Zhu G, Sui S, Shi F, Wang Q

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 ...

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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-? 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-?, the cell oxidative stress level and the Fe2+ 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.

Date: May. 12, 2022

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