Ferrostatin-1 Reversed Chronic Intermittent Hypoxia-Induced Ferroptosis in Aortic Endothelial Cells via Reprogramming Mitochondrial Function

Abstract

Purpose

Chronic intermittent hypoxia (CIH) related arterial endothelium injury is a common cause of cardiovascular system injury. However, the mechanism still needs to be clarified. In this study, we aimed to clarify the role and mechanism of ferrostatin-1 (Fer-1) in CIH-related rat arterial endothelial cells (ROAEC) ferroptosis.

Methods

ROAEC was divided into control group, CIH group, and CIH+ Fer-1 group. Cell viability was detected by cell counting kit 8 kits (CCK8). The apoptotic rate, reactive oxygen species (ROS) levels, Fe2+ levels, and lipid ROS levels were detected by flow cytometry. Malondialdehyde (MDA) levels and nicotinamide adenine dinucleotide (NAD+)/NADH ratio were detected via Elisa kits. The mRNA and protein levels of cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were detected by qRT-PCR and Western blot. Mitochondrial structure and function were observed by transmission electron microscope (TEM) and mitochondrial membrane potential (MMP). Central carbon metabolism was measured to compare metabolites among each group.

Results

After the CIH exposure, ROAEC cell viability decreased; The levels of cell apoptosis, ROS, Fe2+, MDA, and lip ROS increased; The levels of NAD+/NADP ratio decreased; The mRNA and protein levels of GPX4 and SLC7A11 decreased (all p<0.05). Co-cultured with Fer-1 reversed the levels of apoptosis rate, cell viability, ROS, Fe2+, MAD, lipid ROS, NAD+/NADH ratio and the mRNA and protein expression of GPX4 and SLC7A11 (all p<0.05). The TEM results showed that damaged mitochondrial membrane and the matrix spillover in the CIH group. The results of the JC-1 assay showed decreased MMP in the CIH group. Fer-1 treatment ameliorated the mitochondrial injury. The results of central carbon metabolism found that CIH altered the metabolites in the TCA cycle, which were reversed by Fer-1 treatment.

Conclusion

CIH-induced ferroptosis in ROAEC, which were reversed by Fer-1 via reprogramming mitochondrial function.

Keywords:

• obstructive sleep apnea
• chronic intermittent hypoxia
• arterial endothelium injury
• ferroptosis
• mitochondrial function

Abbreviations

OSA, obstructive sleep apnea; CIH, chronic intermittent hypoxia; Fer-1, ferrostatin-1; ROAEC, Rat arterial endothelial cells; SLC7A11, cystine/glutamate antiporter solute carrier family 7 member 11; GPX4, glutathione peroxidase 4; CCK-8, cell counting kit-8; ROS, reactive oxygen species; COPD, chronic obstructive pulmonary disease; DMEM, Dulbecco’s Modified Eagle’s Medium; DCFH-DA, 2’,7’-Dichlorodihydrofluorescein diacetate; TEM, transmission electron microscope; RIPA, Radio Immunoprecipitation Assay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST, Tris Buffer Solution Tween; PBS, phosphate-buffered saline; TCA, tricarboxylic acid cycle; MMP, mitochondrial membrane potential; αKG, alpha-Ketoglutaric acid; NAD+, nicotinamide adenine dinucleotide; MDA, Malondialdehyde; C11-BODIPY 581/591, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diazas-indacene-3-undecanoic acid; PPP, pentose phosphate pathways.

Data Sharing Statement

The data used in this study can be obtained through the corresponding author.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This study was funded by the Natural Science Foundation of Fujian Province (No. 2023J01554), Joint Funds for the Fujian Provincial Finance Project (No: BPB-LNF2021), National Natural Science Foundation of China (No. 82170101).