Xuzhou City Key Laboratory of Modern AgroBiotechnology, Xuzhou Vocational College of Bioengineering, No. 297 of Sanhuan West Road, Quanshan District, Xuzhou City, 221006, Jiangsu Province, China.
Hereditas. 2022 May 12;159(1):21. doi: 10.1186/s41065-022-00235-y.
Ferroptosis, a novel manner of cell death depended on iron ion, contributed to goat mammary epithelial cell dysfunction. Interleukin-6 (IL-6) is a major pro-inflammatory factor during many inflammation-related diseases including mastitis, and a quite recently identified ferroptosis inducer. This study aims to explore the role of IL-6 in the dysfunction of goat mammary epithelial cells (GMECs) and how the level of IL-6 was regulated.
Primary GMECs were isolated, cultured and treated with lipopolysaccharide (LPS) alone or together with Ferrostatin-1 (Fer-1), a well-known ferroptosis inhibitor. CCK-8 was used to detect cell viability, ELISA was used to detect TNF-α content, and the levels of ROS, GSH and MDA were analyzed with DCFDA-cell ROS detection kit, GSH assay kit and MDA assay kit, respectively. The iron ion level was measured with an iron assay kit.
The expression level of IL-6 protein in GMECs was up-regulated in response to LPS treatment, and the secretion of TNF-α, the cell oxidative stress level and the Fe ion content was robustly increased, which could be reversed by Fer-1 treatment. Knockdown of IL-6 decreased cell oxidative stress level and inhibited ferroptosis in LPS-treated GMECs. Further, ubiquitin experiment and co-immunoprecipitation assay showed that USP14 upregulated IL-6 protein expression by reducing the ubiquitination of IL-6, and overexpression of IL-6 reversed the inhibitory effect of USP14 shRNA on LPS-treated GMECs ferroptosis. The NRF2 inhibitor Brusatol reversed the inhibitory effect of IL-6 shRNA on LPS-treated ferroptosis.
IL-6 protein is deubiquitinated by USP14 and upregulated in LPS-treated GMECs, further promoting ferroptosis and inflammation through the NRF2 signaling pathway.
铁死亡是一种依赖铁离子的新型细胞死亡方式，导致山羊乳腺上皮细胞功能障碍。白细胞介素-6（IL-6）是乳腺炎等许多炎症相关疾病中的主要促炎因子，也是最近发现的一种铁死亡诱导剂。本研究旨在探讨 IL-6 在山羊乳腺上皮细胞（GMEC）功能障碍中的作用以及 IL-6 水平如何被调节。
分离、培养原代 GMECs，用脂多糖（LPS）单独或与 Ferrostatin-1（Fer-1）（一种著名的铁死亡抑制剂）共同处理。CCK-8 法检测细胞活力，ELISA 法检测 TNF-α 含量，DCFDA-细胞 ROS 检测试剂盒、GSH 测定试剂盒和 MDA 测定试剂盒分别检测 ROS、GSH 和 MDA 水平，铁测定试剂盒检测铁离子水平。
GMECs 中 IL-6 蛋白表达水平随 LPS 处理而上调，TNF-α 分泌、细胞氧化应激水平和 Fe 离子含量显著增加，Fer-1 处理可逆转上述变化。IL-6 敲低降低 LPS 处理的 GMECs 中的细胞氧化应激水平并抑制铁死亡。此外，泛素实验和共免疫沉淀实验表明，USP14 通过减少 IL-6 的泛素化来上调 IL-6 蛋白表达，而过表达 IL-6 逆转了 USP14 shRNA 对 LPS 处理的 GMECs 铁死亡的抑制作用。NRF2 抑制剂 Brusatol 逆转了 IL-6 shRNA 对 LPS 处理的铁死亡的抑制作用。
USP14 使 LPS 处理的 GMECs 中的 IL-6 蛋白去泛素化而上调，通过 NRF2 信号通路进一步促进铁死亡和炎症。
