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花生四烯酸Alox15/12-HETE代谢通路抑制血管钙化的发生

韩迎春, 张继超, 张聪聪, 杜杰^*

首都医科大学附属北京安贞医院血管生物研究室，北京市心肺血管疾病研究所，北京 100029

摘要

本研究旨在探索血管钙化中花生四烯酸脂氧酶代谢产物的变化及作用。采用5/6肾切除及高磷饲喂的方法建立小鼠血管钙化的模型。在造模6周后，检测主动脉全长血管钙含量，主动脉弓部进行茜素红染色和Von Kossa染色检测钙沉积情况。收集对照血管和钙化血管组织进行花生四烯酸代谢产物的质谱检测，分析脂氧酶通路代谢小分子的变化。通过实时定量PCR方法检测钙化血管脂氧酶的表达改变。使用脂氧酶抑制剂明确脂氧酶代谢通路对血管钙化的影响。结果显示，造模6周后，肾切除组小鼠血管钙含量比假手术组显著升高(P < 0.05)，茜素红染色和Von Kossa染色显示肾切除小鼠主动脉弓部有明显的钙沉积，表明小鼠血管钙化造模成功。收取造模6周的钙化血管和对照血管，通过液相色谱-质谱(LC-MS)方法检测到9种花生四烯酸脂氧酶代谢产物，多种代谢产物(12-HETE、11-HETE、15-HETE等)的含量在钙化血管中显著升高，其中12-HETE含量最高并且升高最显著。进一步检测钙化血管中产生12-HETE的代谢酶的mRNA水平，发现花生四烯酸脂氧酶15 (arachidonate 15-lipoxygenase, Alox15)表达增加。Alox15特异性抑制剂PD146176可显著降低血浆12-HETE水平，促进主动脉弓部的钙沉积、增加血管钙含量。这些结果显示花生四烯酸脂氧酶代谢在钙化血管中活化，Alox15/12-HETE通路可能对血管钙化发挥保护作用。

关键词： 血管钙化; 花生四烯酸; 脂氧酶-15; 12-HETE

分类号：R339.4

Arachidonic acid Alox15/12-HETE signaling inhibits vascular calcification

HAN Ying-Chun, ZHANG Ji-Chao, ZHANG Cong-Cong, DU Jie^*

Vascular Biology Department of Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung and Vascular Diseases, Beijing 100029, China

Abstract

This study aims to explore the effects of arachidonic acid lipoxygenase metabolism in vascular calcification. We used 5/6 nephrectomy and high-phosphorus feeding to establish a model of vascular calcification in mice. Six weeks after nephrectomy surgery, vascular calcium content was measured, and Alizarin Red S and Von Kossa staining were applied to detect calcium deposition in aortic arch. Control aortas and calcified aortas were collected for mass spectrometry detection of arachidonic acid metabolites, and active molecules in lipoxygenase pathway were analyzed. Real-time quantitative PCR was used to detect changes in the expression of lipoxygenase in calcified aortas. Lipoxygenase inhibitor was used to clarify the effect of lipoxygenase metabolic pathways on vascular calcification. The results showed that 6 weeks after nephrectomy surgery, the aortic calcium content of the surgery group was significantly higher than that of the sham group (P < 0.05). Alizarin Red S staining and Von Kossa staining showed obvious calcium deposition in aortic arch from surgery group, indicating formation of vascular calcification. Nine arachidonic acid lipoxygenase metabolites were quantitated using liquid chromatography/mass spectrometry (LC-MS) analysis. The content of multiple metabolites (12-HETE, 11-HETE, 15-HETE, etc.) was significantly increased in calcified aortas, and the most abundant and up-regulated metabolite was 12-HETE. Furthermore, we examined the mRNA levels of metabolic enzymes that produce 12-HETE in calcified blood vessels and found the expression of arachidonate lipoxygenase-15 (Alox15) was increased. Blocking Alox15/12-HETE by Alox15 specific inhibitor PD146176 significantly decreased the plasma 12-HETE content, promoted calcium deposition in aortic arch and increased vascular calcium content. These results suggest that the metabolism of arachidonic acid lipoxygenase is activated in calcified aorta, and the Alox15/12-HETE signaling pathway may play a protective role in vascular calcification.

Key words: vascular calcification; arachidonic acid; lipoxygenase-15; 12-HETE

收稿日期：2021-01-04　　录用日期：2021-03-11

通讯作者：杜杰　　E-mail: jiedu@ccmu.edu.cn

DOI: 10.13294/j.aps.2021.0051

引用本文：

韩迎春, 张继超, 张聪聪, 杜杰. 花生四烯酸Alox15/12-HETE代谢通路抑制血管钙化的发生[J]. 生理学报 2021; 73 (4): 571-576.

HAN Ying-Chun, ZHANG Ji-Chao, ZHANG Cong-Cong, DU Jie. Arachidonic acid Alox15/12-HETE signaling inhibits vascular calcification. Acta Physiol Sin 2021; 73 (4): 571-576 (in Chinese with English abstract).