热量限制通过HNF3γ下调NOX4表达来抑制内皮细胞的衰老

遗传 ›› 2012, Vol. 34 ›› Issue (5): 573-583.

热量限制通过HNF3γ下调NOX4表达来抑制内皮细胞的衰老

刘强¹, 李虹², 陈怀红¹, 王静¹

1. 浙江大学医学院附属第二医院老年病科, 杭州 310009 2. 杭州市第一人民医院, 南京医科大学附属杭州医院, 杭州 310006

收稿日期:2011-09-27 修回日期:2012-03-06 出版日期:2012-05-20 发布日期:2012-05-25
通讯作者: 刘强 E-mail:liuqianghare@126.com
基金资助:
浙江省科技厅公益性技术应用研究计划项目(编号：2011C23016)资助

Caloric restriction suppressing endothelial cells senescence via down-regulation NOX4 by HNF3γ

LIU Qiang¹, LI Hong², CHEN Huai-Hong¹, WANG Jing¹

1. Department of Gerontology, the Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China 2. Department of Cardiology, the Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China

Received:2011-09-27 Revised:2012-03-06 Online:2012-05-20 Published:2012-05-25

摘要/Abstract

摘要： 为了观察热量限制对主动脉内皮细胞中HNF3γ及NOX4基因表达的影响, 揭示HNF3γ-NOX4-活性氧通路介导热量限制抗内皮细胞衰老的分子机制, 文章将主动脉内皮细胞分为5组：对照组、高热量组、低热量组、siRNA+低热量组、siRNA+高热量组。应用逆转录实时定量PCR(Real-time quantitative PCR, RT-qPCR)、Western blotting分析各组HNF3γ、NOX4 mRNA及蛋白水平变化, 并检测各组细胞内活性氧产量及细胞衰老程度变化。采用染色质免疫共沉淀分析HNF3γ蛋白与NOX4基因启动子区域结合情况, 萤光素酶报告基因检测HNF3γ蛋白结合后对NOX4基因启动子活性的影响。结果显示：与对照组比较, 低热量组HNF3γ mRNA和总HNF3γ蛋白表达水平、磷酸化/总HNF3γ比值显著升高(P<0.05), NOX4 mRNA和蛋白表达水平、细胞内活性氧产量及细胞衰老程度显著降低(P<0.05); 高热量组HNF3γ mRNA和总HNF3γ蛋白表达水平、磷酸化/总HNF3γ比值显著降低(P<0.05), NOX4 mRNA和蛋白表达水平、细胞内活性氧产量及细胞衰老程度显著升高(P<0.05); siRNA+低热量组及siRNA+高热量组中NOX4 mRNA和蛋白表达水平、细胞内活性氧水平及细胞衰老程度显著升高(P< 0.05)。染色质免疫共沉淀证实HNF3γ蛋白可与NOX4基因启动区域4个结合位点(-6 bp、-76 bp、-249 bp、-954 bp)结合。萤光素酶报告基因检测显示HNF3γ蛋白与NOX4启动子区域1个位点(-6 bp)、2个位点(-6、-76 bp)、3个位点(-6、-76、-249 bp)、4个位点(-6、-76、-249、-954 bp)结合, 可使NOX4启动子活性分别降低至对照组的80.15±4.64%、40.02.±2.15%、16.46±2.24%、12.13±1.46%, P<0.05。上述结果提示热量限制可上调HNF3γ基因表达, 增强HNF3γ蛋白活性, 促进HNF3γ蛋白同NOX4基因启动子区域结合, 抑制NOX4基因表达, 进而减少细胞内活性氧产生而延缓动脉内皮细胞衰老。

关键词: 热量限制, 内皮细胞, HNF3γ, NOX4, 衰老

Abstract: The aim of current study is to investigate the molecular mechanism behind caloric restriction (CR) suppressing endothelial cells senescence via HNF3γ (hepatocyte nuclear factor 3 gamma)-NOX4 (NADPH oxidase 4)–ROS（reactive oxygen species）signaling pathway. HAECs (human aortic endothelial cells) were divided into 5 groups: control group, high caloric group (about 1.5 times caloric intake of control group), low caloric group (about 0.5 times caloric intake of control group), siRNA plus low caloric group (before low caloric treatment pretreated with special siRNA targeting HNF3γ), and siRNA plus high caloric group (before high caloric treatment pretreated with special siRNA targeting HNF3γ). Expression of HNF3γ or NOX4 was quantified by qRT-PCR and Western blot. Intracellular ROS production was measured by flow cytometer. Endothelial cells senescence was assayed by senescence associated β-galactosidase staining. After verifying the binding of HNF3γ to NOX4 promoters region by chromatin immunoprecipitation assay (Chip), NOX4 promoter activity was assayed by dual-luciferase reporter system. Compared with the control group, the level of HNF3γ mRNA, total HNF3γprotein and ratio of phosphor/total HNF3γ protein increased significantly (P<0.05) in low caloric group, and decreased significantly (P<0.05) in high caloric group and siRNA plus low or high caloric group; whereas the level of NOX4 mRNA, NOX4 protein, intracellular ROS and endothelial cells senescence decreased significantly (P<0.05) in low caloric group and increased significantly (P<0.05) in high caloric group and siRNA plus low or high caloric group. Chip result showed there were 4 HNF3γ binding sites in NOX4 gene promoter region ((-6, -76, -249 and -954bp) and HNF3γ could bind to all 4 those predicted sites. According to the results of dual-luciferase reporter system, HNF3γ binding to 1 site (-6bp), 2 sites (-6 and -76bp), 3 sites (-6, -76 and -249bp) and 4 sites(-6, -76, -249 and -954bp) successively could suppress NOX4 promoter activity to 80.15±4.64%, 40.02.±2.15%, 16.46±2.24% and 12.13±1.46% that of baseline respectively (all P<0.05). In a word, through up-regulating HNF3γ, promoting HNF3γ binging to NOX4 promoter region and suppressing NOX4 gene expression，low caloric intake decreases production of intracellular ROS and therefore endothelial cells senescence.

Key words: 热量限制, 内皮细胞, HNF3γ, NOX4, 衰老

刘强，李虹，陈怀红，王静. 热量限制通过HNF3γ下调NOX4表达来抑制内皮细胞的衰老[J]. 遗传, 2012, 34(5): 573-583.

LIU Qiang, LI Hong, CHEN Huai-Hong, WANG Jing. Caloric restriction suppressing endothelial cells senescence via down-regulation NOX4 by HNF3γ[J]. HEREDITAS, 2012, 34(5): 573-583.

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