[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。
Changes in iron content,lipid peroxidation,and ferroptosis-related gene expression in vascular endothelial cells in vitro induced by vibration
[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 en-dothelial cell dysfunction and cell damage.Cell ferroptosis may be one of the important mecha-nisms 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 vibration groups 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 colori-metric 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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