糖基化终产物对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响及二甲双胍的保护作用

目的 探讨糖基化终产物及二甲双胍对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响.方法 用胶原酶法分离人脐静脉内皮细胞并加以培养.将内皮细胞与不同浓度的糖基化终产物和二甲双胍分别孵育3、6、12、24 h,CCK-8法测定人脐静脉内皮细胞增殖活性.硝酸还原酶法测定一氧化氮含量,分光光度法测定一氧化氮合酶活性,蛋白免疫印迹法检测内皮型一氧化氮合酶蛋白表达水平.结果 糖基化终产物抑制人脐静脉内皮细胞增殖,二甲双胍促进人脐静脉内皮细胞增殖.糖基化终产物抑制人脐静脉内皮细胞的一氧化氮生成和一氧化氮合酶活性(P<0.01),呈剂量、时间依赖关系.二甲双胍(与对照组相比)或与糖基化终产物共同干预(与糖基化终产物组相比)均增加人脐静脉内皮细胞一氧化氮生成和一氧化氮合酶活性(P<0.01).糖基化终产物与人脐静脉内皮细胞共同孵育24 h后,内皮型一氧化氮合酶表达水平明显下降;二甲双胍上调内皮型一氧化氮合酶的表达;与糖基化终产物组相比,糖基化终产物与二甲双胍共同干预组内皮型一氧化氮合酶表达上调(P<0.01).结论 二甲双胍能够改善糖基化终产物导致的人脐静脉内皮细胞损伤.

Effects of Advanced Glycation End Products on Activity and Expression of Nitric Oxide Synthase in Human Umbilical Vein Endothelial Cells and the Protective Effects of Metformin

Aim To investigate the effects of advanced glycation end products (AGE) and metformin on activity and expression of endothelial nitric oxide synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVEC). Methods HUVEC were co-incubated with different concentrations of AGE-BSA and metformin for 3,6,12 and 24 hours respectively. HUVEC viability was measured by CCK-8 ; nitric oxide (NO) was measured by the technique of nitrate re-ductase; the activity of NOS was determined by the spectrophotography and protein expression of eNOS was measured by Western Blotting. Results AGE-BSA inhibited the proliferation of HUVEC, while metformin promoted the prolifera-tion of HUVEC. AGE-BSA significantly inhibited the generation of NO and the activity of NOS in a concentration and time-dependent manner (P < 0.01). Mefformin (compared with the control group) or with AGE-BSA co-interventions (compared with the AGE group) significantly increased the generation of NO and the activity of NOS (P < 0.01). The expression of eNOS had a significant reduction when HUVEC were incubated with AGE-BSA for 24 hours, while the expres-sion of eNOS had a significant increase when incubated with metformin. Compared with AGE-BSA group, the expression of eNOS up-regulated in metformin intervention group (P < 0.01). Conclusion Metformin ameliorates the dysfunction of HUVEC induced by AGE.