Targeting GCS regulates the NRF2 axis to inhibit hepatocyte endoplasmic reticulum stress-ferroptosis improving immune-mediated liver injury.

BACKGROUND

Ferroptosis is a regulated, iron-dependent form of cell death triggered by toxic accumulation of lipid peroxides on cell membranes. Although it is established that glycosphingoid metabolites can activate the ferroptosis signaling pathway, whether they are involved in regulating endoplasmic reticulum stress (ERS)-ferroptosis in immune-mediated liver injury requires further investigation.

METHODS

This study used the GCS inhibitor GENZ-123346 (GENZ) to treat ConA-induced immune liver injury in mice. Inflammatory factor levels and liver histology were analyzed to evaluate GENZ's effects. Additionally, the antioxidant and lipid peroxidation capacity of GENZ was tested in vivo and in vitro. Based on the regulation of NFE2-related factor 2 (NRF2) signaling pathway expression (including si-NRF2, NRF2 inducer sulforaphane (SFN), and inhibitor ML385), the regulatory effect of GENZ on ERS-ferroptosis was investigated.

RESULTS

GENZ administration improved the survival rate of mice with immune-mediated liver injury, decreased serum transaminase activity, inhibited inflammatory factor production, reduced hepatocyte ferroptosis. In in vitro experiments, ferroptosis in AML12 cells was accompanied by the activation of ERS-related proteins Glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) and mild alterations in endoplasmic reticulum ultrastructure. Inhibition of ERS by 4-phenylbutyric acid (4-PBA) attenuated ferroptosis in AML12 cells. However, when the cells were treated with the ferroptosis inducer RSL3 in combination with the ERS inducer thapsigargin (Tg), GENZ could not effectively suppress ferroptosis. Silencing NRF2 exacerbated cellular ferroptosis and ERS while diminishing the expression of downstream effectors heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). Conversely, SFN treatment alleviated these effects and upregulated NRF2 signaling, increasing HO-1 and NQO1 expression. Furthermore, ML385 administration reversed the hepatoprotective and antioxidant effects of GENZ on immune-mediated liver injury in mice.

CONCLUSIONS

GCS inhibition exerts hepatoprotective effects by modulating the NRF2 antioxidant pathway to suppress ERS and hepatocyte ferroptosis, revealing a novel role of glycosphingolipid metabolism in immune-mediated liver injury and programmed hepatocyte death.