IRG1/Itaconate inhibits hepatic stellate cells ferroptosis and attenuates TAA-induced liver fibrosis by regulating SLC39A14 expression.

This study aimed to elucidate the protective roles of Immune Response Gene-1 (IRG1) and exogenous itaconate in murine models of hepatic fibrosis and to delineate the underlying mechanistic pathways using both wild-type and IRG1-deficient (IRG1) mice. Primary murine stellate cells (mHSC) and bone marrow-derived macrophages (BMDM) were isolated and cocultured. Hepatocellular fibrosis was induced in vitro using Transforming Growth Factor-beta (TGF-β) to evaluate the protective efficacy of IRG1/itaconate. Histopathological damage in the hepatic tissues was assessed using Hematoxylin and Eosin (H&E), Masson's trichrome, and Sirius red staining, followed by hepatic fibrosis scoring. The levels of released inflammatory cytokines were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Immunohistochemistry was used to detect 4-Hydroxynonenal (4-HNE) levels and Perls staining was used to assess ferroptosis. RNA sequencing and gene enrichment analyses were performed to identify implicated molecular entities and signaling pathways. IRG1 and SLC39A14 knockdown and overexpression cell lines were generated. Quantitative real-time PCR (qRT-PCR) and western blotting (WB) were used to measure the mRNA and protein expression levels in hepatic tissues and cells. Kits were used to assess reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and the concentrations of liver enzymes, iron, GSH, and GSSG within hepatic tissues and cells.4-octyl itaconate (4-OI) significantly attenuated the histopathological damage in hepatic tissues, preserved the normal hepatic function, effectively reduced the release of inflammatory cytokines, and mitigated oxidative stress markers such as ROS and MDA in Thioacetamide (TAA)-induced fibrotic mice. Notably, this study is the first to reveal the pivotal role of SLC39A14 in the pathogenesis of hepatic fibrosis in murine models and elucidate how IRG1/itaconate mediates downstream ferroptosis-related signaling pathways by targeting SLC39A14, thereby inhibiting ferroptosis-induced hepatic fibrosis. IRG1/itaconate can alleviate the TAA-induced hepatic fibrosis in mice by regulating the expression of SLC39A14, consequently suppressing hepatic stellate cell ferroptosis.