白藜三醇对人脐静脉内皮细胞血管生成的影响及其可能机制

Effect of Resveratrol on Angiogenesis of Human Umbilical Vein Endothelial Cells With the Possible Mechanisms

目的：探究白藜三醇（Res）对正常状态下人脐静脉内皮细胞（HUVEC）血管生成的影响及其可能机制。 方法：体外培养HUVEC，实验分为：DMEM高糖（〈4500 mg/L）培养基培养组（正常组）、白藜三醇组（药物组）、白藜三醇＋磷脂酰肌醇3-激酶（PI3K）阻断剂组（LY294002，药物阻断剂组1）、LY294002组（阻断剂组1）、白藜三醇＋NG-硝基-L-精氨酸甲酯盐酸盐组（一氧化氮合酶阻断剂L-NAME，药物阻断剂组2）、L-NAME组（阻断剂组2），其中LY294002为磷脂酰肌醇3-激酶阻断剂。采用CCK-8法检测细胞增殖情况；免疫印迹（Western blot）法检测蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）、内皮型一氧化氮合酶（eNOS）、磷酸化内皮型一氧化氮合酶（p-eNOS）蛋白的表达；亚硝酸还原酶法检测一氧化氮（NO）的水平；迁移小室（transwell chamber）检测内皮细胞的迁移能力；小管形成实验检测内皮细胞的体外小管形成能力。 结果：①药物组（1、5μmol/L）与正常组相比细胞增殖增加（P〈0.01）；药物组（5μmol/L）与药物组（0.2、10、20μmol/L）相比细胞增殖增加（P〈0.01）；药物组（20μmol/L）对细胞增殖有抑制作用（P〈0.01），差异均有统计学意义。②药物组（5μmol/L ）较正常组p-Akt、p-eNOS蛋白表达增加（P〈0.05~0.01），NO水平增加（P〈0.05），差异均有统计学意义；药物阻断剂组1 p-Akt、p-eNOS蛋白表达与药物组比较均减低（P均〈0.01），NO水平减低（P〈0.05）；药物阻断剂组1（5μmol/L）较阻断剂组1 p-Akt蛋白、p-eNOS蛋白及NO差异均无统计学意义（P均〉0.05）。③细胞迁移及小管形成实验：药物组（5μmol/L）内皮细胞迁移率及体外小管形成数量大于正常组（P〈0.01），药物阻断剂组2内皮细胞迁移率及体外小管形成数量小于药物组（P〈0.01）。 结论：白藜三醇可能通过激活PI3K/Akt/eNOS信号通路诱导正常状态下内皮细胞NO水平的增加，从而促进内皮细胞增殖、迁移及体外小管形成。

Objective： To explore the effect of resveratrol （Res） on angiogenesis of human umbilical vein endothelial cells （HUVEC） with the possible mechanisms in vitro. Methods： The HUVECs were cultured in 6 groups.①Control group, HUVEC were cultured with high glucose in DMEM,②Res group, the cell were cultured with Res at different concentrations,③Res with PI3K blocker LY294002 （Res＋Blocker 1） group, ④Blocker 1 group, HUVEC were cultured with LY294002 alone, ⑤Res with eNOS blocker L-NAME （Res ＋Blocker 2） group and ⑥Blocker 2 group. The effect of Res on HUVEC proliferation was detected by CCK-8 kit, the protein expressions of p-Akt, p-eNOS were examined by Westin blot analysis, nitric oxide （NO） level was measured by nitrate reduction method and the endothelial cell migration was assayed by transwell chamber method. Results： ① Compared with Control group, HUVEC proliferation increased in Res （1, 5μmol/L ） group, P〈0.01, the proliferation in Res （5μmol/L） group was higher than those in Res （0.2, 10, 20μmol/L） group, P〈0.01, while Res （20 μmol/L） group could inhibit the proliferation P〈0.01. ②Compared with Control group, Res （5μmol/L） group had the higher protein expressions of p-Akt, p-eNOS, P〈0.05-0.01, higher NO level, P〈0.05.③Compared with Res group, Res＋Blocker 1 group had lower expressions of p-Akt, p-eNOS, P〈0.01, lower NO, P〈0.05; the expressions of p-Akt, p-eNOS and NO level were similar between Res＋Blocker 1 group and Blocker 1 group, all P〉0.05.④Compared with Control group, the cell migration and tubing formation were higher in Res （5μmol/L） group, P〈0.01;compared with Res group, the cell migration and tubing formation were lower in Res＋Block2 group, P〈0.01. Conclusion： Res could up-regulate NO level via activating PI3-K/Akt/eNOS signaling and therefore, improving the proliferation, migration and tubing formation of HUVEC in vitro.