吴思恒, 陈青松, 曾颖珊, 夏云, 杨虹雨. 振动诱导体外血管内皮细胞铁含量、脂质过氧化及铁死亡相关基因表达的改变[J]. 环境与职业医学, 2024, 41(5): 560-566. DOI: 10.11836/JEOM23383
引用本文: 吴思恒, 陈青松, 曾颖珊, 夏云, 杨虹雨. 振动诱导体外血管内皮细胞铁含量、脂质过氧化及铁死亡相关基因表达的改变[J]. 环境与职业医学, 2024, 41(5): 560-566. DOI: 10.11836/JEOM23383
WU Siheng, CHEN Qingsong, ZENG Yingshan, XIA Yun, YANG Hongyu. Changes in iron content, lipid peroxidation, and ferroptosis-related gene expression in vascular endothelial cells in vitro induced by vibration[J]. Journal of Environmental and Occupational Medicine, 2024, 41(5): 560-566. DOI: 10.11836/JEOM23383
Citation: WU Siheng, CHEN Qingsong, ZENG Yingshan, XIA Yun, YANG Hongyu. Changes in iron content, lipid peroxidation, and ferroptosis-related gene expression in vascular endothelial cells in vitro induced by vibration[J]. Journal of Environmental and Occupational Medicine, 2024, 41(5): 560-566. DOI: 10.11836/JEOM23383

振动诱导体外血管内皮细胞铁含量、脂质过氧化及铁死亡相关基因表达的改变

Changes in iron content, lipid peroxidation, and ferroptosis-related gene expression in vascular endothelial cells in vitro induced by vibration

  • 摘要: 背景

    血管内皮损伤是振动引发手臂振动病(HAVD)的重要环节,长时间振动暴露可导致血管内皮细胞功能障碍及细胞损伤,细胞铁死亡有可能是振动诱导血管内皮细胞损伤并导致HAVD的重要机制之一。

    目的

    探究振动是否可诱导体外血管内皮细胞铁死亡相关指标发生改变。

    方法

    将人脐静脉内皮细胞(HUVEC)分为振动组以及对照组。振动组是对HUVEC施加125 Hz、6.5 m·s−2频段的振动,并且分为四组:1 d 2 h组、1 d 4 h组、2 d 2 h组、2 d 4 h组;对照组除了不接触振动外,其他条件均与实验组相同。待细胞融合至80%时,对照组与同组别实验组同时收获。采用细胞亚铁比色法测试盒、还原型谷胱甘肽(GSH)比色法测试盒、微量丙二醛(MDA)测试盒分别检测不同振动时间和频次对HUVEC内铁、还原型GSH、MDA含量的影响;使用实时荧光定量聚合酶链反应(RT-PCR)检测细胞铁死亡相关基因酰基辅酶A合成酶长链家族成员4(ACSL4)、肿瘤蛋白53(P53)、重组人铁蛋白重链(FTH1)和谷胱甘肽过氧化酶4(GPX4)mRNA的表达情况;使用蛋白免疫印迹法(WB)检测HUVEC铁死亡相关蛋白表达水平。

    结果

    与对照组相比,振动可诱导HUVEC铁含量增加且存在剂量-效应趋势。与对照组相比,振动组的HUVEC细胞内还原型GSH含量随着振动时间和频次的增加而减少,并且存在剂量-效应趋势。与对照组相比,1 d 2 h、1 d 4 h和2 d 4 h振动组的HUVEC细胞内MDA含量增加,且1 d 2 h和1 d 4 h振动组中MDA含量随时间增加而增加。RT-PCR结果显示,与1 d 2 h组相比,1 d 4 h组ACSL4、P53表达水平上升。与2 d对照组相比,振动2 d 2 h组和2 d 4 h组ACSL4表达水平上升,振动2 d 4 h组P53 mRNA表达水平上升。与1 d对照组相比,振动1 d 2 h组内皮细胞FTH1、GPX4 mRNA的表达水平下降。WB结果显示,与对照组相比,振动1 d 2 h组内皮细胞铁死亡相关蛋白ACSL4的表达水平上升;1 d 2 h组和2 d 4 h组中P53的表达水平上升;振动1 d 4 h组、2 d 2 h组GPX4的表达水平下降,且振动2 d 2 h组相对于振动1 d 2 h组的下降更加明显。上述差异均具有统计学意义(P<0.05)。

    结论

    振动可诱导体外血管内皮细胞中的铁含量升高,GSH的减少和MDA的增加,以及铁死亡相关基因ACSL4、P53、FTH1、GPX4 mRNA和蛋白表达改变。

     

    Abstract: Background

    Vascular endothelial injury is an important pathogenic step of vibration-induced hand arm vibration disease (HAVD), and long-term vibration exposure can lead to vascular endothelial cell dysfunction and cell damage. Cell ferroptosis may be one of the important mechanisms of vibration-induced vascular endothelial cell injury and HAVD.

    Objective

    To explore whether vibration can induce changes in ferroptosis-related indicators in vascular endothelial cells in vitro.

    Methods

    Human umbilical vein endothelial cells (HUVEC) were divided into four vibrationgroups and two control groups. The vibration groups were exposed to an vibration setting of 125 Hz, 6.5 m·s−2 frequency band and for different durations: 1 d 2 h (total 1 d, 2 h per day), 1 d 4 h (total 1 d, 4 h per day), 2 d 2 h (total 2 d, 2 h per day), and 2 d 4 h (total 2 d, 4 h per day), respectively. All control groups were treated the same as the experimental groups except no vibration exposure. When the cells were 80% confluent, the control groups and the corresponding experimental groups were harvested at the same time. The effects of subgroup treatments on iron, reduced glutathione (GSH), and malondialdehyde (MDA) in HUVEC were detected with a cell ferrous colorimetric test kit, a reduced GSH colorimetric test kit, and a trace MDA test kit, respectively. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expressions of ferroptosis-related genes acyl-CoA synthetase long chain family member 4 (ACSL4), tumor protein 53 (P53), recombinant human ferritin heavy chain (FTH1), and glutathione peroxidase 4 (GPX4). Western blot (WB) was used to detect the expression levels of ferroptosis-related proteins in HUVEC.

    Results

    Compared with the control groups, the vibration induced an increase in the iron content of HUVEC with a dose-response trend. Compared with the control groups, the reduced GSH content of HUVEC in the vibration group decreased with the increase of vibration time and frequency, and there was a dose-response trend. Compared with the control groups, the intracellular MDA content of HUVEC in the 1 d 2 h, 1 d 4 h, and 2 d 4 h vibration groups increased, and the MDA content in the 1 d 2 h and 1 d 4 h vibration group increased with time. The RT-PCR results showed that the mRNA expression levels of ACSL4 and P53 in the 1 d 4 h group increased compared with the 1 d 2 h group. Compared with the 2 d control group, the mRNA expression levels of ACSL4 in the 2 d 2 h vibration group and the 2 d 4 h vibration group increased, and the mRNA expression level of P53 in the 2 d 4 h vibration group increased. Compared with the 1 d control group, the mRNA expression levels of FTH1 and GPX4 in endothelial cells in the vibration 1 d 2 h group decreased. The WB results showed that compared with the control groups, the expression level of ferroptosis-related protein ACSL4 in endothelial cells increased in the vibration 1 d 2 h group; the expression levels of P53 in the 1 d 2 h and 2 d 4 h vibration groups increased; the expression levels of GPX4 decreased in the 1 d 4 h and 2 d 2 h vibration group, and the decrease was more obvious in the 2 d 2 h vibration group than in the 1 d 2 h vibration group; the above differences were statistically significant (P<0.05).

    Conclusion

    Vibration induces an increase in iron content, a decrease in GSH, and an increase in MDA in vascular endothelial cells in vitro, as well as mRNA and protein expressions of ferroptosis-related genes ACSL4, P53, FTH1, and GPX4.

     

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