Aging-associated GSK3β overexpression exacerbates hepatic ischemia-reperfusion injury through Nrf2 deficiency-induced hepatocyte ferroptosis.

Aging leads to significant downregulation of Nrf2 in human liver specimens from elderly donors compared to young donors.

• Human liver specimens were collected from young donors (25-35 years) and elderly donors (65-75 years).
• Nrf2 downregulation was observed at both protein and transcript levels in elderly human livers.
• This age-associated Nrf2 suppression was also confirmed in aged C57BL/6 mice and radiation-induced senescent L02 hepatocytes.

Age-related Nrf2 suppression was associated with impaired antioxidant defenses and increased ferroptotic markers following hepatic ischemia-reperfusion injury.

• Ferroptosis markers measured included elevated lipid peroxidation, iron overload, malondialdehyde (MDA) accumulation, glutathione (GSH) depletion, and transferrin receptor (TFR) upregulation.
• These changes were more pronounced in aged mice compared to young mice subjected to HIRI.
• Senescent L02 hepatocytes also exhibited similar ferroptotic responses consistent with Nrf2 deficiency.

Genetic Nrf2 deficiency in Nrf2-knockout mice exacerbated pathological changes associated with hepatic ischemia-reperfusion injury.

• Nrf2-KO mouse models were subjected to HIRI and compared to wild-type controls.
• Nrf2 deficiency worsened ferroptotic responses including increased lipid peroxidation, iron overload, MDA accumulation, GSH depletion, and TFR upregulation in both cellular and animal models.
• These findings confirmed the functional role of Nrf2 in protecting against ferroptosis during HIRI.

Treatment with the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively reduced liver damage biomarkers and ferroptotic responses in Nrf2-knockout systems.

• Fer-1 treatment reduced liver damage biomarkers ALT and AST in Nrf2-KO mice subjected to HIRI.
• Ferroptotic responses including lipid peroxidation and iron overload were attenuated by Fer-1 in Nrf2-deficient models.
• These results indicate that ferroptosis is a downstream effector of Nrf2 deficiency in HIRI.

Aging-induced GSK3β overexpression drives Nrf2 dysfunction, establishing a GSK3β-Nrf2-ferroptosis axis in ischemia-reperfusion injury progression.

• GSK3β expression was found to be upregulated in aged mice and senescent hepatocytes.
• GSK3β overexpression was mechanistically linked to Nrf2 suppression, connecting aging-related kinase dysregulation to antioxidant pathway impairment.
• This GSK3β-Nrf2-ferroptosis axis was identified as a novel aging-related pathogenic mechanism in HIRI.

Therapeutic targeting of GSK3β with microdose lithium (LiCl) alleviated hepatic ischemia-reperfusion injury in aged mice.

• LiCl was administered as a GSK3β inhibitor intervention in aged C57BL/6 mice subjected to HIRI.
• Microdose lithium treatment resulted in alleviation of HIRI, consistent with restoration of the GSK3β-Nrf2-ferroptosis axis.
• LiCl treatment was tested alongside ferrostatin-1 as a pharmacological strategy targeting the identified pathogenic pathway.

The study identified a comparative model framework linking human aging liver pathology to murine and cellular models of senescence-associated HIRI.

• Three comparative systems were used: human liver specimens from young (25-35 years) and elderly (65-75 years) donors; young and aged C57BL/6 mice and Nrf2-KO mice subjected to HIRI; and radiation-induced senescent L02 hepatocytes.
• Consistent findings across all three systems supported the translational relevance of the GSK3β-Nrf2-ferroptosis axis.
• Radiation-induced senescence was used as an in vitro model to replicate aging-associated cellular changes in hepatocytes.