Objective:To explore the effects of neutrophilic granule protein (NGP) on the expression of lipocalin 2 (LCN2) in inflammatory macrophages and its mechanism.Methods:NGP-high-expressed RAW264.7 cells (NGP/RAW cells) and negative control RAW264.7 cells (NC/RAW cells) were cultured in vitro. Primary peritoneal macrophages of NGP-high-expressed mice and wild-type C57BL/6 mice were extracted, then cultured in vitro. The cell inflammatory model was established by stimulating with 10 mg/L lipopolysaccharide (LPS, LPS group), and the phosphate buffer solution (PBS) control group was set up. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of LCN2 in different types of cells. The protein expression of phosphorylated signal transduction and activator of transcription 1 (p-STAT1) was detected with Western blotting. Other NGP/RAW cells and NC/RAW cells were treated with 10 mg/L LPS, 5 mg/L STAT1 pathway inhibitor (fludarabine)+10 mg/L LPS, respectively. The PBS control group was set up. ELISA was used to detect the level of LCN2. Results:In different types of cells, the levels of LCN2 were increased significantly after LPS stimulation in the LPS group as compared with those in the PBS control group, and peaked at 24 hours (μmol/L: 25.61±1.02 vs. 0.46±0.02 in NC/RAW cells, 74.51±2.14 vs. 0.25±0.04 in NGP/RAW cells, 10.13±0.22 vs. 0.01±0.01 in primary macrophages of wild-type C57BL/6 mice, 28.35±0.61 vs. 0.08±0.01 in primary macrophages of NGP-high-expressed mice, all P < 0.05), indicating that the expression of LCN2 in macrophages altered during inflammation reaction. The level of LCN2 in NGP/RAW cells was found significantly increased at different time points after LPS stimulation comparing with that in NC/RAW cells (μmol/L: 8.32±0.22 vs. 3.12±0.11 at 6 hours, 23.12±0.86 vs. 8.12±0.32 at 12 hours, 74.51±2.14 vs. 25.61±1.02 at 24 hours, all P < 0.05), along with the expression of p-STAT1 was significantly up-regulated. The level of LCN2 in the primary macrophages of NGP-high-expressed mice was also significantly increased at 24 hours after LPS stimulation comparing with that in the primary macrophages of wild-type C57BL/6 mice (μmol/L: 28.35±0.61 vs. 10.13±0.22, P < 0.05). However, after pretreated with STAT1 pathway inhibitors, the production of LCN2 in NGP/RAW cells was decreased significantly comparing with that in the LPS group (μmol/L: 6.81±0.19 vs. 22.54±0.58, P < 0.05). But the inhibitors had no significant effect on LCN2 production in NC/RAW cells showing no significant difference as compared with LPS group (μmol/L: 8.04±0.20 vs. 7.86±0.15, P > 0.05), indicating that NGP could up-regulate the expression of LCN2 in macrophages stimulated by LPS by promoting STAT1 activation. Conclusion:NGP could positively regulate LCN2 expression in inflammatory macrophages by activating STAT1 pathway.

Herein, we define the role of ferroptosis in the pathogenesis of diabetic cardiomyopathy (DCM) by examining the expression of key regulators of ferroptosis in mice with DCM and a new ex vivo DCM model. Advanced glycation end-products (AGEs), an important pathogenic factor of DCM, were found to induce ferroptosis in engineered cardiac tissues (ECTs), as reflected through increased levels of Ptgs2 and lipid peroxides and decreased ferritin and SLC7A11 levels. Typical morphological changes of ferroptosis in cardiomyocytes were observed using transmission electron microscopy. Inhibition of ferroptosis with ferrostatin-1 and deferoxamine prevented AGE-induced ECT remodeling and dysfunction. Ferroptosis was also evidenced in the heart of type 2 diabetic mice with DCM. Inhibition of ferroptosis by liproxstatin-1 prevented the development of diastolic dysfunction at 3 months after the onset of diabetes. Nuclear factor erythroid 2-related factor 2 (NRF2) activated by sulforaphane inhibited cardiac cell ferroptosis in both AGE-treated ECTs and hearts of DCM mice by upregulating ferritin and SLC7A11 levels. The protective effect of sulforaphane on ferroptosis was AMP-activated protein kinase (AMPK)-dependent. These findings suggest that ferroptosis plays an essential role in the pathogenesis of DCM; sulforaphane prevents ferroptosis and associated pathogenesis via AMPK-mediated NRF2 activation. This suggests a feasible therapeutic approach with sulforaphane to clinically prevent ferroptosis and DCM.