The Effect Of High Glucose On NO Secretion And SIRT1 Expression In Human Umbilical Vein Endothelial Cell

ObjectiveDiabetes is a major independent risk factor for cardiovascular disease and stroke. Previous studies showed that high glucose induced endothelial dysfunction by affecting NO secretion directly or indirectly. Recent studies point to silence information regulator 1 (SIRT1) as a key regulator of vascular endothelial homeostasis, controlling angiogenesis, vascular tone and endothelial dysfunction, and a positive feedback mechanism exists between SIRT1 and NO. The aim of this study is to investigate the effect of high glucose on the secretion of NO and the expression of SIRT1 in human umbilical vein endothelial cells (HUVEC). ContentsPrimary HUVECs were isolated and cultured, then they were identified by detecting human VIII factor associated antigen with immunohistochemical method. Subculture the cells when they are over 80% confluence. Experiments are done with cells between 2-4 passages. HUVEC were treated with glucose at different concentrations (5.5, 11.1, 22.2, 33.3mmol/L) for 48h. NO content in medium were detected by nitrate reductase test.SIRT1 mRNA expression levels were determined by reverse transcription-polymerase chain reaction (RT-PCR)and the protein expression levels were evaluated by Western blotting.Then HUVEC were treated with high glucose(33.3mmol/L) for different time(0h,24h,48h and 72h),NO content in medium and SIRT1 mRNA and protein level were detected by the times.Methods1,Experimental methods included: (1) Primary cultivation and serial subcultivation of HUVEC. (2) Immunocytochemistry. (3)NO detected by nitrogen oxide kit. (4) Extraction of total RNA and Protein,RT-PCR and western blotting.2,Statistic methods: All analysis were performed with SPSS11.5 software package.Normal distribution of the data was tested using kolmogorov-Smirnov nonparametric test.Groups comparions were performed with one way analysis of variance (post levene test).comparion of any two group among the groups were performed with SNK-q test. Difference were considered statistically significent at P<0.05.Results1,Primary HUVECs were isolated and cultured, immunocytochemical stain show human VIII factor related antigen positive.2,NO content in mediun significantly decreased by different glucose concentration (P<0.05) .In contrast to normal glucose, high glucose (22.2 mmol/L,33.3 mmol/L) significantly decreased SIRT1 expression in both protein and mRNA levels (P<0.05).3,In contrast to 0h , the NO content in medium increased after culturing 24h(P<0.05). The expression of SIRT1 in both RNA and protein level ,as well as the NO content in medium, significantly decreased after culturing 48h and 72h (P<0.05).ConclusionsHigh glucose may decrease NO secretion and SIRT1 expression in a dose-and time-dependent manner in HUVEC. SIRT1-eNOS axis may be a pathway that connecting high glucose and endothelial dysfunction.