Oxidative stress levels and antioxidant defense mechanisms (Nrf2-Keap1 signaling pathway) in the Harderian glands of hibernating Daurian ground squirrels

Cyclic hibernation bouts in Daurian ground squirrels (Spermophilus dauricus) lead to repeated suppression and recovery of mitochondrial respiratory function across multiple organs, potentially impacting reactive oxygen species (ROS) dynamics. The Harderian gland (HG) plays an important role in endocrine regulation through porphyrin secretion. However, the influence of hibernation on oxidative pressure and associated antioxidant pathways in the HG remains inadequately understood. In the current study, we investigated the morphological changes, secretory activity, ROS levels, and underlying mechanisms in the HG of Daurian ground squirrels at distinct circannual stages of hibernation. Results indicated that: (1) Protoporphyrin levels in the HG increased during hibernation compared to the summer active (SA) phase, with a reduction in acinar lumen during torpor, potentially related to hibernation in a low-light environment. (2) Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content during hibernation and post-hibernation (POST) did not exceed the levels observed in SA, indicating that the HG effectively mitigated oxidative pressure and lipid peroxidation during these periods. (3) Superoxide dismutase (SOD) activity increased while glutathione peroxidase (GPx) activity decreased during Inter-bout arousal (IBA) compared to both SA and torpor, although total antioxidant capacity (T-AOC) remained stable across all stages. (4) Overall fluorescent intensity of nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) was significantly lower than in SA. These findings demonstrate that the HG in Daurian ground squirrels maintains a favorable oxidative status through the regulation of antioxidant enzyme activities during hibernation and even post-hibernation.