体外研究胰岛素及磷脂酰肌醇3-激酶途径对一氧化氮生成的影响

目的探讨胰岛素及磷脂酰肌醇3-激酶(PI3-K)途径对NO生成的影响。方法检测胰岛素、葡萄糖以及PI3-K活性不可逆的抑制剂(Wortmannin)对培养的人脐静脉内皮细胞(HUVECs)PI3-K表达以及NO、超氧阴离子(O_2~-)产生和内皮型一氧化氮合酶(eNOS)活性的影响。实验分为对照组、10 mU/L胰岛素组、100 mU/L胰岛素组、甘露醇组、5 mmol/L葡萄糖+10 mU/L胰岛素组(5 mmol/L G1组)、25 mmol/L葡萄糖+100 mU/L胰岛素组(25 mmol/L G2组)、50 nmol/L Wortmannin组(50 nmol/L W组)、50 nmol/L Wortmannin+10 mU/L胰岛素组(50 nmol/L W1组)和50 nmol/L Wortmannin+100 mU/L胰岛素组(50 nmol/L W2组)。结果与对照组比较,不同浓度胰岛素组eNOS活性及NO水平显著升高(P<0.01);25 mmol/L G2组、50 nmol/L W组、50 nmol/LW1组和50 nmol/L W2组eNOS活性及NO水平均显著降低,O_2~-生成明显增加(P<0.01);与对照组比较,不同浓度胰岛组、50 nmol/L W组、50 nmol/L W1组和50 nmol/L W2组PI3-K蛋白表达显著升高(P<0.05,P<0.01)。结论 PI3-K信号途径对于促进NO产生、维持血管内皮细胞的正常功能具有重要作用,在高糖、高胰岛素状态下该条途径受损并由此引发内皮功能障碍。

Effect of insulin/PI3-K pathway on NO production in vitro

Objective To elucidate the effect of insulin/phosphatidylinositol 3-kinase(PI3-K) sig-naling pathway on endothelial nitric oxide(NO) production. Methods The effects of insulin,glu-cose and wortmannin(inhibitor of PI3-K) on the PI3-K expression, the generation of NO and su-peroxide anion(O2-) in human umbilical vein endothelial cells(HUVECs) were investigated. The experiment included 9 groups, control group, 10 mU/L insulin, 100 mU/L insulin, mannitol, 5 mmol/L glucose+10 mU/L insulin,25 mmol/L glucose+ 100 mU/L insulin, 50 nmol/L wort-mannin, 50 nmol/L wortmannin+ 10 mU/L insulin and 50 nmol/L wortmannin+100 mU/L insu-lin. Results Different dosages of insulin(10 mU/L, 100 mU/L) significantly increased NO pro-duction and endothelial nitric oxide synthase(eNOS) activity in HUVECs as compared with con-trol group. In the presence of 25 mmol/L glucose+100 mU/L insulin,wortmannin and wortman-nin+insulin(10 mU/L, 100 mU/L), NO production and eNOS activity significantly decreased (P < 0.01 vs controls,respectively) accompanied by elevated O2- production (P < 0.01 vs con-trols, respectively). Compared with controls, insulin (10 mU/L, 100 mU/L)and wortmannin,wortmannin+insulin(10 mU/L,100 mU/L) promoted PI3-K expression. Conclusions PI3-K sig-naling pathway plays an important role in NO production and maintenance of endothelial func-tion. In insulin-resistant state, this pathway is damaged, leading to endothelial dysfunction.