Jia Chen,1– 4,* Xiaoyu Deng,1– 4,* Ting Lin,1– 4 Jiefeng Huang,1– 4 Yisong Yang,1– 4 Ningfang Lian1– 4 

1Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, People’s Republic of China; 2Fujian Provincial Sleep-Disordered Breathing Clinic Center, Fuzhou, Fujian, People’s Republic of China; 3Institute of Respiratory Disease, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China; 4Department of Respiratory and Critical Care Medicine, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, People’s Republic of China

*These two authors contributed equally to this work

Correspondence: Ningfang Lian, Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Fujian Medical University, No. 20, Chazhong Road, Taijiang District, Fuzhou, Fujian Province, 350005, People’s Republic of China, Tel +86-591-87981698, Email 1533532863@qq.com

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