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.
Hidradenitis Suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent nodules, abscesses, and dermal tracts. We recently reported a high prevalence of anemia in HS patients (Soliman et al, 2018). This data led to the hypothesis that chronic inflammation in HS predominantly causes anemia of chronic disease (ACD) through upregulation of IL-6 and hepcidin, a key regulator of iron homeostasis. Hepcidin is widely accepted as a marker distinguishing ACD from iron deficiency anemia (IDA) in inflammatory conditions. The purpose of our study was to measure serum hepcidin in HS patients to classify the type of anemia observed in this cohort. We measured hematologic data, inflammatory markers, and serum hepcidin using an enzyme-linked immunosorbent assay (ELISA, R&D Systems Inc, Minneapolis) in 55 patients with varying degrees of HS severity. Independent sample t-tests and one-way ANOVA were used to compare hepcidin levels in HS patients with anemic and non-anemic states. Of 55 HS patients evaluated in this study, 42 (76%) were female and the average age was 37.6 AE 13.2 years. The mean hemoglobin (Hb) was 12.2 AE 2.0 g/dL and mean hepcidin was 19.5 AE 12.9 ng/mL. Anemic patients (n¼26) with reduced iron stores (ferritin 30 ng/mL) had significantly lower hepcidin than anemic patients with adequate iron stores (9.7 AE 15.8 ng/mL vs. 23.8 AE 11.1, p ¼ 0.03). Elevated hepcidin in this subset suggests that ACD is the predominant cause of anemia. Analysis by ANOVA also found that hepcidin levels were significantly greater in patients with more severe HS, as measured by HS-Physician Global Assessment scores (p¼.005). Hepcidin may serve as a surrogate biomarker for active disease and inflammation in HS. These findings affirm the utility of hepcidin as a promising tool for distinguishing IDA from ACD; in turn, identifying HS patients likely to benefit from iron supplementation.
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