Background: Ischemia-reperfusion (I/R) injury involves oxidative stress, inflammation, and mitochondrial dysfunction. Galectin-3 (Gal-3) plays a key regulatory role in these processes; however, its association with the Mitochondrial Transcription Factor A/Peroxisome Proliferator-Activated Receptor Beta (TFAM/PPARβ) signaling pathway remains unclear. Therefore, this study aims to investigate the role of Gal-3 in I/R injury, focusing on its impact on oxidative stress, inflammatory responses, and the TFAM/PPARβ signaling pathway. Furthermore, it explores the role of TFAM in Gal-3-mediated I/R injury.

Methods: A mouse I/R model was established, and recombinant Gal-3 protein or anti-Gal-3 antibody was injected to modulate Gal-3 expression. mRNA and protein levels of Gal-3 in mouse serum, as well as relevant oxidative stress and inflammation markers, were evaluated using quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), Western blotting, and histological analysis. The human myocardial AC16 cell line was used to establish an in vitro I/R model, and Sh-Gal-3 or Gal-3 overexpression plasmids were transfected to regulate Gal-3 expression, further assessing the effects of Gal-3 on oxidative stress and inflammatory response. Additionally, Sh-Gal-3 and Sh-TFAM were co-transfected into the I/R in vitro cell model to explore the role of TFAM in Gal-3-mediated effects.

Results: In the in vivo I/R model, compared to the Sham group, Gal-3 mRNA and protein levels were significantly increased in the serum of I/R mice (p < 0.05). Recombinant Gal-3 protein treatment aggravated myocardial injury, as evidenced by increased levels of Creatine Kinase-MB (CK-MB), Creatine Kinase (CK), Left Ventricular End-Diastolic Diameter (LVEDD), and Left Ventricular End-Systolic Diameter (LVESD), with a substantial increase in cardiac pathological damage. In contrast, anti-Gal-3 antibody treatment significantly alleviated these adverse effects. Masson staining revealed a significant increase in myocardial fibrosis in the I/R group, and Gal-3 further exacerbated fibrosis, which was counteracted by anti-Gal-3 treatment. Furthermore, the levels of pro-inflammatory cytokines and oxidative stress markers were significantly elevated in the I/R group, with administration of Gal-3 recombinant protein further enhancing these changes, while anti-Gal-3 treatment reduced them. In the in vitro model, Gal-3 overexpression significantly increased the levels of inflammatory factors and reactive oxygen species (ROS), whereas Gal-3 knockdown reduced these levels. After co-transfection of Sh-Gal-3 and Sh-TFAM, compared to the Sh-Gal-3 group, there was a significant increase in ROS fluorescence intensity, Malondialdehyde (MDA) levels, and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) disaggregation levels, while Superoxide Dismutase (SOD) levels were significantly reduced (p < 0.05). Additionally, the levels of Interleukin-6 (IL-6), Interferon-gamma (IFN-γ), and Tumor Necrosis Factor-alpha (TNF-α) in the Sh-Gal-3+Sh-TFAM group were significantly higher than those in the Sh-Gal-3 group.

Conclusions: This study demonstrates that Gal-3 plays a crucial role in I/R injury, promoting oxidative stress, inflammation, and myocardial damage, by modulating the TFAM/PPARβ signaling pathway. Furthermore, TFAM plays a critical regulatory role in Gal-3-mediated oxidative stress and inflammation. These findings provide new insights into treating I/R injury, and targeting Gal-3 and TFAM may offer a potential therapeutic strategy for I/R-induced injury.