Artesunate Induces Ferroptosis in Hepatic Stellate Cells and Alleviates Liver Fibrosis via the ROCK1/ATF3 Axis.

BACKGROUND AND AIMS: Development of fibrosis in chronic liver disease requires activation of hepatic stellate cells (HSCs) and leads to a poor outcome. Artesunate (Art) is an ester derivative of artemisinin that can induce ferroptosis in HSCs, and activated transcriptional factor 3 (ATF3) is an ATF/CREB transcription factor that is induced in response to stress. In this study, we examined the role of the Rho-associated protein kinase 1 (ROCK1)/ATF3 axis in Art-induced ferroptosis in HSCs.

METHODS: HSC activation and ferroptosis were studied in vitro by western blotting, polymerase chain reaction, immunofluorescence, and other assays. ATF3 electrophoretic mobility and ROCK1 protein stability were assayed by western blotting. Immunoprecipitation was used to detect the interaction of ROCK1 and ATF3, as well as ATF3 phosphorylation. A ubiquitination assay was used to verify ROCK1 degradation. Atf3-interfering and Rock1-overexpressing mice were constructed to validate the anti-hepatic fibrosis activity of Art in vivo .

RESULTS: Art induced ferroptosis in HSCs following glutathione-dependent antioxidant system inactivation resulting from nuclear accumulation of unphosphorylated ATF3 mediated by ROCK1-ubiquitination in vitro . Art also decreased carbon tetrachloride-induced liver fibrosis in mice, which was reversed by interfering with Atf3 or overexpressing Rock1.

CONCLUSIONS: The ROCK1/ATF3 axis was involved in liver fibrosis and regulation of ferroptosis, which provides an experimental basis for further study of Art for the treatment of liver fibrosis.