<i>Klebsiella pneumoniae</i> induced ferroptosis by inhibition of the Nrf2/xCT/GPX4 pathway in bovine mastitis: <i>In vivo</i> and <i>in vitro</i>

<i>Klebsiella pneumoniae</i> (<i>K. pneumoniae</i>), a prominent causative agent of mastitis in dairy cattle, remains enigmatic in its pathogenic mechanisms. This study aimed to reveal the effects of <i>K. pneumoniae</i> on mammary glands via the induction of ferroptosis, as well as the protective role of Ferrostatin-1 (Fer-1) against this pathogen-mediated damage in bovine mammary epithelial cells (BMECs). Holstein cows were used to establish an intramammary infection model of <i>K. pneumoniae</i>. <i>In vitro</i>, primary BMECs were treated with 10 μM Fer-1 and <i>K. pneumoniae</i> alone or in combination. The results showed that mammary glands infected with <i>K. pneumoniae</i> exhibited increased transcriptional levels of <i>interleukin</i> (<i>IL</i>)<i>-1β</i>, <i>IL-6</i>, <i>IL-8</i>, and <i>tumor necrosis factor-α</i> (<i>TNF-α</i>). Concurrently, significant elevations in iron, 4-hydroxynonenal, and reactive oxygen species (ROS) levels were observed. Conversely, <i>K. pneumoniae</i> infection downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), cystine/glutamate antiporter (xCT), glutathione peroxidase 4 (GPX4), and glutathione levels. Following <i>K. pneumoniae</i> invasion, intracellular Fe²⁺ and lipid ROS accumulated in the BMECs, impeding activation of Nrf2/xCT/GPX4 signal transduction. Additionally, Fer-1 facilitated the nuclear translocation of Nrf2 protein, upregulating the protein levels of Nrf2/xCT/GPX4 while downregulation transcriptional levels of <i>IL-1β</i>, <i>IL-6</i>, <i>IL-8</i>, and <i>TNF-α</i>. In conclusion, Fer-1 alleviates <i>K. pneumoniae</i>-induced inflammatory factor activation and ferroptosis in BMECs via upregulation of the Nrf2/xCT/GPX4 pathway, supporting ferroptosis inhibition holds promise as a feasible therapeutic agent for the control of mastitis.