下调NAD(P)H氧化酶4加重缺氧复氧心肌细胞损伤:线粒体SIRT3信号的关键作用

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

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。

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 meas- ured 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 mito chondrial ROS production. More importantly, the increased NAD /NADH following H/R was reversed by Nox4 inhibition, whereas SIRT3 protein expression was further suppressed. CONCLUSION： Down regulation of Nox4 exacerbates H/R-induced cardiomyocyte injury by increasing mitochondrial ROS pro duction and inhibiting mitochondrial energetics. The protective role of Nox4 against H/R injury may be attributed to the upregulated NAD ＋/NADH-SIRT3 signaling.