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戢力维, 邓艳, 李涛. 酮体β-羟基丁酸减轻炎症状态下血管内皮细胞线粒体氧化应激的机制研究[J]. 四川大学学报(医学版), 2021, 52(6): 954-959. DOI: 10.12182/20211160202
引用本文: 戢力维, 邓艳, 李涛. 酮体β-羟基丁酸减轻炎症状态下血管内皮细胞线粒体氧化应激的机制研究[J]. 四川大学学报(医学版), 2021, 52(6): 954-959. DOI: 10.12182/20211160202
JI Li-wei, DENG Yan, LI Tao. Effect of Ketone Body β-Hydroxybutyrate to Attenuate Inflammation-Induced Mitochondrial Oxidative Stress in Vascular Endothelial Cells[J]. Journal of Sichuan University (Medical Sciences), 2021, 52(6): 954-959. DOI: 10.12182/20211160202
Citation: JI Li-wei, DENG Yan, LI Tao. Effect of Ketone Body β-Hydroxybutyrate to Attenuate Inflammation-Induced Mitochondrial Oxidative Stress in Vascular Endothelial Cells[J]. Journal of Sichuan University (Medical Sciences), 2021, 52(6): 954-959. DOI: 10.12182/20211160202

酮体β-羟基丁酸减轻炎症状态下血管内皮细胞线粒体氧化应激的机制研究

Effect of Ketone Body β-Hydroxybutyrate to Attenuate Inflammation-Induced Mitochondrial Oxidative Stress in Vascular Endothelial Cells

  • 摘要:
      目的   探讨酮体β-羟基丁酸(β-OHB)对炎症状态下人脐静脉血管内皮细胞(HUVECs)线粒体氧化应激的调控作用及机制。
      方法   用脂多糖(LPS)和三磷酸腺苷(ATP)诱导巨噬细胞释放促炎因子,收集培养基上清液作为条件培养基(macrophage-conditioned medium, MCM)培养HUVECs。研究共设置7组HUVECs细胞。①对照组:正常培养的HUVECs;②MCM组:MCM培养的HUVECs;③~⑦组为加入不同试剂的HUVECs共培养组,包括:③MCM+β-OHB组,④MCM+N-乙酰半胱氨酸(NAC)组,⑤MCM+β-OHB+NAC组,⑥MCM+β-OHB+组蛋白去乙酰化酶激动剂ITSA1组,⑦MCM+β-OHB+组蛋白去乙酰化酶抑制剂Entinostat组。MitoSOX免疫荧光染色分析线粒体超氧化物水平,实时荧光定量聚合酶链式反应(RT-qPCR)检测抗氧化基因mRNA表达,并以Seahorse线粒体能量分析仪测定线粒体有氧呼吸能力。
      结果   与对照组相比,MCM培养的HUVECs细胞线粒体超氧化物生成明显增多,而β-OHB处理可明显抑制线粒体超氧化物生成,并伴随抗氧化基因mRNA表达水平上升,以及线粒体基础耗氧率和呼吸储备能力的显著提高。NAC处理并未进一步提高β-OHB对线粒体功能的保护作用。并且,ITSA1处理可完全取消β-OHB的抗氧化和线粒体保护作用,而Entinostat处理后上述作用仍然维持。
      结论   酮体β-OHB通过抑制组蛋白去乙酰化酶活性,激活抗氧化通路,减轻血管内皮细胞的线粒体氧化应激。

     

    Abstract:
      Objective   To investigate the regulatory function and mechanism of β-hydroxybutyrate (β-OHB), a ketone body, on the mitochondrial oxidative stress of inflammatory human umbilical vein endothelial cells (HUVECs).
      Methods   Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) were used to induce macrophages to release proinflammatory factors, and the culture supernatant was collected as a macrophage-conditioned medium (MCM) to culture HUVECs. A total of 7 groups of cells were used in the study: ①control group, or normal cultured HUVECs; ②MCM group, or the MCM-cultured HUVECs; groups ③ to ⑦ were all HUVECs co-cultured with different reagents, including ③MCM+β-OHB group, ④MCM+N-acetylcysteine (NAC) group, ⑤MCM+β-OHB+NAC group, ⑥MCM+β-OHB+histone deacetylase agonist ITSA1 group, and ⑦MCM+β-OHB+histone deacetylase inhibitor Entinostat group. MitoSOX immunofluorescence staining was conducted to analyzes the mitochondrial superoxide levels, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was performed to examine the mRNA expression of antioxidant genes, and Seahorse mitochondrial energy analyzer was used to measure mitochondrial aerobic respiration capacity.
      Results   Compared with the control group, mitochondrial superoxide production was significantly increased in the MCM cultured HUVECs cells, while β-OHB treatment significantly inhibited mitochondrial superoxide production, which was accompanied by an increase in the mRNA expression of antioxidant genes, and significant increase in the basal mitochondrial oxygen consumption rate and respiratory reserve capacity. NAC treatment did not further enhance the protective effect of β-OHB on mitochondrial functions. In addition, ITSA1 treatment could completely offset the antioxidant and mitochondrial protective effects of β-OHB, and these stated effects were still maintained after Entinostat treatment.
      Conclusion   The ketone body β-OHB attenuates the mitochondrial oxidative stress of vascular endothelial cells through activating the antioxidant pathway and inhibiting histone deacetylase activity.

     

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