白藜芦醇通过PI3K/Akt/SVV信号通路改善大鼠心肌微血管内皮细胞缺血再灌注损伤

Resveratrol attenuates hypoxia-reperfusion injury induced rat myocardium microvascular endothelial cell dysfunction through upregulating PI3K/Akt/SW pathways

目的 探讨生存素（survivin,SVV）在白藜芦醇抗心肌微血管内皮细胞（cardiac microvascular endothelial cells,CMEC）缺血再灌注损伤中的作用.方法 体外培养大鼠心肌微血管内皮细胞,建立缺血再灌注损伤模型,完全随机法分为正常对照组（对照组）、缺血再灌注组、缺血再灌注＋白藜芦醇组（白藜芦醇组）、缺血再灌注＋白藜芦醇＋ LY294002组（LY294002组）.采用MTT比色法检测CMEC的增殖数量,Transwell法检测CMEC的迁移数量,PI-AnnexinV检测CMEC的凋亡率.Western blot法检测细胞SVV的蛋白表达水平.结果 缺血再灌注组CMEC增殖和迁移数量均显著低于对照组[0.19±0.03比0.42±0.07和（28±2）个/5个视野比（50±3）个/5个视野,P均＜0.01],凋亡率则显著高于对照组[（19.7±0.8）％比（5.4±0.3）％,P＜0.02].白藜芦醇组CMEC增殖和迁移数量均显著高于缺血再灌注组[0.36±0.07比0.19±0.03和（55±3）个/5个视野比（28±2）个/5个视野,P均＜0.05],凋亡率则显著低于缺血再灌注组[（9.6±0.7）％比（19.7±0.8）％,P＜0.05],SVV蛋白表达水平则高于缺血再灌注组（P＜0.05）.而LY294002组CMEC增殖和迁移数量均显著低于白藜芦醇组[0.25±0.05比0.36±0.07和（34±3）个/5个视野比（55±3）个/5个视野,P均＜0.05],凋亡率则显著高于白藜芦醇组[（16.2±0.6）％比（9.6±0.7）％,P＜0.05],SVV蛋白表达水平低于白藜芦醇组（P＜0.05）.结论 白藜芦醇可抑制缺血再灌注诱导的心肌微血管内皮细胞凋亡,其机制可能与PI3K/Akt介导的SVV上调相关.

Objective To detect the role of surviving （SVV） in the protective effect of resveratrol against hypoxia/reperfusion injury （H/RI） of cardiac microvascular endothelial cells （CMECs）.Methods CMECs isolated from the hearts of adult rats were exposed to hypoxia （94％ N2,5％ CO2,1％ O2） for 2 h followed by 4 h reoxygenation （95％ O2,5％ CO2）.The cell proliferation of CMECs was measured by MTT assay and Transwell method was used to detect migration ability of CMEC,PI-AnnexinV double staining and flow cytometry technique were employed to observe the apoptotic rate of CMECs.The SVV protein expression was detected with Western blot method.Results Compared to control group,the proliferation （0.19 ± 0.03vs.0.42 ±0.07,P ＜0.01） and migration （（28 ± 2）/5HPF vs.（50 ±3）/5 HPF,P ＜0.01） abilities were impaired and the apoptosis index （（19.7 ± 0.8） ％ vs.（5.4 ± 0.3） ％,（P ＜ 0.05） of CMEC was increased after H/RI.The proliferation （0.36 ± 0.07 vs.0.19 ± 0.03,P ＜ 0.05） and migration （（55 ± 3）/5 HPF vs.（28 ± 2）/5 HPF,P ＜ 0.05） abilities of CMEC were significantly improved while the apoptosis index （（9.6 ±0.7） ％ vs.（19.7 ± 0.8） ％,P ＜ 0.05） was significantly decreased in H/RI ＋ resveratrol group compared to H/RI group.SVV protein expression was also upregulated in H/RI ＋ resveratrol group compared to H/RI group （P ＜0.05）.To further ascertain the role of SVV in the protective effects of resveratrol,PI3K specific inhibitor LY294002 was added to H/RI ＋ resveratrol group,the proliferation（0.25 ± 0.05 vs.0.36 ± 0.07,P ＜0.05） and migration （（34 ± 3）/5HPF vs.（55 ± 3）/5HPF,P ＜ 0.05） abilities were significantly decreased,the apoptosis index （（16.2 ± 0.6） ％ vs.（9.6 ± 0.7） ％,P ＜ 0.05） was increased and the protein expression of SVV was downregulated （P ＜ 0.05） in LY294002 ＋ H/RI ＋ resveratrol group compared to H/RI ＋ resveratrol group.Conclusion Resveratrol could significantly reduce H/RI induced apoptosis and attenuate H/RI induced cardiac microvascular endothelial cells dysfunction through up-regulating PI3K/Akt/SVV pathways.