SIRT1对高糖高脂培养胰岛微血管内皮细胞eNOS表达及活性的影响

Effects of SIRT1 on the expression and activity of eNOS in high fat and high glucose cultured islet en- dothelial cells

目的分析高表达组蛋白去乙酰化酶（sirtuin 1，SIRTl）对高糖高脂培养胰岛微血管内皮细胞（islet endothelialcells，IEC）内皮型一氧化氮合酶（eNOS）表达及活性的影响。方法合成含绿色荧光蛋白报告基因的小鼠SIRTl基因重组质粒，以Lipofectamine2000转染IEC后以高脂或高糖高脂处理48h。高脂组以0．5mmol／L棕榈酸处理；高脂高糖组以33．3mmol／L葡萄糖＋0．5mmol／L棕榈酸处理。应用Westem印迹法检测SIRrll基因转染效果；应用实时荧光定量PCR及Western印迹法检测eNOS的基因及蛋白水平；应用硝酸还原酶法检测培养细胞上清中NO的水平。结果相对于正常对照组，高脂培养IECeNOS基因及蛋白水平无明显变化；但高脂高糖环境下，eNOS的基因及蛋白水平明显下降。高表达SIRT1不仅可升高高脂培养内皮细胞eNOS蛋白表达，且可升高高脂高糖培养内皮细胞eNOSmRNA及蛋白表达。相对于正常对照组，高脂培养使内皮细胞分泌NO水平明显下降，高糖高脂培养使内皮细胞NO水平进一步下降，高表达SIRT1不仅可升高基础状态下内皮细胞的NO水平，且可升高高脂、高脂高糖培养内皮细胞的NO水平，差异具有统计学意义。结论SIRT1可改善高糖高脂培养胰岛微血管内皮细胞的eNOS表达及活性，使内皮源性NO分泌增加，因而可能有助于改善胰岛B细胞功能，在糖尿病的药物开发、基因治疗及胰岛移植治疗中具有广阔的前景。

Objective To investigate the effects of Sirtuin 1 （SIRT1） on expression and activity of islet endothelial cells （IEC） cultured by palmitic acid and high glucose. Methods Recombinant mouse SIRTI plasmid including green fluorescent protein report gene was synthesized and transfected to islet endothelial cells using Lipofectamine 2000. Then, ceils were treated by high level lipid with or without high level glucose for 48 hours. Cells in high glucose and lipid group were cultured in 33.3 mmol/L glucose and 0.5 mmol/L palmitic acid for 48 hours.Cells in high lipid group were cultured in 0.5 mmol/L palmitic acid for 48 hours. Western blot was used to test the transfecting efficacy of SIRT1 recombinant plasmid. Real time quantitative-PCR and Western blot were used to exam the expression of endothelial nitric synthases（eNOS） mRNA and protein, respectively. Nitric oxide reductase method was used to test NO level in supernatant of cell culture. Results Compared with the control group, there was no change on eNOS mRNA and protein level in high lipid group; however, the expression of eNOS mRNA and protein level decreased significantly in cells cultured in high level lipid and glucose group. SIRT1 overexpression increased the protein level of eNOS in high lipid group, and also increased mRNA and protein level in high lipid and glucose group. Compared with the control group, NO decreased significantly in high level lipid cultured endothelial cells, and decreased further in high lipid and high glucose cultured group. SIRT1 overexpression increased NO not only in normal ceils but also in high level lipid with or without high glucose cells. Conclusions SIRT1 improves the expression and activity of eNOS in IEC and can increase the release of NO by endothelial ceils, which may be helpful in improving the function of islet beta cells. SIRT1 may have great promise in new medicine development, gene therapy and islet transplantation in diabetes.