Chinese medical journal · 2025
H3K9 acetylation-dependent CHAC1 transcription in dihydroartemisinin-induced hepatic stellate cell ferroptosis.
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Shen Min, Han Wenshu, Cai Liangliang, Gong Weijuan, Qian Li
Аннотация
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: In vitro experiments were conducted using human hepatic stellate cell line HSC-LX2 and mouse hepatic stellate cell line mHSC, with DHA treatment to induce ferroptosis, and ferrostatin-1 as a ferroptosis inhibitor for control. RNA sequencing was performed to analyse differentially expressed genes in DHA-treated HSCs, and real-time polymerase chain reaction, Western blotting, and immunofluorescence were used to verify glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) expression. Chromatin immunoprecipitation quantitative polymerase chain reaction was used to detect histone acetylation at the CHAC1 promoter, and luciferase reporter assays with wild-type or mutated CHAC1 promoter were used to confirm activating transcription factor 4 (ATF4) binding sites. In vivo experiments used male C57BL/6J mice induced with carbon tetrachloride (CCl 4 ) to establish a liver fibrosis model. Histopathological staining, serum biochemical index detection, and ferroptosis-related assays in isolated liver cells were performed to evaluate the therapeutic effect of DHA and the roles of CHAC1 and ATF4.
RESULTS: DHA inhibited HSC activation through the ferroptosis pathway, 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 in HSC-LX2 and mHSC cells. 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. 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.
METHODS: In vitro experiments were conducted using human hepatic stellate cell line HSC-LX2 and mouse hepatic stellate cell line mHSC, with DHA treatment to induce ferroptosis, and ferrostatin-1 as a ferroptosis inhibitor for control. RNA sequencing was performed to analyse differentially expressed genes in DHA-treated HSCs, and real-time polymerase chain reaction, Western blotting, and immunofluorescence were used to verify glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) expression. Chromatin immunoprecipitation quantitative polymerase chain reaction was used to detect histone acetylation at the CHAC1 promoter, and luciferase reporter assays with wild-type or mutated CHAC1 promoter were used to confirm activating transcription factor 4 (ATF4) binding sites. In vivo experiments used male C57BL/6J mice induced with carbon tetrachloride (CCl 4 ) to establish a liver fibrosis model. Histopathological staining, serum biochemical index detection, and ferroptosis-related assays in isolated liver cells were performed to evaluate the therapeutic effect of DHA and the roles of CHAC1 and ATF4.
RESULTS: DHA inhibited HSC activation through the ferroptosis pathway, 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 in HSC-LX2 and mHSC cells. 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. 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.