作者：蔡荟芝，罗玲

单位：

710000陕西省西安市，西安交通大学第一附属医院心血管内科

Units：

Department of Cardiology, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710000, China

关键词：

动脉粥样硬化；缺氧诱导因子1α；铁死亡

Keywords：

Atherosclerosis; Hypoxia-inducing factor 1α; Ferroptosis

CLC：

R 543.5

DOI：

10.12114/j.issn.1008-5971.2024.00.014

Funds：

摘要：

目的　探讨缺氧诱导因子1α（HIF-1α）通过介导铁死亡影响动脉粥样硬化的机制。方法　本实验 时间为2022年2月—2023年6月。人体试验：选取西安交通大学第一附属医院收治的6例行颈动脉内膜切除术的颈动脉 粥样硬化患者的手术标本为颈动脉粥样硬化组，6例因糖尿病动脉闭塞所致下肢坏死而截肢者的手术标本为糖尿病动 脉闭塞组，6例下肢动脉粥样硬化患者的手术标本为下肢动脉粥样硬化组，6例先天性四肢动静脉瘘患者的手术标本 为正常组。对四组手术标本进行苏木素-伊红（HE）染色、油红O染色、透射电子显微镜检查，采用 Western blotting 法检测谷胱甘肽过氧化物酶4（GPX4）、HIF-1α表达水平。动物实验：选取ApoE -/- C57BL/6小鼠18只，采用简单随 机法将小鼠分为对照组、高脂肪饮食（HFD）组、PX-478组，每组6只。对照组小鼠采用正常饲料喂养； HFD组和 PX-478组小鼠采用HFD连续喂养5个月。之后PX-478组小鼠给予PX-478 5 mg·kg -1·d-1，每2 d腹腔注射1次；对照组 和HFD组小鼠给予等量0.9%氯化钠溶液，每2 d腹腔注射1次，均连续给药2个月。腹腔注射结束后处死小鼠，取其主 动脉。对三组小鼠主动脉窦进行HE染色、油红O染色、Russell-Movall五色染色，采用Western blotting法检测HIF-1α 表达水平。细胞实验：选取THP-1细胞并诱导其向巨噬细胞分化，将THP-1巨噬细胞分为THP-1巨噬细胞组、氧化型 低密度脂蛋白（ox-LDL）组和ox-LDL＋PX-478组，其中ox-LDL组加入ox-LDL并孵育48 h，ox-LDL＋PX-478组加入 ox-LDL和PX-478并孵育48 h。采用Western blotting法检测GPX4、HIF-1α表达水平，采用Mito-Tracker染色法计数活 性氧（ROS）阳性细胞数，测定铁含量、谷胱甘肽（GSH）水平。结果　人体试验结果：HE染色结果显示，与正常 组相比，颈动脉粥样硬化组、糖尿病动脉闭塞组和下肢动脉粥样硬化组动脉中可见内膜增厚、纤维帽和核心坏死的动 脉粥样硬化斑块。油红O染色结果显示，与正常组相比，颈动脉粥样硬化组、糖尿病动脉闭塞组和下肢动脉粥样硬化 组动脉内膜存在广泛的脂质沉积。透射电子显微镜检查结果显示，与正常组相比，颈动脉粥样硬化组、糖尿病动脉闭 塞组和下肢动脉粥样硬化组动脉中线粒体普遍较小，线粒体嵴模糊。下肢动脉粥样硬化组、糖尿病动脉闭塞组和颈动 脉粥样硬化组GPX4表达水平低于正常组，HIF-1α表达水平高于正常组（P＜0.05）。动物实验结果：HE染色结果显 示，与对照组相比，HFD组小鼠主动脉窦中斑块形成，胶原纤维沉积；与HFD组相比，PX-478组小鼠主动脉窦中斑块 形成、胶原纤维沉积减少。油红O染色结果显示，与对照组相比，HFD组小鼠主动脉窦中脂质沉积；与HFD组相比， PX-478组小鼠主动脉窦中脂质沉积减少。HFD组斑块面积、脂质面积、黏蛋白面积大于对照组和PX-478组，HIF-1α 表达水平高于对照组和PX-478组（P＜0.05）。细胞实验结果：ox-LDL组和ox-LDL＋PX-478组GPX4表达水平、 GSH水平低于THP-1巨噬细胞组，HIF-1α表达水平、ROS阳性细胞数、铁含量高于THP-1巨噬细胞组（P＜0.05）； ox-LDL＋PX-478组GPX4表达水平、GSH水平高于ox-LDL组，HIF-1α表达水平、ROS阳性细胞数、铁含量低于 ox-LDL组（P＜0.05）。结论　动脉粥样硬化过程中存在铁死亡现象，HIF-1α表达水平升高；下调HIF-1α表达可减 少ROS生成，升高GSH水平，抑制铁死亡，从而减轻动脉粥样硬化。

Abstract：

　Objective To investigate the mechanism of hypoxia-inducing factor 1α (HIF-1α) influencing atherosclerosis mediated by ferroptosis. Methods The experiment time was from February 2022 to June 2023. Human trial: 6 surgical specimens from patients with carotid atherosclerosi undergoing carotid endarterectomy and admitted to the First Affiliated Hospital of Xi'an Jiaotong University were selected as the carotid atherosclerosis group, 6 surgical specimens from patients with amputation due to lower limb necrosis caused by diabetes artery occlusion were selected as the diabetes artery occlusion group, 6 surgical specimens from patients with lower extremity atherosclerosis were selected as the lower extremity atherosclerosis group, 6 surgical specimens from patients with congenital arteriovenous fistula in the limbs were selected as the normal group. Surgical specimens in four groups were subjected to hematoxylin-eosin (HE) staining, oil red O staining, and transmission electron microscopy examination, Western blotting method was used to detect glutathioneperoxidase 4 (GPX4) and HIF-1 α expression levels. Animal experiment: 18 C57BL/6 mice with ApoE -/- were selected and divided into the control group, the high fat diet (HFD) group, and the PX-478 group by simple randomization, with 6 mice in each group. The mice in control group were fed with normal feed; the mice in HFD group and PX-478 group were continuously fed with HFD for 5 months. Afterwards, the mice in PX-478 group were given PX-478 5 mg kg -1·d-1, intraperitoneal injection every 2 days; the mice in control group and HFD group were given an equal amount of 0.9% sodium chloride solution, intraperitoneal injection every 2 days, administered continuously for 2 months. After intraperitoneal injection, the mice were euthanized and their aortas were taken. Aortic sinus of mite in 3 groups were subjected to HE staining, oil red O staining, and Russell-Movall five color staining, Western blotting method was used to detect HIF-1α expression level. Cell experiment: THP-1 cells were selected and induced to differentiation into macrophages. THP-1 macrophages cells were divided into the THP-1 macrophage group, oxidized low-density lipoprotein (ox-LDL) group, and ox-LDL ＋PX-478 group. The ox-LDL group was added to ox-LDL and incubated for 48 hours, the ox-LDL＋PX-478 group was added to ox-LDL and PX-478 and incubated for 48 hours. Western blotting method was used to detect GPX4 and HIF-1α expression levels, Mito-Tracker staining was used to count the number of reactive oxygen species (ROS) positive cells, and iron content and glutathione (GSH) level were measured. Results Human trial results: HE staining results showed that compared with the normal group, atherosclerotic plaques with thickening intima, fibrous cap and core necrosis were observed in carotid atherosclerosis group, diabetes artery occlusion group and lower extremity atherosclerosis group. Oil red O staining results showed that compared with the normal group, the carotid atherosclerosis group, diabetes artery occlusion group and lower extremity atherosclerosis group had extensive lipid deposition in the intima of the arteries. Transmission electron microscopy examination results showed that compared with the normal group, mitochondria of the carotid atherosclerosis group, diabetes artery occlusion group and lower extremity atherosclerosis group were generally smaller and the mitochondrial ridge was fuzzy. GPX4 expression level in lower extremity atherosclerosis group, diabetes artery occlusion group and carotid atherosclerosis group was lower than that in normal group, HIF-1α expression level was higher than that in the normal group (P < 0.05) . Animal experiment results: HE staining results showed that compared with the control group, plaques formed in the aortic sinus and collagen fibers were deposited in the HFD group; compared with the HFD group, the formation of plaques and deposition of collagen fibers in the aortic sinus of PX- 478 group were reduced. Oil red O staining results showed that compared with the control group, lipid deposition was observed in the aortic sinuses of mice in the HFD group; compared with the HFD group, lipid deposition decreased in the aortic sinuses of mice in the PX-478 group. The plaque area, lipid area, and mucin area in the HFD group were larger than those in the control group and PX-478 group, and HIF-1α expression level was higher than that in the control group and PX-478 group (P < 0.05) . Cell experiment results: the GPX4 expression level and GSH level in the ox-LDL group and ox-LDL＋PX-478 group were lower than those in the THP-1 macrophage group, HIF-1α expression level, the number of ROS positive cells, and iron content were higher than those in the THP-1 macrophage group (P < 0.05) ; the GPX4 expression level and GSH level in the ox-LDL＋PX-478 group were higher than those in the ox-LDL group, HIF-1 α expression level, the number of ROS positive cells, and iron content were lower than those in the ox-LDL group (P < 0.05) . Conclusion During atherosclerosis, ferroptosis is activated, HIF-1 α expression level is increased. Down-regulating HIF-1α expression can reduce ROC production, increase GSH level, inhibit ferroptosis, and thus reduce atherosclerosis.

ReferenceList：