铁抑制素-1抑制丙酮醛诱导的小鼠肺泡巨噬细胞铁死亡

doi:10.12122/j.issn.1673-4254.2024.12.21

摘要/Abstract

摘要：

目的探讨丙酮醛（MG）对小鼠肺泡巨噬细胞的损伤及铁死亡抑制剂铁抑制素-1（FER-1）在其中的作用机制。方法采用小鼠肺泡巨噬细胞来源的MH-S细胞体外培养，分别用MG及FER-1进行处理，建立细胞损伤及损伤挽救模型。分别设置对照组：MH-S正常培养、MG组：90 μg/mL MG、MG+FER-1组：90 μg/mL MG+10 μmol/mL FER-1、FER-1组：10 μmol/mL FER-1。用双氯荧光素（DCFH-DA）检测细胞内ROS水平，脂质氧化（MDA）试剂盒检测MDA含量，细胞亚铁比色法检测亚铁离子（Fe²⁺）含量；线粒体膜电位检测试剂盒（JC-1）检测线粒体膜电位变化，Western blotting检测谷胱甘肽过氧化物酶4（GPX4）、长链酰基辅酶A合酶4（ACSL4）的表达水平。结果 90 μg/mL MG处理MH-S细胞24 h，GPX4的表达水平降低（P<0.001），ACSL4的表达水平升高（P<0.01），同时出现细胞内亚铁离子浓度升高，线粒体膜电位丢失，胞内活性氧水平升高，丙二醛含量增多等现象，最终导致细胞存活率降低（P<0.01）。通过FER-1共同干预后，GPX4的表达升高、ACSL4的表达降低，胞内铁离子浓度降低、线粒体膜电位恢复、活性氧水平降低，丙二醛含量减少（P<0.001）。结论 MG能够剂量依赖性诱导MH-S细胞发生铁死亡，而铁死亡抑制剂FER-1能够挽救MG所导致的铁死亡。

关键词: 铁抑制素-1, 小鼠肺泡巨噬细胞, 丙酮醛, 铁死亡

Abstract:

Objective To investigate the inhibitory effect of FER-1 on methylglyoxal-induced ferroptosis in cultured mouse alveolar macrophages. Methods MH-S cells derived from mouse alveolar macrophages treated with 90 μg/mL methylglyoxal, 10 μmol/mL FER-1MG+FER-1, or both were examined for intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and ferrous ion (Fe²⁺) levels and changes in mitochondrial membrane potential. Western blotting was performed to detect the protein expression levels of glutathione peroxidase 4 (GPX4) and long-chain acyl-CoA synthase 4 (ACSL4). Results Methylglyoxal treatment of MH-S cells for 24 h significantly decreased the protein expression level of GPX4, upregulated the protein expression of ACSL4, increased intracellular concentrations of ferrous ions, ROS and MDA, caused loss of mitochondrial membrane potential, and decreased cell viability. Treatment of the cells with FER-1 effectively attenuated these detrimental effects of methylglyoxal in MH-S cells by increasing GPX4 expression, reducing ACSL4 expression and intracellular ferrous ions, ROS and MDA levels, and restoring the mitochondrial membrane potential. Conclusion Methylglyoxal can induce ferroptosis in MH-S cells in a dose-dependent manner, and FER-1 can rescue the cells from methylglyoxal-induced ferroptosis.

Key words: ferroptosis inhibitor ferroptin-1, mouse alveolar macrophages, methylglyoxal, ferroptosis

张琪, 计泽朝, 加沙尔∙阿拜, 王友达, 阿不都许库尔∙阿不力米提null. 铁抑制素-1抑制丙酮醛诱导的小鼠肺泡巨噬细胞铁死亡[J]. 南方医科大学学报, 2024, 44(12): 2443-2448.

Qi ZHANG, Zezhao JI, Abai JIASHAER∙, Youda WANG, ABUDUXUKUER∙Abulimiti. FER-1 inhibits methylglyoxal-induced ferroptosis in mouse alveolar macrophages in vitro[J]. Journal of Southern Medical University, 2024, 44(12): 2443-2448.

图/表 6

图1 不同浓度丙酮醛对小鼠肺泡巨噬细胞GPX4、ACSL4表达的影响

Fig.1 Effects of different concentrations (0-120 μg/mL) of methylglyoxal (MG) for 24 h on protein expressions of GPX4 and ACSL4 in mouse alveolar macrophages (Mean±SD, n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control group.

图2 FER-1抑制MG诱导的小鼠肺泡巨噬细胞铁死亡中GPX4、ACSL4的表达

Fig.2 FER-1 inhibits MG-induced changes in expressions of GPX4 and ACSL4 in mouse alveolar macrophages (Mean±SD, n=5). ##P<0.01 vs control group; *P<0.05, **P<0.01 vs 90 μg/mL MG group.

图3 FER-1抑制丙酮醛对小鼠肺泡巨噬细胞内ROS生成的促进作用

Fig.3 FER-1 ameliorates MG-induced increase of intracellular ROS production in MH-S cells (Mean±SD, n=4). ####P<0.0001 vs control group; ****P<0.0001 vs 90 μg/mL MG group.

图4 FER-1抑制丙酮醛对小鼠肺泡巨噬细胞内脂质过氧化的水平

Fig.4 FER-1 ameliorates MG-induced increase in lipid peroxidation levels in MH-S cells (Mean±SD, n=4). ###P<0.001 vs control group, ****P<0.0001 vs 90 μg/mL MG group.

图5 FER-1抑制MG诱导的小鼠肺泡巨噬细胞线粒体膜电位丢失

Fig.5 FER-1 inhibits MG-induced loss of mitochondrial membrane potential (MMP) in MH-S cells. A: Fluorescent intensity of MMP (red) of MH-S cells treated with MG, FER-1 or both (Original magnification: ×40). B: Quantitative analysis of fluorescence intensity of MMP (Mean±SD, n=4). ####P<0.0001 vs control group, ***P<0.001 vs 90 μg/mL MG group.

图6 FER-1抑制丙酮醛诱导的小鼠肺泡巨噬细胞亚铁离子增多

Fig.6 FER-1 inhibits MG-induced increase of ferrous iron ions in MH-S cells (Mean±SD, n=4). ###P<0.001 vs control group, ***P<0.001 vs 90 μg/mL MG group.

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