N-acetylneuraminic acid promotes ferroptosis of H9C2 cardiomyocytes with hypoxia/reoxygenation injury by inhibiting the Nrf2 axis

JI Chunfei; ZUO Zongchao; WANG Jun; LI Miaonan

doi:10.12122/j.issn.1673-4254.2025.01.10

Abstract：Objective To investigate the mechanism through which N-acetylneuraminic acid(Neu5Ac)exacerbates hypoxia/reoxygenation(H/R)injury in rat cardiomyocytes(H9C2 cells).Methods H9C2 cells were cultured in hypoxia and glucose deprivation for 8 h followed by reoxygenation for different durations to determine the optimal reoxygenation time.Under the optimal H/R protocol,the cells were treated with 0,5,10,20,30,40,50,and 60 mmol/L Neu5Ac during reoxygenation to explore the optimal drug concentration.The cells were then subjected to H/R injury followed by treatment with Neu5Ac,Fer-1(a ferroptosis inhibitor),or both.The changes in SOD activity,intracellular Fe2+and lipid ROS levels in the cells were evaluated,and the cellular expressions of Nrf2,GPX4,HO-1,FSP1,and xCT proteins were detected using Western blotting.Results Following hypoxia and glucose deprivation for 8 h,the cells with reoxygenation for 6 h,as compared with other time lengths of reoxygenation except for 9 h,showed the lowest expression levels of Nrf2,GPX4,HO-1,and FSP1 proteins(P＜0.001).Neu5Ac treatment of dose-dependently decreased the viability of the cells with H/R injury with an IC50 of 30.07 mmol/L.Reoxygenation for 3 h with normal glucose supplementation and a Neu5Ac concentration of 30 mmol/L were selected as the optimal conditions in the subsequent experiments.The results showed that Neu5Ac could significantly increase SOD activity,Fe2+and lipid ROS levels and reduce Nrf2,GPX4,HO-1,and FSP1 protein expressions in H9C2 cells with H/R injury,but its effects were significantly attenuated by treatment with Fer-1.Conclusion Neu5Ac exacerbates ferroptosis of myocardial cells with H/R injury by inhibiting the Nrf2 axis to promote the production of ROS and lipid ROS.