Inhibition of USP14 suppresses ferroptosis and inflammation in LPS-induced goat mammary epithelial cells through ubiquitylating the IL-6 protein

Abstract

Background 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. Methods 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. Results 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 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. Conclusion IL-6 protein is deubiquitinated by USP14 and upregulated in LPS-treated GMECs, further promoting ferroptosis and inflammation through the NRF2 signaling pathway.

Construction of a ferroptosis-related eight gene signature for predicting the prognosis and immune infiltration of thyroid cancer

“…A previous study found that FABP4 was upregulated in recurrent human breast cancer samples; FABP4 protects cancer cells from oxidative stress-induced ferroptosis and is associated with a worse prognosis in cancer patients (24). IL-6 reversed ferroptosis and growth inhibition induced by xCT knockdown or erastin in head and neck squamous cell carcinoma (HNSCC) (25) but promoted ferroptosis in bronchial and mammary epithelial cells (26,27). The association of these eight genes with ferroptosis has been clearly validated in other tumors and diseases; these associations will be the focus of future research on the relationship between THCA and ferroptosis.…”

Emerging Roles of the Iron Chelators in Inflammation

“…In recent years, it has been demonstrated that there is a relation between ferroptosis and inflammation. Several pro-inflammatory cytokines are involved in ferroptosis by regulation of GPX4 levels and activity in cancer cells [ 40 , 41 ]. A strong downregulation of GPX4 was observed in these cells treated with TNF, inducing ferroptosis initiation [ 42 ].…”

Quercetin Suppresses Ferroptosis in Chondrocytes via Activating the Nrf2/ GPX4 Signaling Pathway

“…12 Decreased protein levels of GPX4, which is substantially lower in OA-affected cartilage compared to healthy cartilage, has been associated with worsened OA progression. 13 Moreover, nuclear factor-erythroid 2-related factor 2 (Nrf2), which positively regulates GPX4 protein levels, 14 has shown chondroprotective effects in OA therapies. 15 Therefore, targeting the Nrf2-mediated ferroptosis pathway holds potential as a therapeutic approach for treating OA.…”