解耦联蛋白2在血管紧张素Ⅱ致线粒体活性氧生成中的作用

Effect of Uncoupling Protein-2 on Angiotensin Ⅱ Induced Reactive Oxygen Species Generation in Mitochondria

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 study the effects of angiotensin Ⅱ （Ang Ⅱ） on endothelial cell mitochondria membrane potential （MMP）,reactive oxygen species （ROS） production,and the roles of uncoupling protein-2 （UCP2）.Methods This work includes three parts：1）Investigating the impact of Ang Ⅱ on MMP and the production of ROS.Human umbilical vein endothelial cells （HUVEC） at 3-4 passages were cultured in vitro with control （DMEM,or Ang Ⅱ at 0.1,1 and 10 μmol/L respectively for 24 hours.Intracellular ROS and MMP were detected by flow cytometry.UCP2 mRNA expressions were quantitated by RT-PCR.2）Investigating the effects of UCP2 on MMP and the production of ROS by down-,or up-regulating its expression using UCP2 antisense-oligonucleotides or exogenous uncoupling agents carbonylcyanide-m-chlorophenylhydrazone （CLCCP）.3）Investigating the impact of exogenous oxidizing agent （H2O2） on UCP2 mRNA expression.Results Ang Ⅱ increased ROS,MMP and UCP2 expression in concentration-dependent manner in HUVEC.Compared to control group,intervention with UCP2 antisense oligonucleotides significantly increased ROS production and MMP level（from 90.0±2.50 to 165.3±2.2 and from 125.0±4.6 to 205.1±5.8,respectively,all P〈0.01）,or CLCCP for 24 hours significantly decreases ROS production and MMP level（from 210.3±5.2 to 156.2±3.5 and from 307.2±4.1 to 220.0±9.2,respectively,all P〈0.01）.H2O2 increased UCP2 mRNA expression （P〈0.01）.Conclusion Ang Ⅱ can stimulate ROS production and protectively up-regulate UCP2 expression in HUVEC,which suggests that upregulating of UCP2 might be an important underlying anti-oxidative mechanism during oxidative stress.