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线粒体分裂抑制剂Mdivi-1改善血管紧张素II介导的内皮功能紊乱

陈宇, 林静蓉, 高平进

中国科学院上海生命科学研究院健康科学研究所血管生物学实验室，中国科学院大学，上海 200025；上海交通大学医学院附属瑞金医院，上海市高血压研究所，医学基因组学国家重点实验室，上海 200025

摘要

动力相关蛋白(dynamin-related protein 1, Drp1)与线粒体的动态变化有着密切的联系，是介导线粒体分裂的主要功能蛋白。本研究旨在探讨血管紧张素II (angiotensin II, AngII)对血管内皮细胞线粒体融合和分裂的影响以及Drp1功能抑制剂Mdivi-1对AngII介导的内皮损伤是否有减轻作用。AngII或联合Drp1抑制剂Mdivi-1处理人脐静脉内皮细胞(human umbilical vascular endothelial cells, HUVECs)后，用Western blot检测Drp1、eNOS和凋亡相关酶的蛋白表达，用MitoTracker Red染色观察细胞内线粒体形态，用JC-1探针染色检测线粒体膜电位变化，用DCFH-DA染色检测细胞活性氧簇(reactive oxygen species, ROS)生成，用Transwell实验检测细胞迁移情况，用Annexin V/PI染色检测细胞凋亡情况。结果显示，AngII处理12 h后，HUVECs的Drp1的表达水平显著升高，内皮细胞迁移、凋亡及ROS的生成显著增加，eNOS表达量显著降低；同时，AngII处理还诱导了线粒体形态的改变，使网状的线粒体变成了短管状，并伴随着线粒体膜电位的下降。Mdivi-1可以显著逆转AngII对内皮功能的上述损伤作用，提高内皮细胞线粒体膜电位及eNOS的表达量，降低细胞内ROS水平，抑制内皮细胞凋亡及迁移能力。以上结果提示，Drp1抑制剂Mdivi-1可以减轻AngII介导的内皮损伤。

关键词： 血管紧张素II; 人脐静脉内皮细胞; Mdivi-1; 内皮功能紊乱; 活性氧

分类号：Q25

Mitochondrial division inhibitor Mdivi-1 ameliorates angiotensin II-induced endothelial dysfunction

CHEN Yu, LIN Jing-Rong, GAO Ping-Jin

Laboratory of Vascular Biology, Institute of Health Science, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), University of Chinese Academy of Sciences, Shanghai 200025, China； State Key Laboratory of Medical Genomics and Shanghai Key Laboratory of Vascular Biology at Ruijin Hospital and Shanghai Institute of Hypertension, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Abstract

Mitochondrial fission can occur via activation of dynamin-related protein 1 (Drp1), which participates in the mitochondrial membrane scission process. The present study was designed to investigate the effect of angiotensin II (AngII) on mitochondrial fission and fusion in human umbilical vascular endothelial cells (HUVECs). And we further inquire into whether Mdivi-1, a newly identified pharmacological inhibitor of Drp1, can prevent endothelial dysfunction induced by AngII. The HUVECs were treated with AngII alone or in combination with Mdivi-1. Western blot was used to detect protein expressions of Drp1, endothelial nitric oxide synthase (eNOS) and apoptosis-related enzymes. MitoTracker Red and JC-1 dye were used to detect mitochondrial morphology and membrane potential, respectively. DCFH-DA probe was used to access intracellular reactive oxygen species (ROS) generation. Transwell assay was used to evaluate cell migration. Annexin V/PI staining was used to assess cellular apoptosis. The results showed that, in cultured HUVECs, AngII (1 × 10−7 mol/L, 12 h) treatment significantly upregulated the expression of Drp1 followed by increased apoptosis and decreased eNOS expression. The treatment of AngII resulted in a change in mitochondrial morphology from elongated to uniformly punctate organelles, which was accompanied by decreased mitochondrial membrane potential. Furthermore, Mdivi-1 significantly protected against AngII-induced endothelial dysfunction, as shown by increased mitochondrial membrane potential and eNOS expression, reduced ROS level, decreased apoptosis and migration ability. Taking together, our data suggest that inhibition of Drp1 with Mdivi-1 can restore AngII-induced endothelial dysfunction.

Key words: angiotensin II; human umbilical vascular endothelial cells; Mdivi-1; endothelial dysfunction; reactive oxygen species

收稿日期：2016-03-02　　录用日期：2016-08-01

通讯作者：林静蓉　　E-mail: jingrong.lin@163.com

引用本文：

陈宇, 林静蓉, 高平进. 线粒体分裂抑制剂Mdivi-1改善血管紧张素II介导的内皮功能紊乱[J]. 生理学报 2016; 68 (5): 669-676.

CHEN Yu, LIN Jing-Rong, GAO Ping-Jin. Mitochondrial division inhibitor Mdivi-1 ameliorates angiotensin II-induced endothelial dysfunction. Acta Physiol Sin 2016; 68 (5): 669-676 (in Chinese with English abstract).