N-乙酰神经氨酸中通过抑制Nrf2轴促进缺氧/复氧损伤的H9C2心肌细胞发生铁死亡

doi:10.12122/j.issn.1673-4254.2025.01.10

摘要/Abstract

摘要：

目的基于铁死亡探讨N-乙酰神经氨酸（Neu5Ac）加剧H9C2大鼠心肌细胞缺氧/复氧损伤（H/RI）的影响及作用机制。方法以大鼠心肌细胞株H9C2细胞作为研究对象，将细胞分为Control组、H/R 0 h组、H/R 3 h组、H/R 6 h组、H/R 9 h组、H/R 12 h组和H/R 15 h组，在缺氧缺糖8 h，分别复氧复糖0、3、6、9、12、15 h建立细胞缺氧/复氧损伤模型模拟心肌缺血再灌注损伤，通过实验选择最佳复氧时间进行后续实验。根据最佳复氧时间，复氧时给予不同Neu5Ac浓度的完全培养基作为复氧液，将细胞分为Control、0、5、10、20、30、40、50、60 mmol/L组，通过实验选择最佳药物浓度进行后续实验。根据前述最佳复氧时间和最佳给药浓度探讨Neu5Ac对H9C2心肌细胞加剧损伤的机制，将H9C2心肌细胞分为5组：Control组、H/R组、H/R+Neu5Ac组、H/R+Fer-1（铁死亡抑制剂）组、H/R+Neu5Ac+Fer-1组。通过检测5组细胞超氧化物歧化酶（SOD）活性，判断各组细胞氧化应激水平；使用FerroOrange荧光探针和C11 BODIPY 581/591荧光探针分别检测细胞内Fe²⁺和脂质过氧化物（Lipid ROS）水平；此外，通过Western blotting检测Neu5Ac在H9C2心肌细胞H/RI中对核因子E2相关因子2（Nrf2）、谷胱甘肽过氧化物酶4（GPX4）、血红素加氧酶-1（HO-1）、铁死亡抑制蛋白1（FSP1）、胱氨酸/谷氨酸反向转运体（xCT）蛋白表达的影响。结果 H9C2心肌细胞缺氧缺糖8 h后，H/R 6 h组和除H/R 9 h组之外的其他组相比，Nrf2、GPX4、HO-1、FSP1蛋白表达最少，且差异有统计学意义（P<0.001）。与Control组相比，不同浓度药物的实验组细胞活力均明显降低（P<0.0001），且随药物浓度逐渐增大，细胞活力逐渐减小，并测得该药物的半抑制浓度IC₅₀=30.07 mmol/L。选择心肌细胞未发生明显铁死亡的时间，即缺氧缺糖8 h合并复氧复糖3 h，且复氧复糖时Neu5Ac浓度为30 mmol/L进行后续实验。结果显示，Neu5Ac可以提高SOD活性，增加Fe²⁺和Lipid ROS水平，降低Nrf2、GPX4、xCT、HO-1、FSP1等5种蛋白的表达（P<0.05）。在Neu5Ac的基础上使用Fer-1后，与H/R+Neu5Ac组相比，SOD活性下降，Fe²⁺和Lipid ROS水平减少，Nrf2、GPX4、xCT、HO-1、FSP1等5种蛋白的表达升高（P<0.0001）。结论 Neu5Ac在H/RI中加剧心肌细胞发生铁死亡可能是通过抑制Nrf2轴进而产生过多的活性氧和脂质活性氧而导致。

关键词: N-乙酰神经氨酸, 心肌细胞, 缺氧/复氧损伤, 铁死亡

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.

Key words: N-acetylneuraminic acid, myocardial cells, hypoxia/reoxygenation injury, ferroptosis

季春斐, 左宗超, 王钧, 李妙男. N-乙酰神经氨酸中通过抑制Nrf2轴促进缺氧/复氧损伤的H9C2心肌细胞发生铁死亡[J]. 南方医科大学学报, 2025, 45(1): 72-79.

Chunfei JI, Zongchao ZUO, Jun WANG, Miaonan LI. N-acetylneuraminic acid promotes ferroptosis of H9C2 cardiomyocytes with hypoxia/reoxygenation injury by inhibiting the Nrf2 axis[J]. Journal of Southern Medical University, 2025, 45(1): 72-79.

图/表 6

图1 Western blotting检测分析复氧不同时间对Nrf2、GPX4、HO-1、FSP1蛋白的表达的影响

Fig.1 Western blotting of the expression of Nrf2, GPX4, HO-1, and FSP1 in H9C2 cells with hypoxia and glucose deprivation for 8 h, followed by reoxygenation with normal glucose supplementation for 0, 3, 6, 9, 12, and 15 h.***P<0.001, ****P<0.0001 vs H/R 6h group.

图2 不同Neu5Ac浓度对细胞H/RI存活率的影响

Fig.2 Effect of different Neu5Ac concentrations on survival rate of H9C2 cells with H/R injury at 3 h during reoxygenation (Mean±SD, n=3). A-D: Other groups were compared with Control Group, 10 mmol/L group, 20 mmol/L group and 30 mmol/L group separately. **P<0.01, ****P<0.0001, ****P<0.0001.

图3 Neu5Ac对H9C2细胞H/RI中SOD活力的影响

Fig.3 Effect of Neu5Ac on SOD activity in H9C2 cells with H/R injury. ***P<0.001 vs H/R group,####P<0.0001 vs H/R+N group.

图4 FerroOrange荧光探针检测Neu5Ac对H9C2细胞H/RI中Fe2+产生的影响

Fig.4 FerroOrange fluorescent probe for detecting the effect of Neu5Ac on Fe2+production in H9C2 cells with H/R injury.

图5 Neu5Ac对H9C2细胞H/RI中Lipid ROS产生的影响

Fig.5 Effect of Neu5Ac on the production of lipid ROS in H9C2 cells with H/R injury. A: Fluorescence images of the 5 groups of cells in Oxidation state, Reduction state, and Merge. B: Mean fluorescence intensity of the 5 groups of cells in the oxidized state. C: Mean fluorescence intensity of the 5 groups of cells in the reduced state. D: Ratio of the mean fluorescence intensity in the oxidized and reduced states in the 5 groups. ***P<0.001, ****P<0.0001 vs H/R group, ###P<0.001, ####P<0.0001 vs H/R+N group.

图6 Neu5Ac在H9C2细胞中对铁死亡相关蛋白表达的影响

Fig.6 Effect of Neu5Ac on expression of ferroptosis related proteins in H9C2 cells with H/R injury. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 vs H/R group,####P<0.0001 vs H/R+N group.

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