Department of Endocrinology and Metabolism, First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, China.
Chin Med J (Engl). 2009 Dec 20;122(24):3055-61.
Endothelial cell senescence is accelerated under high glucose condition, which may contribute to the vascular complications in the diabetics. It has been proved that aspirin has multiple cytoprotective effects. This study aimed to investigate the effect of aspirin on high glucose-induced endothelial cell senescence and its possible mechanism.
Human umbilical venous endothelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with different treatments including the normal glucose (5.5 mmol/L), high glucose (33 mmol/L) and aspirin (0.01 - 1.00 mmol/L) with high glucose. And 300 micromol/L L-NAME was added to the culture medium when needed. After 48 hours, SA-beta-gal staining was used to evaluate the senescence. Total nitric oxide (NO) production and NO synthase (NOS) activity were measured using Griess reaction and molecular probes of 3-amino-4-aminomethyl-2', 7'-difluorescein, diacetate. The level of intracellular reactive oxygen species was monitored by flow cytometry using 2', 7'-dichlorofluorescein diacetate. Endothelial NOS (eNOS), caveolin-1 protein expressions and caveolin-1/eNOS interaction were analyzed by immunoblotting and immunoprecipitation respectively. Asymmetric dimethylarginine (ADMA) concentration was determined by high-performance liquid chromatography.
Exposure to 33 mmol/L glucose for 48 hours significantly increased the number of SA-beta-gal positive cells. Co-incubation with aspirin markedly inhibited SA-beta-gal activity dose-dependently. Aspirin increased NOS activity with eNOS protein expression unchanged and increased NO levels and alleviated oxidative stress. Consistent with these findings, caveolin-1 expression, caveolin-1/eNOS interaction and ADMA accumulation were also decreased. All the inhibitory effects of aspirin on senescence were completely obliterated by L-NAME, the NOS inhibitor.
The anti-senescent effects of aspirin are fulfilled by increasing NO production via the up-regulation of NOS activity and preventing caveolin-1 expression, caveolin-1/eNOS interaction and ADMA accumulation.
高糖条件下内皮细胞衰老加速，可能导致糖尿病患者血管并发症。已有研究证实阿司匹林具有多种细胞保护作用。本研究旨在探讨阿司匹林对高糖诱导的内皮细胞衰老的作用及其可能机制。
人脐静脉内皮细胞在含不同处理因素的 Dulbecco's modified Eagle's 培养基（DMEM）中培养，包括正常葡萄糖（5.5mmol/L）、高葡萄糖（33mmol/L）和阿司匹林（0.01-1.00mmol/L），高糖时需加入 300μmol/L L-NAME。48 小时后，用 SA-β-半乳糖苷染色法评价细胞衰老。采用格里斯反应和 3-氨基-4-氨甲基-2',7'-二氟荧光素二乙酸酯分子探针测定总一氧化氮（NO）生成和一氧化氮合酶（NOS）活性。用 2',7'-二氯荧光素二乙酸酯通过流式细胞术监测细胞内活性氧水平。用免疫印迹和免疫沉淀分别分析内皮型一氧化氮合酶（eNOS）、窖蛋白-1 蛋白表达和窖蛋白-1/eNOS 相互作用。用高效液相色谱法测定不对称二甲基精氨酸（ADMA）浓度。
暴露于 33mmol/L 葡萄糖 48 小时后，SA-β-半乳糖苷阳性细胞数明显增加。阿司匹林浓度依赖性地显著抑制 SA-β-半乳糖苷活性。阿司匹林增加了 NOS 活性，同时 eNOS 蛋白表达不变，NO 水平增加，氧化应激减轻。与这些发现一致，窖蛋白-1 表达、窖蛋白-1/eNOS 相互作用和 ADMA 积累也减少。NOS 抑制剂 L-NAME 完全消除了阿司匹林对衰老的抑制作用。
阿司匹林通过增加 NOS 活性、抑制窖蛋白-1 表达、窖蛋白-1/eNOS 相互作用和 ADMA 积累来增加 NO 生成，从而发挥抗衰老作用。
