Abstract: Objective To investigate the effect of nuclear factor-erythroid 2-related factor 1(Nrf1) on ferroptosis of murine cardiomyocyte cell line HL-1 induced by high-fat (HF) and its underlying mechanism. Methods HL-1 cells were incubated with different doses of 0 palmitic acid(PA) as 100, 200, 300, 400, and 500 μmol/L PA for 24 hours, and 300 μmol/L PA acid was selected as the subsequent HF intervention concentration. The cells were divided into the following groups: control group, vehicle group (HF + solvent), ferrostatin-1 group (HF+ferroptosis inhibitor), tert-butylhydroquinone (tBHQ) group (HF+Nrf1 agonist), Erastin group (HF+ferroptosisagonist), and tBHQ+Erastin group (HF + Nrf1 agonist + ferroptosis agonist). Cell viability of HL-1 cells was determined by the CCK-8 assay. Intracellular reactive oxygen species (ROS) level was measured by DCFH-DA fluorescent probe assay. The concentration of malondialdehyde(MDA),glutathione(GSH),and glutathione peroxidase 4(GPX4)was determined by commercial available kits. The expression level of ferroptosis marker prostaglandin-endoperoxide synthase 2(PTGS2) and Nrf1 was detected by RT-qPCR and Western blot. Results With the increase of palmitic acid concentration, the viability of HL-1 cells decreased significantly, and after intervention with 300 μmol/L PA, the viability of HL-1 cells was significantly lower than that of the control group(P＜0.001). Under high-fat conditions, the level of myocardial ferroptosis marker PTGS2 increased significantly, while the level of GSH and GPX4 decreased significantly(all P＜0.05 or P＜0.01). On the contrary, after incubation with ferrostatin-1, a specific inhibitor of ferroptosis, cell viability was significantly improved(P＜0.01), and high-fat-induced ferroptosis was significantly reversed(P＜0.05). At the same time, the expression of Nrf1(both mRNA and protein levels) in high-fat group was significantly decreased(P＜0.01). After adding Nrf1 agonist(tBHQ), cell viability increased significantly(P＜0.01), and the degree of ferroptosis decreased significantly(P＜0.01). Further use of the ferroptosis agonist Erastin reversed the myocardial protective effect of Nrf1 agonist(P＜0.05). Conclusions High-fat can aggravate ferroptosis of cardiomyocytes by causing oxidative stress and reducing antioxidant capacity. Nrf1 can play a myocardial protective role in high-fat environment by inhibition of ferroptosis.

Key words: mouse cardiomyocyte cell line HL-1, high-fat, ferroptosis, nuclear factor-erythroid 2-related factor 1(Nrf1)