Mastitis is one of the diseases that are common in the development of the dairy industry and causes major economic losses. The occurrence and development of mastitis is a process of interaction between pathogenic microorganisms and breast defense system, which is determined by the pathogenicity of the pathogen and the strength of breast defense function. Mammary epithelial cells are the main parenchymal cells in the breast, the dysfunction of which is a main pathological mechanism of mastitis. Recently, it was revealed that ferroptosis, a new type of programmed cell death, which is iron dependent and different from apoptosis and cell necrosis and pyroptosis, was involved in the dysfunction of mammary epithelial cells. Inhibition of ferroptosis by biological or chemical means has been demonstrated to improved functions of mammary epithelial cells, including cell viability, oxidative stress and inflammation. In this study, we also found that LPS treatment increased oxidative stress, inflammation and ferroptosis of mammary epithelial cells, and inhibition of ferroptosis by USP14 knockdown or IL-6 knockdown could alleviate dysfunction of mammary epithelial cells17, 18).
Cells with ferroptosis ultimately die due to mitochondrial dysfunction and lipid peroxidation toxicity 19). In the biochemical process, the depletion of intracellular GSH, the decrease of GPX4 activity, the failure of lipid peroxides to be metabolized by GPX4 catalyzed reduction reaction, the accumulation of Fe2+ and a large amount of ROS produced by lipid peroxidation leading to the occurrence of ferroptosis 20). Cell survival rate, the activities of GPX, SOD, CAT and the content of MDA reflect the ability of free radical scavenging and the degree of lipid peroxidation damage, which can be used as important indicators to determine whether cells are experiencing oxidative stress 21–23). The secretion of cytokines interleukin (IL) and TNF-α can also reflect the degree of oxidative damage of cells to a certain extent, and can be used as a reference index for judging the degree of cell damage 24, 25). In this study, we found that IL-6 protein expression was up-regulated in GMECs treated with LPS. The expression of oxidative stress and ferroptosis markers were further detected, and it was found that Fe2+ content was increased, and oxidative stress levels were up-regulated, which was relieved after Fer-1 treatment. The above changes indicated that LPS stimulated ferroptosis of GMECs, and knocking down IL-6 in LPS-treated GMECs inhibited ferroptosis induced by LPS.
IL-6 is a famous pro-inflammatory factor involved in a large number of acute and chronic inflammatory diseases. As a major pro-inflammatory cytokine induced by LPS, IL-6 was recently reported to play an important role in ferroptosis. For example, in mice with hemochromatosis, IL-6/hepcidin signaling was activated and contributed to ferroptosis of mouse hepatocytes, which could be suppressed by the anti-inflammation drug auranofin26). IL-6 and its receptor were aberrantly expressed in injured cartilage tissues of patients with intervertebral disc degeneration, and miR-10a-5p suppressed IL-6 expression and inhibited IL-6 mediated cartilage cell oxidative stress and ferroptosis15). Moreover, IL-6 was reported to regulate ferroptosis of epithelial cells in several organs27, 28). In our study, we found that IL-6 could induce ferroptosis of mammary epithelial cells, and inhibition of IL-6 by USP14 knockdown suppressed ferroptosis, suggesting a novel contribution manner of IL-6 in mastitis.
The ubiquitin-proteasome system mediates the degradation of most proteins in eukaryotic cells, and has various biological functions, such as regulation of inflammation, cell proliferation, signal transduction, transcription regulation, and apoptosis. USP14 is an important deubiquitinating enzyme. It is involved in the process of ubiquitin-mediated proteasome degradation of protein. It has been widely studied in the research of tumors, neurological diseases and aging, but its role in inflammatory response has been rarely reported. In breast cancer cells, knocking down USP14 reduced the protein expression of CDK1 by increasing the ubiquitination level, thereby arresting the cell cycle in the G2/M phase, and significantly reducing cell proliferation 29). In this study, we found that the expression level of USP14 was up-regulated in LPS-treated GMECs, and USP14 up-regulated the expression of IL-6 protein by deubiquitinating IL-6. Knockdown of USP14 inhibited the occurrence of LPS-induced ferroptosis. And overexpression of IL-6 could reverse the inhibitory effect of knockdown of USP14 on LPS-induced ferroptosis.
NRF2 is an important antioxidant and anti-inflammatory regulator. It combines with downstream anti-oxidation response element (ARE) to control the transcription of GPX4, heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1) and other genes 30). NRF2 can also inhibit the degradation of IκB-α proteasome and the activation of NF-κB signaling pathway 31). It is closely related to multiple ferroptosis-related genes, such as antioxidant and iron metabolism, and is currently considered to be an important negative regulator of ferroptosis. NRF2 reduces toxic iron ions by regulating transferrin receptor (TFR1), FPN and FTH1, thereby enhancing the storage capacity of iron 32, 33). On the other hand, GSH, GPX4 and xCT are regulated to increase the reduction capacity and inhibit lipid peroxidation 34, 35). In the study, we found that knockdown of USP14 or IL-6 could activate the NRF signaling pathway, and NRF2 pathway inhibitor Brusatol reversed the inhibitory effect of knockdown of IL-6 on LPS-induced ferroptosis.
In conclusion, IL-6 protein expression can be regulated by deubiquitylation of USP14, and further influence the occurrence of LPS-induced ferroptosis through the NRF2 signaling pathway.