Methionine alleviates heat stress-induced ferroptosis in bovine mammary epithelial cells through the Nrf2 pathway

Heat stress (HS) triggers mammary gland degradation, accompanied by Apoptosis and Autophagy in bovine mammary epithelial cells, negatively affecting milk performance and mammary gland health. Ferroptosis is iron-mediated regulated cell death caused by over production of lipid peroxides, however, the relationship between Ferroptosis and HS in bovine mammary epithelial cells has not been clarified. Methionine (Met) plays a notable role in alleviating HS affecting the mammary glands in dairy cows, but the underlying mechanisms require further exploration. Therefore, we evaluated the regulatory effect and mechanism of Met in alleviating HS-induced Ferroptosis by using bovine mammary epithelial cell line (MAC-T) as an in vitro model. The results showed that Met improved cell vitality, restored mitochondrial function; reduced the content of various Reactive Oxygen Species (ROS), especially hydrogen peroxide (H₂O₂) and superoxide anion (O_2·-); had positive effects on antioxidant Enzyme activity, namely Glutathione Peroxidase (GSH-Px) and superoxide dismutase (SOD). More importantly, Met reduced labile iron protein (LIP) levels; increased iron storage and simultaneously decreased the levels of lipid Reactive Oxygen Species (lipid ROS) and malondialdehyde (MDA), which all caused by HS in MAC-T. Mechanistically, Met increased the protein expression levels of Glutathione Peroxidase 4 (GPX4), solute carrier family 7, member 11 (SLC7A11) and ferritin heavy chain 1 (FTH1) by activating nuclear factor E2-related factor 2 (Nrf2) expression. Additionally, the protection effect of Met was cut off in MAC-T cells after interference with Nrf2, manifesting in decresing the protein expression levels of GPX4, SLC7A11 and FTH1,and increasing the levels of LIP and lipid ROS. Our findings indicate that Met eases HS-induced Ferroptosis in MAC-T through the Nrf2 pathway, revealing that Met produces a marked effect on easing HS-induced bovine mammary gland injury in dairy cows.

Ferroptosis; Heat stress; Iron metabolism; Lipid peroxidation; Methionine.