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目的观察血管紧张素Ⅱ(AngⅡ)对内皮细胞线粒体膜电位(MMP)和反应性氧族(ROS)的影响,探讨解耦联蛋白2(UCP2)在该过程中的作用。方法整个研究分3个部分:①观察AngⅡ对内皮细胞线粒体MMP和ROS产生的影响。分别用不同浓度AngⅡ(0.1、1、10μmol/L)干预体外原代培养并传代3～4代的人脐静脉内皮细胞(HUVEC)24h,用流式细胞仪检测MMP和ROS的生成,逆转录聚合酶链式反应(RT-PCR)定量UCP2 mRNA的表达;②分别用UCP2反义寡核苷酸和外源性解耦联剂羰基氰化物间氯苯腙(CLCCP)下调或上调UCP2,观察UCP2对内皮细胞线粒体ROS及MMP的影响;③观察外源性氧化剂过氧化氢对UCP2mRNA表达的影响。结果 AngⅡ呈浓度依赖性增加HUVEC中ROS及MMP的水平及UCP2的表达;和对照组比较,UCP2反义寡核苷酸干预24h,可使内皮细胞ROS和MMP明显升高(分别从90.0±2.5增加至165.3±2.2和从125.0±4.6增加至205.1±5.8,均P<0.01);而外源性解耦联剂CLCCP干预则使ROS和MMP明显下降(分别从210.3±5.2降至156.2±3.5和从307.2±4.1降至220.0±9.2,均P<0.01)。过氧化氢能明显增加UCP2mRNA的表达(P<0.01)。结论 AngⅡ可增加线粒体ROS的产生,同时UCP2表达呈保护性上调;提示氧化应激过程中,UCP2的作用可能是细胞重要的抗氧化机制。
Objective To investigate the effects of angiotensin Ⅱ on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) in endothelial cells and investigate the role of uncoupling protein 2 (UCP2) in this process. Methods The whole study was divided into three parts: ① To observe the effects of AngⅡon mitochondrial MMP and ROS production in endothelial cells. The human primary culture and passage of 3-4 generations of human umbilical vein endothelial cells (HUVECs) were induced by different concentrations of AngⅡ (0.1, 1, 10μmol / L) for 24 hours respectively. The production of MMPs and ROS, UCP2 mRNA expression was quantified by polymerase chain reaction (RT-PCR); ② UCP2 was down-regulated or UCP2 was up-regulated by UCP2 antisense oligodeoxynucleotide and exogenous decoupling agent carbonyl cyanide m-chlorophenyl hydrazone (CLCCP) UCP2 on endothelial cells mitochondrial ROS and MMP; ③ observe the exogenous oxidant hydrogen peroxide on UCP2mRNA expression. Results AngⅡ increased the levels of ROS and MMP and the expression of UCP2 in HUVECs in a concentration-dependent manner. Compared with the control group, UCP2 antisense oligonucleotides could significantly increase the ROS and MMPs of endothelial cells (90.0 ± 2.5 Increased to 165.3 ± 2.2 and increased from 125.0 ± 4.6 to 205.1 ± 5.8, all P <0.01), whereas ROS and MMP were significantly decreased by CLCCP intervention (from 210.3 ± 5.2 to 156.2 ± 3.5 And from 307.2 ± 4.1 to 220.0 ± 9.2, all P <0.01). Hydrogen peroxide significantly increased UCP2 mRNA expression (P <0.01). Conclusions AngⅡ can increase the production of mitochondrial ROS, while the expression of UCP2 is protectively increased. It suggests that the role of UCP2 may be an important antioxidant mechanism in the process of oxidative stress.