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

Klebsiella pneumoniae (K. pneumoniae), a prominent causative agent of mastitis in dairy cattle, remains enigmatic in its pathogenic mechanisms. This study aimed to reveal the effects of K. pneumoniae 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 K. pneumoniae. In vitro, primary BMECs were treated with 10 μM Fer-1 and K. pneumoniae alone or in combination. The results showed that mammary glands infected with K. pneumoniae exhibited increased transcriptional levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Concurrently, significant elevations in iron, 4-hydroxynonenal, and reactive oxygen species (ROS) levels were observed. Conversely, K. pneumoniae infection downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), cystine/glutamate antiporter (xCT), glutathione peroxidase 4 (GPX4), and glutathione levels. Following K. pneumoniae 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 IL-1β, IL-6, IL-8, and TNF-α. In conclusion, Fer-1 alleviates K. pneumoniae-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.

肺炎克雷伯菌（Klebsiella pneumoniae, K. pneumoniae）是奶牛乳腺炎的主要致病菌，但其致病机制仍未完全阐明。本研究旨在通过诱导铁死亡（ferroptosis）揭示肺炎克雷伯菌对乳腺的影响，以及铁死亡抑制剂Ferrostatin-1（Fer-1）在牛乳腺上皮细胞（bovine mammary epithelial cells, BMECs）中对抗该病原体介导的损伤的保护作用。本研究以荷斯坦奶牛为实验对象，建立肺炎克雷伯菌乳腺内感染模型。体外实验中，原代牛乳腺上皮细胞分别以10 μM Fer-1、肺炎克雷伯菌单独处理，或二者联合处理。结果显示，感染肺炎克雷伯菌的乳腺组织中，白细胞介素（interleukin, IL）-1β、IL-6、IL-8及肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）的转录水平显著升高；同时，铁离子、4-羟基壬烯醛（4-hydroxynonenal）与活性氧（reactive oxygen species, ROS）的水平亦明显上升。与之相反，肺炎克雷伯菌感染会下调核因子E2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）、胱氨酸/谷氨酸反向转运体（cystine/glutamate antiporter, xCT）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）的表达及谷胱甘肽水平。肺炎克雷伯菌入侵后，牛乳腺上皮细胞内的Fe²+与脂质ROS发生蓄积，阻碍了Nrf2/xCT/GPX4信号通路的激活。此外，Fer-1可促进Nrf2蛋白的核转位，上调Nrf2/xCT/GPX4的蛋白水平，同时下调IL-1β、IL-6、IL-8及TNF-α的转录水平。综上，Fer-1可通过上调Nrf2/xCT/GPX4通路，缓解肺炎克雷伯菌诱导的牛乳腺上皮细胞炎症因子活化与铁死亡，提示抑制铁死亡有望成为防控奶牛乳腺炎的可行治疗策略。