罗格列酮对高胰岛素培养的内皮细胞一氧化氮的影响及其机制研究

目的观察罗格列酮（RGZ）对高胰岛素培养的人脐静脉内皮细胞（HUVEC）NO浓度和内皮型一氧化氮合酶（eNOS）、磷酯酰肌醇3激酶（P13K）和蛋白激酶B（PKB）表达的影响，探讨RGZ改善高胰岛素状态下内皮功能障碍的信号转导机制。方法高浓度胰岛素培养HUVEC72h，并用不同浓度的RGZ进行干预。检测NO浓度，PI3K mRNA的表达，PKB、eNOS总蛋白和PKB丝氨酸473（PKB-Ser473）、eNOS丝氨酸1177（eNOS-Ser1177）的磷酸化表达。结果高浓度胰岛素培养HUVEC能呈剂帚和时间依赖性地降低N0的浓度，抑制内皮细胞P13KmRNA表达和PKB-Ser473、eNOS-Ser1177的磷酸化。用RGZ干预能硅著升高高胰岛素培养的内皮细胞NO的浓度和PKB、eNOS的磷酸化，增强PI3KmRNA表达；eNOS和P13K阻断剂均能阻断RGZ对高胰岛素培养的内皮细胞中NO浓度的升高，PI3K阻断剂还能阻断RGZ对高胰岛素培养内皮细胞PKB、eNOS的磷酸化。结论高胰岛素能下调P13K／PKB／eNOs信号通路而抑制内皮细胞NO的产生，RGZ能通过上调PI3K／PKB通路而增强高胰岛素培养的内皮细胞eNOS的活性和NO的产生。

Effect produced by rosiglitazone on nitric oxide produced by the high insulin- cultured human umbilical vein endothelial cells and its mechanism

Objective To investigate the effects of rosiglitazone on endothelium-produced nitric oxide （NO） and on the expressions of phosphatidylinositol 3 kinase （PI3K）/protein kinase B（PKB） /the endothelial nitric oxide synthase （eNOS） in high insulin cultured human umbilical vein endothelial cells （HUVEC）. Methods HUVEC cultured with 100nmd/L insulin were treated with various concentrations of rosiglitazone. NO was measured using Griess reaction in cell culture supernatants, expression of PI3K mRNA was measured using RT-PCR, expresstions of PKB, eNOS and phosphorylation of PKB-Ser473, eNOS-Ser1177 were measured using Western blot. Results In HUVEC, High insulin inhibited endothelial NO production and eNOS-Ser1177 phosphorylation without significant effect on eNOS expression. High insulin also inhibited expression of PI3K mRNA and phosphorylation of PKB-Ser473. Rosiglitazone increased NO production and the expressions of PI3K mRNA and phosphorylation of PKB, eNOS in HUVEC cultured with high insulin. L-NAME blocked the rosiglitazone-induced NO formation, and LY294002 prevented the increases of NO concentration and eNOS-Serl177 and PKB-Ser473 phosphorylation induced by rosigtitazone in HUVEC cultured with high insulin. Conclusions High insulin inhihites phosphorylation of eNOS by down-regulating PI3K/PKB signaling pathways. Treatment with rosiglitazone stimulates eNOS phosphorylation through a PI3K/PKB-mediated mechanism in high insulin cultured HUVEC.