摘要
目的 探究N-myc下游调控基因1(N-myc downstream regulated gene 1,NDRG1)对氧糖剥夺(oxygen-glucose deprivation, OGD)诱导的小鼠脑微血管内皮细胞(brain microvascular endothelial cells, BMVEC)铁死亡的影响。方法 提取并培养小鼠原代BMVEC,电镜观察氧糖剥夺后原代BMVEC线粒体形态。体外培养bEnd.3细胞,分为对照组、模型组(构建OGD模型)、小干扰RNA阴性对照(si-NC)组、NDRG1干扰RNA(si-NDRG1)组、阴性对照造模(OGD+si-NC)组、NDRG1干扰造模(OGD+si-NDRG1)组(n=3)。模型组、OGD+si-NC组和OGD+si-NDRG1组构建OGD模型,si-NC组和OGD+si-NC组进行si-NC转染,si-NDRG1组和OGD+si-NDRG1组进行si-NDRG1转染,检测各组细胞活力、丙二醛、谷胱甘肽、Fe^(2+)水平、脂质过氧化水平、NDRG1、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)及酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long-chain family member 4,ACSL4)蛋白水平。结果 与对照组比较,模型组OGD处理后的细胞贴壁数量显著减少,胞质出现肿胀现象,细胞活力显著降低[(37.68±2.43)%vs(96.34±12.08)%,P<0.05],GPX4明显下调(0.78±0.02 vs 1.15±0.01,P<0.01),ACSL4(1.45±0.04 vs 0.78±0.12,P<0.01)、NDRG1蛋白表达明显上调(1.22±0.01 vs 0.13±0.01,P<0.01)。si-NDRG1组GPX4、NDRG1蛋白表达明显低于si-NC组,ACSL4、谷胱甘肽蛋白表达明显高于si-NC组,差异有统计学意义(P<0.05)。OGD+si-NC组GPX4蛋白表达及谷胱甘肽水平明显低于si-NC组和si-NDRG1组,NDRG1、ACSL4蛋白表达、丙二醛、Fe^(2+)相对荧光强度、氧化型脂质相对荧光强度明显高于si-NC组和si-NDRG1组,差异有统计学意义(P<0.05)。OGD+si-NDRG1组GPX4蛋白表达及谷胱甘肽水平明显低于si-NC组和si-NDRG1组,NDRG1明显低于si-NC组,明显高于si-NDRG1组,ACSL4、Fe^(2+)相对荧光强度明显高于si-NC组和si-NDRG1组(P<0.05)。OGD+si-NDRG1组NDRG1、ACSL4、丙二醛、Fe^(2+)相对荧光强度、氧化型脂质相对荧光强度明显低于OGD+si-NC组,差异有统计学意义(P<0.05)。结论 降低NDRG1可以通过改善铁代谢和脂质过氧化,减轻OGD诱导的微血管内皮细胞铁死亡。
Objective To investigate the effect of N-myc downstream regulated gene 1(NDRG1)on ferroptosis in mouse brain microvascular endothelial cells(BMVEC)induced by oxygen-glu-cose deprivation(OGD).Methods After primary BMVEC were isolated and cultured from mice,electron microscopy was used to observe the mitochondrial morphology of the cells after OGD.bEnd.3 cells were cultured and divided into six groups:control group,model group(OGD),si-NC(negative control)group,si-NDRG1(NDRG1 interference RNA)group,OGD+si-NC(negative.control modeling)group,and OGD+si-NDRG1(NDRG1 interference)group(n=3).The model group,OGD+si-NC group,and OGD+si-NDRG1 group were subjected to the OGD model.Si-NC transfection was performed in the si-NC and OGD+si-NC groups,while si-NDRG1 transfection was carried out in the si-NDRG1 and OGD+si-NDRG1 groups.Cell viability,MDA,glutathione,Fe^(2+),lipid peroxidation levels,and protein levels of NDRG1,glutathione peroxidase 4(GPX4),and acyI-CoA synthetase long-chain family member 4(ACSL4)were detected in each group.Results Compared with the control group,the model group showed a significant reduction in the number of cells adhering to the surface after OGD treatment,swollen cytoplasm and decrease in:ell viability[(37.68±2.43)%us(96.34±12.08)%,P<0.05],down-regulation of GPX4 and up-regulation of ACSL4 and NDRG1 expression(0.78±0.02 Us 1.15±0.01,P<0.01;1.45±0.04 Us 0.78±0.12,P<0.01;1.22±0.01 Us 0.13±0.01,P<0.01).In the si-NDRG1 group,the protein levels of GPX4 and NDRG1 were significantly lower,while the protein levels of ACSL4 and gluta-thione were significantly higher than the si-NC group(P<0.05).The OGD+si-NC group showed significantly lower GPX4 expression and glutathione level,while obviously higher NDRG1,ACSL4 expression,MDA,and relative fluorescence intensities of Fe2+and oxidized lipid levels when compared to the si-NC and si-NDRG1 groups(P<0.05).The OGD+si-NDRG1 group showed significantly lower GPX4 expression and glutathione level,and higher ACSL4 and relative fluorescence intensity of Fe^(2+)than the si-NC group and the si-NDRG1 group,while lower NDRG1 expression than the si-NC group but higher than the si-NDRG1 group,and lower NDRG1,ACSL4,MDA,and relative fluorescence intensities of Fe2+and oxidized lipids when compared with the OGD+si-NC group(P<0.05).Conclusion Knockdown of NDRG1 can alleviate OGD-induced ferroptosis in microvascular endothelial cells by improving iron metabolism and lipid per-oxidation.
作者
黄海锋
高玉元
段青蕊
王丽娟
Huang Haifeng;Gao Yuyuan;Duan Qingrui;Wang Lijuan(Department of Neurology,Guangdong Provincial People's Hospital·Guangdong Academy of Medical Sciences,Southern Medical University,Guangzhou 510080,Guangdong Province,China)
出处
《中华老年心脑血管病杂志》
北大核心
2025年第6期798-803,共6页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
国家自然科学基金(81974195,82101377,82371249,82471257)
广东省科技计划项目(2020A0505140006)
广州市科技计划项目(202201000005)
广东医学科学技术研究基金项目(A2021308,A2022092)。
关键词
卒中
铁死亡
内皮细胞
缺氧缺血
脑
基因
MYC
stroke
ferroptosis
endothelial cells
hypoxia ischemia,brain
genes,myc
