白藜三醇对人脐静脉内皮细胞增殖及NO表达的影响

摘要

Objective To investigate the regulating effects of resveratrol on the expressions of phospho -Akt ( p-Akt), phospho-eNOS (p-eNOS) and nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) .The effects of resveratrol on the proliferation of HUVECs and its possible mechanisms were also investigated .Methods Grouping:high glucose (<4 .5 mg/L) DMEM medium group (normal group), resveratrol group (including 0.2, 1, 5, 10, 20 μmol/L subgroups), resveratrol -LY294002 group, LY294002 group (blocker group).LY294002 is the bloc-ker of phosphatidylinositol 3-kinase ( PI3-K) .CCK8 assay was used to detect the effects of reaveratrol on the prolifer-ation of HUVECs .Western blot was used to detect the p -Akt and the p-eNOS protein level respectively .NO concen-trations in conditioned media were determined using the nitrate reductase methods .Results ①CCK8:resveratrol group (1, 5 μmol/L) increased cell proliferation compared with normal group (P<0.01);resveratrol (5μmol/L) increased cell proliferation compared with the drug group (0.2, 10, 20μmol/L) (P<0.01);resveratrol (20μmol/L) decreased cell proliferation compared with the normal group (P<0.01).②Western blot:p-Akt (P<0.01), p-eNOS (P<0.05) expressions were increased significantly in resveratrol group compared with normal group ; and p -Akt ( P <0.01), p-eNOS (P<0.01) expressions were decreased in resveratrol -blocker group compared with resveratrol group .③Nitric oxide assays:the culture media NO content of HUVECs was increased significantly in resveratrol group com -pared with normal group (P<0.05), and it was decreased in resveratrol -blocker group compared with resveratrol group (P<0.01).Conclusion Resveratrol can increase of expression of NO in HUVECs via PI 3-K/Akt/eNOS pathway, and further promote the proliferation of HUVECs .%目的：观察白藜三醇对正常状态下人脐静脉内皮细胞（ human umbilical vein endothelial cells ， HU-VECs）磷酸化蛋白激酶B（p-Akt）、磷酸化内皮型一氧化氮合酶（p-eNOS）及一氧化氮（NO）表达的调节作用，探讨白藜三醇对HUVECs增殖的影响及可能机制。方法实验分组：DMEM高糖（＜4．5 mg/L）培养基培养组（正常组）、白藜三醇组（分为0．2、1、5、10、20μmol/L 5个剂量亚组）、白藜三醇＋LY294002组、LY294002组（阻断剂组）。其中LY294002为磷脂酰肌醇3-激酶（ phosphatidylinositol 3-kinase，PI3-K）抑制剂。应用CCK-8法检测细胞增殖情况；Western blot检测各组细胞内蛋白激酶B（ Akt）、p-Akt、内皮型一氧化氮合酶（ eNOS）、p-eNOS蛋白的表达；硝酸还原酶法检测细胞培养液中NO的浓度。结果①白藜三醇组1、5μmol/L 2个剂量亚组与正常组相比细胞增殖增加（P＜0．01），5μmol/L亚组与0．2、10、20μmol/L亚组相比细胞增殖增加（P＜0．01）；白藜三醇20μmol/L对细胞增殖有抑制作用（P＜0．01）。②与对照组比较，白藜三醇组p-Akt（P＜0．01）、p-eNOS（P＜0．05）蛋白表达增加；与白藜三醇组比较，白藜三醇＋LY294002组p-Akt、p-eNOS蛋白表达降低（P＜0．01）。③与对照组比较，白藜三醇组NO表达增加（P＜0．05）；与白藜三醇组比较，白藜三醇＋LY294002组NO表达降低（ P＜0．01）。结论白藜三醇可能通过PI3-K/Akt/eNOS信号通路诱导正常状态下内皮细胞NO表达的增加，从而促进内皮细胞增殖。