fibrotic effects, but its role in HSC ferroptosis remains unclear. This study aimed to investigate the molecular mechanism by which DHA regulates HSC ferroptosis through histone modifications to suppress liver fibrosis.

Methods: RNA sequencing was employed to analyze gene expression changes in HSCs following DHA treatment. The role of glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) in DHA-induced ferroptosis was assessed using genetic inhibition approaches. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were conducted to examine histone acetylation and transcription factor binding at the CHAC1 promoter. In vivo experiments utilized a murine liver fibrosis model to evaluate the therapeutic effects of DHA, with additional interventions targeting CHAC1 and ATF4 to validate their roles in ferroptosis and fibrosis resolution.

Results: This study reported that DHA inhibited HSC activation through the ferroptosis pathway. We revealed that DHA treatment elevated CHAC1 levels in HSCs, Inhibition of CHAC1 prevented DHA-induced HSC ferroptosis, and DHA regulated the expression of CHAC1 at the transcriptional level rather than at the post-transcriptional level. Mechanistically, upregulated H3K9 acetylation was essential for the DHA-mediated transcriptional upregulation of CHAC1 through increased histone acetyltransferase P300 in HSCs. Inhibiting histone acetylation attenuated DHA-induced CHAC1 upregulation and ferroptosis. Aactivating transcription factor 4 (ATF4) was identified as a key transcription factor in the transcriptional activation of CHAC1. Interfering with ATF4 inhibited the transcriptional upregulation of CHAC1 by DHA. Notably, the -212 to -199 bp and -269 to -257 bp promoter regions in CHAC1 were essential for the initiation of transcription of ATF4. In mice, treatment with DHA alleviated murine liver fibrosis by inducing HSC ferroptosis. Inhibition of CHAC1 or ATF4 impaired DHA-induced HSC ferroptosis in murine liver fibrosis.

Conclusion: The transcriptional activation of CHAC1, which is regulated by H3K9 acetylation, was essential for the ability of DHA to trigger HSC ferroptosis and, consequently, to suppress liver fibrosis.