While ferroptosis is implicated in alcoholic liver disease, its precise mechanisms of action are not fully defined. This study aims to investigate the protective role of the ferroptosis inhibitor Ferrostatin-1 (Fer-1) against alcoholic liver injury, exploring its underlying mechanisms. Using mice models of acute and chronic ethanol exposure, we assessed the effects of Fer-1. We evaluated liver function, histopathological damage, and key molecular markers related to ferroptosis, metabolism, and inflammation. Fer-1 significantly improved liver function and alleviated tissue damage, including lipid accumulation and fibrosis. It enhanced antioxidant capacity and reduced iron overload, oxidative stress, and lipid peroxidation. Furthermore, Fer-1 improved dysregulated iron and lipid metabolism and attenuated inflammation. Notably, these protective effects appear to be independent of the Nrf2/HO-1 pathway, autophagy, and NLRP3 inflammasome. Fer-1 exerts multi-faceted protection against alcoholic liver injury by specifically inhibiting ferroptosis. It blocks the vicious cycle of alcohol-induced metabolic imbalances, oxidative stress, and inflammation, offering new potential strategies for treating alcoholic liver disease.

Alcoholic liver disease (ALD) is pathologically characterized by iron overload, oxidative stress, metabolic dysregulation, and inflammation. However, the central regulatory mechanisms orchestrating these processes remain elusive. This study aimed to explore the role of NADPH oxidases 1 and 4 (NOX1/4) in ethanol-induced liver injury and to evaluate the therapeutic potential of the NOX1/4 inhibitor, Setanaxib. Mice were randomly divided into three groups: pair-fed control, ethanol-fed, and ethanol-fed + Setanaxib treatment. On day 16, the mice were euthanized, and liver tissue and blood samples were collected for comprehensive analyses to explore the involvement and potential mechanisms of NOX1/4 in ethanol-induced liver injury. Hepatic NOX1/4 expression was significantly upregulated in ethanol-fed mice, implicating their role in ALD pathogenesis. Setanaxib treatment markedly attenuated multiple facets of this injury, including liver injury, iron overload, hepatic steatosis, and oxidative stress. Mechanistically, Setanaxib may restore iron homeostasis by suppressing hepcidin/FPN1 signaling and downregulating TFR1 expression, activate antioxidant defenses via the Nrf2/HO-1/SLC7A11/GPX4 axis, and mitigate inflammation by reducing macrophage infiltration and pro-inflammatory cytokine release. Our findings suggest that NOX1/4 may play a central role in coordinating the interconnected pathogenic processes that drive ALD progression, including iron overload, lipid dysregulation, oxidative stress, and inflammation. Our findings establish Setanaxib, a novel NOX1/4 inhibitor, as a promising multi-target therapeutic strategy for ALD.

Background: Alcoholic liver disease (ALD) is the most common liver disease worldwide and its underlying molecular mechanisms are still poorly understood. Moreover, conflicting data have been reported on potentially protective autophagy, the exact role of ethanol-metabolizing enzymes and ROS. Methods: Expression of LC3B, CYP2E1, and NOX4 was studied in a mouse model of acute ethanol exposure by immunoblotting and immunohistochemistry. Autophagy was further studied in primary mouse hepatocytes and huh7 cells in response to ethanol and its major intermediator acetaldehyde. Experiments were carried out in cells overexpressing CYP2E1 and knock down of NOX4 using siRNA. The response to external H2O2 was studied by using the GOX/CAT system. Autophagic flux was monitored using the mRFP-GFP-LC3 plasmid, while rapamycin and chloroquine served as positive and negative controls. Results: Acute ethanol exposure of mice over 24 h significantly induced autophagy as measured by LC3B expression but also induced the ROS-generating CYP2E1 and NOX4 enzymes. Notably, ethanol but not its downstream metabolite acetaldehyde induced autophagy in primary mouse hepatocytes. In contrast, autophagy could only be induced in huh7 cells in the presence of overexpressed CYP2E1. In addition, overexpression of NOX4 also significantly increased autophagy, which could be blocked by siRNA mediated knock down. The antioxidant N-acetylcysteine (NAC) also efficiently blocked CYP2E1-and NOX4-mediated induction of autophagy. Finally, specific and non-toxic production of H2O2 by the GOX/CAT system as evidenced by elevated peroxiredoxin (Prx-2) also induced LC3B which was efficiently blocked by NAC. H2O2 strongly increased the autophagic flux as measured by mRFP-GFP-LC3 plasmid. Conclusion: We here provide evidence that short-term ethanol exposure induces autophagy in hepatocytes both in vivo and in vitro through the generation of ROS. These data suggest that suppression of autophagy by ethanol is most likely due to longer alcohol exposure during chronic alcohol consumption with the accumulation of e.g. misfolded proteins.

BACKGROUND Liver-secreted hepcidin is the systemic master switch of iron homeostasis and decreased levels of hepcidin are considered to cause iron overload not only in hereditary hemochromatosis but also in hemolytic anemia and chronic liver diseases. The regulation of hepcidin is complex and its response to iron is still not completely understood. AIM To study the direct effect of iron on various established hepcidin signaling pathways in hepatoma cells or primary hepatocytes. METHODS Hepcidin mRNA expression was studied by quantitative real-time (qRT)-PCR in the presence of various forms of iron including ferric ammonium citrate (FAC) in hepatoma cells (Huh7), murine primary hepatocytes and an established co-culture model of phorbol myristate acetate-differentiated THP-1 monocytes and Huh7 cells. To analyze hepcidin signaling, the response to bone morphogenetic protein 6 (BMP6), interleukin (IL)-6, IL-1β, hypoxia and lipopolysaccharide (LPS) were studied. Hepcidin and small mothers against decapentaplegic 6 (SMAD6) mRNA levels were assessed by qRT-PCR and the expression of phosphorylated signal transducer and activator of transcription 3 (phospho-STAT3), STAT3, phospho-SMAD1/5/8 and SMAD1 proteins were analyzed by western blot. RESULTS All iron III forms including FAC efficiently blocked hepcidin mRNA expression at non-toxic dosages in Huh7 cells or primary hepatocytes in a time and dose-dependent manner (P < 0.001; P < 0.05). Hepcidin blockage could be efficiently blunted by iron chelators salicylaldehyde isonicotinoyl hydrazone (SIH) and Desferal (P < 0.001). FAC also inhibited BMP6, hypoxia, IL-1β and IL-6-mediated hepcidin induction (P < 0.001; P < 0.001; P < 0.05; P < 0.001), and FAC also inhibited LPS-mediated hepatic hepcidin induction in co-culture model (P < 0.001). Moreover, FAC reduced SMAD6 mRNA and p-SMAD1/5/8 protein expression at basal or upon stimulation by BMP6 (P < 0.05; P < 0.01), and FAC also reduced SMAD6 and p-SMAD1/5/8 expression under hypoxia (P < 0.01; P < 0.05). However, FAC has no significant effect on p-STAT3 protein expression at basal or upon stimulation by various stimuli. Notably, in the presence of the BMP/SMAD signaling pathway inhibitor LDN193189 Hydrochloride (LDN), FAC was unable to further decrease hepcidin, SMAD6 and p-SMAD1/5/8 expression compared with LDN alone. CONCLUSION Iron directly blocks hepatocellular hepcidin signaling through the BMP/SMAD pathway but independent of STAT3. This mechanism may contribute to continued iron overload in many pathophysiological conditions ultimately causing a vicious cycle of continued hepcidin suppression.

Liver sinusoidal endothelial cell-derived bone morphogenetic protein 6 (BMP6) and the BMP6-small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte-endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

Liver-secreted hepcidin is the systemic master switch of iron homeostasis and its dysregulation leads to iron accumulation in most of chronic liver diseases. Hepcidin is regulated by iron, inflammation or H2O2, but the role of NOX1 and its products ROS/H2O2 in monocyte-derived macrophages on hepcidin regulation under (patho)physiological conditions is poorly understood. We here investigate the role of NOX1 on regulating hepcidin and cytokines in inflammatory macrophages and subsequent effects on hepatocytes mimicking (patho)physiological conditions (cell ratios, oxygen levels, and inflammation).