Ubiquitylome analysis reveals the protective effect of α-lipoic acid via the ferroptosis pathway during recovery after hepatic oxidative stress in finishing pigs.

Ubiquitination is a critical post-translational modification that regulates hepatic antioxidant defense. α-Lipoic acid (LA) exerts potent hepatoprotective effects, but its mechanism of regulating ubiquitination in porcine livers with oxidative injury, especially during the recovery period, remains unclear. In this study, 18 castrated male Large White (LW) pigs were randomly assigned to control, diquat (DQ), and diquat + LA (DL, 800 mg/kg diet) groups. On day 15, the DQ and DL groups were injected with diquat (8 mg/kg body weight), and liver tissues were sampled on day 29. Tandem mass tag (TMT)-based proteomic analysis quantified 5,326 of 7,271 identified proteins, which were validated by parallel reaction monitoring (PRM). Label-free ubiquitylome analysis identified 1,651 ubiquitination sites corresponding to 1,446 peptides and 594 ubiquitinated proteins, among which 1,208 sites, 1,058 peptides, and 489 proteins were quantified. Our findings revealed that LA upregulated ferritin heavy chain 1 (FTH1) expression and promoted Poly(rC)-binding protein 1 (PCBP1) ubiquitination at K314 in porcine liver during recovery after hepatic oxidative stress; these two proteins are core regulators of iron storage in ferroptosis pathway. Hepatic antioxidant status and histopathological analysis confirmed that dietary LA effectively balanced the compensatory activation of antioxidant system and alleviated oxidative stress-induced liver injury. Cell-based assays further verified that LA significantly ameliorated the reduction in FTH1 and the elevation in Fe2+ concentration induced by oxidative stress in a concentration-dependent manner. Collectively, we propose that LA protects hepatocytes by enhancing iron sequestration, inhibiting lipid peroxidation, and suppressing ferroptosis. This study provides novel insights into how LA maintains hepatic redox homeostasis by regulating the expression and ubiquitination of key proteins in the ferroptosis pathway.