EXPLORING THE ROLE OF MITOCHONDRIAL ANTIOXIDANT SS-31 IN LIVER HEALTH: IMPLICATIONS FOR ATHLETIC PERFORMANCE AND RECOVERY VIA FERROPTOSIS INDUCTION IN HEPATIC STELLATE CELLS

Abstract

Objective: To investigate the efficacy of the mitochondrial antioxidant SS-31 in ameliorating carbon tetrachloride (CCl4)-induced liver fibrosis in mice, focusing on mechanisms relevant to athletic performance and recovery. Methods: Liver fibrosis was induced in C57BL/6 mice using intraperitoneal injections of a 10% CCl4 olive oil solution. Post-induction, mice were treated with three dosages of SS-31 (5 mg/kg, 10 mg/kg, and 15 mg/kg). Control mice did not undergo modeling. Liver damage was assessed through Hematoxylin-Eosin (HE) staining, while Sirius Red and Masson's trichrome staining evaluated fibrosis. Serum levels of liver enzymes (ALT and AST) and inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured. Liver tissues were analyzed for iron content and mRNA expressions of ACSL4, GPX4, α-Smooth Muscle Actin (α-SMA), Collagen I (Coll I), and Fibronectin. Protein expressions of phosphorylated NF-κB (p-NF-κB), NF-κB, and NLRP3 were assessed using Western blot. Additionally, HSC-T6 cells were treated with SS-31 alone and in combination with Ferrostatin-1 to analyze gene expression changes via qRT-PCR. Results: Liver fibrosis and serum levels of ALT, AST, TNF-α, IL-1β, and IL-6 were significantly elevated in the model group compared to controls (P<0.001). Treatment with SS-31 at all dosages significantly reduced liver fibrosis and serum inflammatory markers (P<0.05). Iron content increased, and mRNA expressions of ACSL4 and GPX4 were up-regulated in SS-31 treated mice. Additionally, α-SMA, Coll I, and Fibronectin mRNA expressions were significantly decreased, alongside reductions in p-NF-κB and NLRP3 expressions (P<0.05). In vitro, SS-31 significantly up-regulated ACSL4 and GPX4, while down-regulating α-SMA, Coll I, and Fibronectin in HSC-T6 cells (P<0.001). Co-treatment with Ferrostatin-1 reversed the effects of SS-31 on ACSL4 and GPX4. Conclusion: SS-31 effectively mitigates CCl4-induced liver fibrosis in mice, potentially through inducing ferroptosis in hepatic stellate cells. This mechanism could be crucial for athletes, as improved liver health supports enhanced metabolic efficiency and recovery, critical for maintaining peak performance.