白藜芦醇抑制缺氧复氧诱导心肌微血管内皮细胞凋亡的研究

摘要

目的 探讨白藜芦醇对缺氧复氧诱导心肌微血管内皮细胞(CMEC)凋亡的抑制作用及其可能的分子机制.方法 体外培养大鼠CMEC,建立缺氧复氧损伤模型,随机分为对照1组、缺氧复氧1组、缺氧复氧+白藜芦醇组(白藜芦醇1组),白藜芦醇分别选择5、10、20、30及40 μmol/L浓度,MTT法检测CMEC增殖能力.20 μmol/L白藜芦醇为最适浓度,使用磷脂酰肌醇3激酶(PI3K)特异性抑制剂LY294002,后续实验又分为对照2组、缺氧复氧2组、缺氧复氧+白藜芦醇2组(白藜芦醇2组)、缺氧复氧+白藜芦醇+LY294002组(LY294002组).PI-AnnexinⅤ检测CMEC的凋亡;Western blot法检测细胞蛋白激酶B(Akt)、磷酸化Akt、缺氧诱导因子1α(HIF-1α)蛋白表达或磷酸化水平.结果 与缺氧复氧1组比较,不同浓度白藜芦醇1组均可增加CMEC增殖能力,呈剂量依赖性,以白藜芦醇1组20 μmol/L保护作用最显著(P<0.05).与白藜芦醇2组比较,LY294002组CMEC凋亡率明显升高,磷酸化Akt和Akt蛋白、HIF-1α蛋白表达明显下降(P<0.05).结论 白藜芦醇可显著抑制缺氧复氧诱导的CMEC凋亡,其机制可能与PI3K/Akt介导的HIF-1α上调相关.%Objective To study the inhibitory effect of resveratrol on hypoxia/reoxygen(H/R)-in-duced apoptosis of cardiac microvascular endothelial cells (CMEC ) in rats and its mechanism . Methods A hypoxia/reoxygen injury model was establish by culturing CM EC in vitro . The CMEC were randomly divided into control group 1,H/R group 1 ,and H/R+resveratrol (at the concentrations of 5,10,20,30 or 40 μmol/L) group 1 .Proliferation of CMEC was detected by MTT .Specific inhibitor LY294002 of PI3K was added into different groups to observe the mechanism of resveratrol at the concentration of 20 μmol/L .The CMEC were then divided into control group 2 , H/R group 2 , and H/R+resveratrol group 2 .Apoptosis of CMEC was detected by flow cytometry with PI-annexin V staining .Expression or phosphorylation of Akt ,p-Akt and HIF-1α protein in CMEC was detected by Western blot .Results The proliferation of CMEC was higher in H /R+ resveratrol group 1 than in H/R group 1(P<0 .05 ) .Resveratrol at different concentrations increased the proliferation of CMEC in a dose-dependent manner, especially at the concentration of 20 μmol/L .The apoptosis level of CMEC was significantly higher whereas the the expression level of PI3K,Akt and HIF-1α was significantly lower in LY294002 treatment group than in H/R+resveratrol group 2(P<0 .05 ) .Conclusion Resveratrol significantly inhibits H/R-induced apoptosis of CMEC by up-regulating the PI3K/Akt-mediated HIF-1α expression .