殷昊, 张泽, 吴柳, 王颖竹, 郑玉新, 晋小婷. 柴油机尾气颗粒物及关键组分苯并[a]芘通过靶向氧感受器调控无氧代谢促进心肌缺血缺氧损伤[J]. 环境与职业医学, 2023, 40(5): 491-499. DOI: 10.11836/JEOM22416
引用本文: 殷昊, 张泽, 吴柳, 王颖竹, 郑玉新, 晋小婷. 柴油机尾气颗粒物及关键组分苯并[a]芘通过靶向氧感受器调控无氧代谢促进心肌缺血缺氧损伤[J]. 环境与职业医学, 2023, 40(5): 491-499. DOI: 10.11836/JEOM22416
YIN Hao, ZHANG Ze, WU Liu, WANG Yingzhu, ZHENG Yuxin, JIN Xiaoting. Regulating anaerobic metabolism and promoting myocardial ischemia-hypoxia injury by diesel particulate matter and its key component benzo[a]pyrene via targeting oxygen sensors[J]. Journal of Environmental and Occupational Medicine, 2023, 40(5): 491-499. DOI: 10.11836/JEOM22416
Citation: YIN Hao, ZHANG Ze, WU Liu, WANG Yingzhu, ZHENG Yuxin, JIN Xiaoting. Regulating anaerobic metabolism and promoting myocardial ischemia-hypoxia injury by diesel particulate matter and its key component benzo[a]pyrene via targeting oxygen sensors[J]. Journal of Environmental and Occupational Medicine, 2023, 40(5): 491-499. DOI: 10.11836/JEOM22416

柴油机尾气颗粒物及关键组分苯并a芘通过靶向氧感受器调控无氧代谢促进心肌缺血缺氧损伤

Regulating anaerobic metabolism and promoting myocardial ischemia-hypoxia injury by diesel particulate matter and its key component benzoapyrene via targeting oxygen sensors

  • 摘要:
    背景 柴油机尾气颗粒物(DPM)及其多环芳烃(PAH)组分暴露与缺血性心脏病(IHD)的发病和死亡密切关联,但DPM暴露作用于心肌缺血缺氧损伤的关键组分、作用机制和靶点尚不清楚。
    目的 探讨DPM作用心肌缺氧损伤的关键PAH组分,阐明氧感受器调控的无氧代谢在DPM及关键PAH组分致缺氧损伤中的作用,以及关键PAH组分作用靶点。
    方法 采用人源心肌细胞系AC16细胞,于2% O2条件下,在完全培养基或低血清培养基中经0、1、5、10 μg·mL−1的DPM暴露12 h,采用蛋白免疫印记法测定缺氧诱导因子-1α(HIF-1α)、Bax、裂解半胱天冬酶3(Cleaved-caspase3)蛋白表达,筛选出DPM促进缺血缺氧效应更加明显的培养基条件。正常条件下PAH暴露12 h,检测细胞活力。在缺血缺氧模型中,选择0、0.005、0.5、5 µg·mL−1 PAH暴露12 h,测定HIF-1α、Bax和Cleaved-caspase3蛋白表达。DPM或PAH暴露12 h后,测量细胞中丙酮酸和乳酸的含量。并使用糖酵解抑制剂GSK2837808A进行预处理,探究糖酵解在DPM及苯并a芘(BaP)诱导缺氧损伤中的作用。采用分子对接技术分析PAH与氧感受器缺氧诱导因子脯氨酰羟化酶(PHD2)和天冬酰胺羟化酶(FIH1)之间的结合亲和力,进一步测定DPM或BaP处理12 h后PHD2、FIH1和羟基化缺氧诱导因子-1α(OH-HIF-1α)蛋白表达。
    结果 缺氧条件下,低血清培养基中DPM暴露后,HIF-1α、Bax和Cleaved-caspase3表达变化明显(P<0.01),选择缺氧低血清培养基作为模拟缺血缺氧的基础条件。蒽未诱导细胞活力降低(P>0.05),其余PAH在1 μg·mL−1以上时诱导细胞活力降低(P<0.05)。与对照组相比,不同浓度BaP暴露均上调HIF-1α蛋白表达(P<0.05),并且0.5和5 µg·mL−1 BaP暴露诱导Bax和Cleaved-caspase3蛋白水平升高(P<0.01)。1、5和10 μg·mL−1 DPM或0.5和5 μg·mL−1 BaP暴露后,均诱导细胞内丙酮酸和乳酸含量增加(P<0.05)。与DPM或BaP暴露12 h相比,糖酵解抑制剂共处理组HIF-1α、Bax和Cleaved-caspase3蛋白水平减少(P<0.05)。5种PAH与氧感受器PHD2与FIH1的结合能力均较强,其中BaP最强。与对照组相比,DPM或BaP暴露不影响PHD2与FIH1蛋白水平(P>0.05),但BaP暴露下调OH-HIF-1α蛋白水平(P<0.01)。
    结论 BaP暴露可以促进心肌细胞缺氧及损伤,是DPM作用于心肌缺血缺氧损伤的关键PAH组分。BaP暴露通过结合PHD2,抑制PHD2对HIF-1α的羟基化作用,减少OH-HIF-1α蛋白水平,诱导HIF-1α积累,继而调控心肌细胞无氧代谢增加,促进心肌细胞缺血缺氧损伤。

     

    Abstract:
    Background The exposure to diesel particulate matter (DPM) and its polycyclic aromatic hydrocarbons (PAH) is closely related to the morbidity and mortality of ischemic heart disease (IHD). However, it is unclear what key components and targets of DPM exposure involve in myocardial ischemia-hypoxia injury and associated mechanisms.
    Objective To identify key PAH components of DPM that act on myocardial hypoxic injury, andclarify the role of oxygen sensors-regulated anaerobic metabolism in DPM and key components-induced hypoxic injury and the targets of the key PAH components.
    Methods Human cardiomyocyte cell line AC16 cells were exposed to 0, 1, 5, and 10 μg·mL−1 DPM in a high glucose DMEM medium with 10% fetal bovine serum (FBS) (HGM) or low FBS (0.5%) in high glucose DMEM medium (LFM), for 12 h under 2% O2, and expression of hypoxia-inducible factor-1α (HIF-1α), Bax, and Cleaved-caspase3 was determined by Western blotting. Under normal condition, the cell viability was detected after PAH exposure for 12 h. Under the condition of ischemia-hypoxia model, cells were exposed to 0, 0.005, 0.5, and 5 µg·mL−1 PAH for 12 h, and the protein expression of HIF-1α, Bax, and Cleaved-caspase3 was determined. After exposure to DPM or PAH for 12 h, the contents of pyruvate and lactate in cells were detected. Pretreatment with glycolysis inhibitor GSK2837808A was used to explore the role of glycolysis in DPM and benzoapyrene (BaP)-induced hypoxia injury. A molecular docking technique was used to analyze the binding affinity between PAH and oxygen sensors (prolyl hydroxylase domain-containing protein 2, PHD2, and factor-inhibiting hypoxia-inducible factor 1, FIH1), and the protein levels of PHD2, FIH1, and hydroxyl-HIF-1-alpha (OH-HIF-1α) after the DPM or BaP treatment were further determined.
    Results Under hypoxia, DPM exposure in the LFM induced the expression of HIF-1α, Bax, and Cleaved-caspase3 (P<0.01). Therefore, hypoxia and LFM were selected as the basic ischemia and hypoxia condition. Except for anthracene (Ant) (P>0.05), other PAH decreased cell viability when the concentration was above 1 μg·mL−1 (P<0.05). All concentrations of BaP induced the expression of HIF-1α protein (P<0.05), and the protein levels of Bax and Cleaved-caspase3 were up-regulated after the 0.5 and 5 µg·mL−1 BaP exposure (P<0.01). After exposure to DPM (1, 5 and 10 μg·mL−1) or BaP (0.5 and 5 μg·mL−1), the intracellular pyruvate and lactate contents increased (P<0.05). The glycolysis inhibitor co-treatment decreased the levels of HIF-1α, Bax, and Cleaved-caspase3 proteins compared with the DPM or BaP exposure group for 12 h (P<0.05). The binding abilities of the five PAHs to the oxygen sensors PHD2 and FIH1 were strong, and BaP was the strongest. Although the DPM or BaP exposure had no effects on the protein levels of PHD2 and FIH1 in AC16 cells (P<0.05), the protein level of OH-HIF-1α was decreased (P<0.01).
    Conclusion BaP exposure can promote hypoxia and injury of myocardial cells and is the key PAH component of DPM that induces myocardial ischemia and hypoxia injury. BaP exposure inhibits the hydroxylation function of PHD2 on HIF-1α by combining with PHD2, decreases the level of OH-HIF-1α and induces HIF-1α accumulation. And then HIF-1α promotes anaerobic metabolism and accelerates ischemia and hypoxia injury of myocardial cells.

     

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