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目的探讨阿司匹林是否通过影响一氧化氮(NO)-一氧化氮合酶(NOS)系统发挥抗高糖诱导的内皮细胞衰老的作用。方法人脐静脉血管内皮细胞(HUVECs)分别于正常糖浓度培养液(5.5mmol/L)、高糖培养液(33 mmol/L)、含阿司匹林(0.01~1 mmol/L)培养液及含L-NAME(300μmol/L)培养液中培养48h。采用β-半乳糖苷酶(SA-β-gal)染色鉴定衰老细胞,流式细胞仪检测细胞内活性氧簇(ROS)水平。Griess法检测细胞总NO水平,荧光酶标仪检测细胞内NOS活性。Western blot分析eNOS蛋白及eNOS人丝氨酸(Ser-1177)蛋白表达。结果高糖作用内皮细胞48h后与正常糖浓度相比,SA-β-gal阳性细胞数明显增多。阿司匹林剂量依赖性地减少SA-β-gal阳性细胞数,降低ROS的水平,升高NO的水平、总NOS活性和eNOS Ser-1177蛋白表达(P<0.05)。L-NAME(NOS的抑制剂)能完全抑制高糖环境下阿司匹林的上述作用(P<0.05)。各组间eNOS总蛋白表达差异无统计学意义(P>0.05)。结论高糖环境下阿司匹林通过提高细胞NOS活性而升高NO水平,降低氧化应激反应而发挥抗衰老作用。
Objective To investigate whether aspirin exerts anti-hypertensive effects on endothelial cell senescence by affecting nitric oxide (NO) -nitrogen monoxide synthase (NOS) system. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in normal glucose medium (5.5 mmol / L), high glucose medium (33 mmol / L), aspirin (0.01-1 mmol / L) -NAME (300μmol / L) medium for 48h. The senescent cells were identified by β-galactosidase (SA-β-gal) staining and the intracellular reactive oxygen species (ROS) levels were detected by flow cytometry. Griess method was used to detect the total NO level in the cells, and fluorescence spectrophotometer was used to detect the intracellular NOS activity. Western blot analysis of eNOS protein and eNOS human serine (Ser-1177) protein expression. Results Compared with the normal glucose concentration, the number of SA-β-gal positive cells in endothelial cells increased significantly after 48h. Aspirin dose-dependently reduced the number of SA-β-gal positive cells, decreased ROS levels, increased NO levels, total NOS activity and eNOS Ser-1177 protein expression (P <0.05). L-NAME (inhibitor of NOS) completely inhibited the above effects of aspirin in high glucose (P <0.05). There was no significant difference in total eNOS protein expression among the three groups (P> 0.05). Conclusion Aspirin can play an anti-aging role by increasing NOS activity and increasing NO level under high glucose and reducing oxidative stress.