白藜三醇对人脐静脉内皮细胞增殖及NO表达的影响

Effects of resveratrol on the proliferation of HUVECs and expression of NO

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-NOS）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（〈4．5mg／L）培养基培养组（正常组）、白藜三醇组（分为0．2、1、5、10、20μmol／L5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，P13-K）抑制剂。应用CCK-8法检测细胞增殖情况；Westernblot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／L2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P13-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。

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, 20μmol/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 （1） CCK8 ： resveratrol group （1, 5 Ixmol/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, 20 μmol/L） （P 〈 0.01 ） ; resveratrol （20μmol/L） decreased cell proliferation compared with the normal group （P 〈 0.01 ）. （2） Western 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. （3） Nitric oxide assays： the culture media NO content of HUVECs was increased significantly in resveratrol group compared 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 PI3 - K/Akt/eNOS pathway, and further promote the proliferation of HUVECs.