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