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《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:

2014年第3期

页码:

259-264

栏目:

基础研究

出版日期:

2014-03-20

文章信息/Info

Title:

Downregulation of NAD(P)H oxidase 4 exacerbates cardiomyocyte injury from hypoxia/reoxygenation: important role of mitochondrial SIRT3 signaling

作者:

何 薇; 曾 超; 邹 倩; 马美娟; 李聪叶; 喻秋珺; 王海昌

(第四军医大学西京医院心内科，陕西 西安710032)

Author(s):

HE Wei;  ZENG Chao;  ZOU Qian;  MA Mei juan;  LI Cong ye;  YU Qiu jun;  WANG Hai chang

(Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi, China)

关键词:

NAD(P)H氧化酶; 心肌细胞; 活性氧; 缺氧复氧损伤; 线粒体

Keywords:

NAD(P)H oxidase;  cardiomyocytes;  reactive oxygen species;  hypoxia/reoxygenation injury;  mitochondria

分类号:

R542.2

DOI:

-

文献标识码:

A

摘要:

目的:明确内源性NAD(P)H氧化酶4（Nox4）在心肌细胞缺氧/复氧(H/R)损伤中的作用并探讨其可能的机制。方法:以H9C2心肌细胞系为研究对象，建立H/R模型，将细胞分为对照组、NC-siRNA组、Nox4-siRNA组、H/R组、H/R+NC-siRNA组和H/R+Nox4-siRNA组。采用RNAi方法下调Nox4的表达,MTT比色法测定相对存活率、荧光素酶化学发光法测量ATP水平，MitoSOX荧光探针检测线粒体ROS，比色法测定NAD+/NADH的比值，Western blot法测定Nox4和SIRT3表达水平。结果:与对照组相比，H/R组H9C2心肌细胞中Nox4蛋白的水平明显增加(P<0.05)，相对存活率、ATP的水平明显降低(P<0.05,P<0.01)，而线粒体ROS生成明显增加(P<0.01)，同时NAD+/NADH的比值明显增加、SIRT3表达量明显降低(P<0.05,P<0.01)。与H/R组相比，H/R+Nox4-siRNA组Nox4蛋白水平显著降低(P<0.05)，相对存活率、ATP的水平进一步降低、线粒体ROS生成进一步增加(P<0.05)，同时，NAD+/NADH的比值明显降低(P<0.01)、SIRT3蛋白水平进一步降低(P<0.05)。结论:下调Nox4可加重H/R诱导的心肌细胞损伤和氧化应激，抑制线粒体能量生成，其心肌细胞保护机制可能是通过上调NAD+/NADH的比值，增加SIRT3的表达而发挥作用。

Abstract:

AIM:To investigate the role of NAD(P)H oxidase 4 (Nox4) in cardiomyocytes exposed to hypoxia/reoxygenation and its potential mechanism. METHODS: H9C2 myocytes were exposed to hypoxia/reoxygenation (H/R) and Nox4 was downregulated by RNA interference. H9C2 myocytes were divided into control group, NC-siRNA group, Nox4-siRNA group, H/R group, H/R+NC-siRNA group and H/R+Nox4-siRNA group. Cell viability was detected by MTT assay. Cellular ATP level was measured by luciferase chemiluminescence assay and NAD+/NADH was assessed by a commercial assay kit followed by colorimetry. Mitochondrial ROS production was detected by MitoSOX fluorescent probe and protein expression of Nox4 and SIRT3 was analyzed by Western blot. RESULTS: Compared with control group, H/R induced increased protein expression of Nox4 and resulted in significant reduction in cell viability and ATP level with a remarkable increase in mitochondrial ROS production. NAD+/NADH ratio increased, whereas SIRT3 protein expression decreased in response to H/R. Compared with H/R group, H/R+Nox4-siRNA group exhibited further decline in cell viability and ATP level, with enhanced mitochondrial ROS production. More importantly, the increased NAD+/NADH following H/R was reversed by Nox4 inhibition, whereas SIRT3 protein expression was further suppressed. CONCLUSION: Downregulation of Nox4 exacerbates H/R-induced cardiomyocyte injury by increasing mitochondrial ROS production and inhibiting mitochondrial energetics. The protective role of Nox4 against H/R injury may be attributed to the upregulated NAD+/NADH-SIRT3 signaling.

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备注/Memo

备注/Memo:

收稿日期：2013-10-28.基金资助：国家自然科学基金项目资助(81200101,31171090)
通讯作者：王海昌，教授，主要从事冠心病治疗研究 Email：Wanghc@fmmu.edu.cn
共同通讯作者：喻秋珺，讲师，主要从事心肌缺血损伤机制及保护策略研究 Email：yuqiujun@gmail.com
作者简介：何薇，硕士生 Email：heweiwei2012@163.com

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更新日期/Last Update: 2014-03-21