Pulsatilla Saponin B4 Alleviates H₂O₂-Induced Oxidative Stress and Apoptosis via the AMPK/Nrf2 Pathway in Bovine Mammary Epithelial Cell Models

The elevated metabolic demands of lactation in dairy cows cause an excess of reactive oxygen species (ROS) in the mammary tissue, which disrupts redox homeostasis and ultimately induces oxidative stress. This oxidative stress directly damages mammary epithelial cells, reduces milk yield and quality, and exacerbates oxidative damage in the mammary gland, ultimately leading to significant economic losses. Therefore, alleviating oxidative stress is essential to safeguard the health of dairy cow mammary glands and ensure farming profitability. Pulsatilla saponin B4 (PSB4), a triterpenoid saponin monomer derived from the roots of Pulsatilla chinensis, possesses antioxidant activities. However, its protective effect against oxidative injury in bovine mammary epithelial cells (BMECs) and the exact mechanisms are not fully elucidated. Therefore, this study aims to elucidate the specific protective effects and mechanisms of PSB4 against oxidative damage induced by hydrogen peroxide (H₂O₂). The results demonstrated that PSB4 effectively alleviates oxidative stress on two fronts: by enhancing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to boost total antioxidant capacity (T-AOC), and by significantly reducing malondialdehyde (MDA) levels and suppressing excessive ROS production. Mechanistically, PSB4 primarily functions by enhancing the nuclear relocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulating antioxidant response genes. Furthermore, PSB4 effectively reduced H₂O₂-induced apoptosis in BMECs, a finding jointly confirmed by JC-1 assay (effectively reversed mitochondrial depolarization) and flow cytometry (showing reduced apoptotic rates). This protective effect was linked to the normalization of apoptosis-associated protein expression, primarily through an increased B-cell lymphoma 2 (BCL2)/BCL2-associated X Protein (Bax) ratio and decreased cysteinyl aspartate-specific proteinase 3 (Caspase-3) expression. Notably, these protective effects of PSB4 could be antagonized by an AMP-activated protein kinase (AMPK)-specific inhibitor (Compound C, CC). Overall, this preliminary study confirms that at the tested concentrations, PSB4 exerts a protective effect against oxidative damage in BMECs, likely through modulation of the AMPK/Nrf2/Caspase-3 signaling axis. These findings provide a rationale for future in vivo studies and support the potential development of PSB4 as a nutritional supplement or therapeutic agent to alleviate oxidative stress and improve mammary health in dairy cows.