Glutathione peroxidase 3 preserves hepatocyte mitochondrial quality control to enhance macrophage pro‐regenerative phenotype during liver regeneration

Abstract Background Precise regulation of mitochondrial function is critical for liver regeneration. However, the underlying regulatory mechanism remains elusive. Here, we aimed to investigate the role of hepatocellular glutathione peroxidase 3 (GPX3) in liver regeneration. Methods In a 70% partial hepatectomy (PH) mouse model, immunostaining and single‐cell RNA sequencing revealed significant enrichment but down‐regulation of mitochondrial oxidative phosphorylation pathways post‐PH, along with up‐regulated hypoxia‐inducible factor 1a (HIF‐1a) and GPX3 in hepatocytes. Single‐cell analysis confirmed peak GPX3 expression in hepatocytes at day 2 post‐PH. Hepatocyte‐specific GPX3 knockout impaired mitochondrial function and delayed liver regeneration. Results Mechanistically, immunoprecipitation–mass spectrometry and MitoCarta3.0 analysis identified voltage‐dependent anion channel 1 (VDAC1) as a direct GPX3‐binding partner. GPX3 interacted with VDAC1 via its A2 domain (residues 75–150), suppressing VDAC1 oligomerisation to restore mitochondrial Ca 2+ homeostasis and preserve mitochondrial quality control (MQC). Notably, GPX3 deficiency promoted mitochondrial DNA (mtDNA) release, activating the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway in macrophages. Persistent STING hyperactivation increased interferon production while suppressing hepatocyte growth factor release, further inhibiting regeneration. Critically, GPX3 overexpression enhanced liver regeneration in both PH and hepatic ischemia–reperfusion injury models, underscoring its central role across regenerative stressors. Conclusions In conclusion, GPX3 promotes liver regeneration by inhibiting VDAC1 oligomerisation to stabilise mitochondrial Ca 2+ dynamics and MQC, while preventing mtDNA‐mediated functional and phenotypic alterations in macrophages, positioning it as a therapeutic target for liver regeneration. Key points GPX3 directly binds VDAC1 via its A2 domain to suppress VDAC1 oligomerisation, restoring mitochondrial Ca 2 + homeostasis and preserving mitochondrial quality control during liver regeneration. GPX3 deficiency promotes mtDNA release, hyperactivating the cGAS–STING pathway in macrophages and suppressing hepatocyte growth factor (HGF) release. GPX3 overexpression enhances liver regeneration in both partial hepatectomy and hepatic ischemia–reperfusion injury models, highlighting its therapeutic potential.