葡萄籽多酚对糖基化终产物诱导的内皮细胞组织因子表达的影响

Inhibitory effect of grape seed polyphenol on tissue factor expression induced by advanced glycation end products in endothelial cells

目的 观察葡萄籽多酚（GSP）对糖基化终产物（AGEs）诱导的人脐静脉内皮细胞（HuVEC）组织因子（TF）蛋白及其mRNA表达的抑制作用。方法 牛血清白蛋白（BSA）与葡萄糖在体外共同孵育以制备糖基化终产物（AGE-BSA），将培养的HUVEC与不同浓度的GSP（5、15、25μg／ml）预孵育4h，再加入200μg／mlAGE-BSA共同培养，分别用流式细胞仪测定TF蛋白表达，用逆转录PCR检测TFmRNA的表达，用激光共聚焦显微镜检测细胞内活性氧簇（ROS）的产生。结果 HUVEC未受刺激时，细胞内活性氧簇（ROS）（0．83±0．23）产生较低，TF蛋白[（4．42±0．69）％]及mRNA（0．0020±0．0008）表达亦较低，而AGE-BSA刺激明显增强了细胞内ROS（3．12±0．31）产生和TF蛋白（42．28±5．80）％及其mRNA（0．2964±0．0403）表达，与空白对照组比较，差异有统计学意义（P〈0．01）。不同浓度GSP预孵育则以剂量依赖性的方式显著抑制了AGE-BSA诱导的细胞内ROS[分别为（2．36±0．34）、（1．88±0．43）、（1．23±0．20）3产生，并下调了TF蛋白[分别为（28．45±2．19）％、（17．49±3．84）％、（8．54±1．57）％）]及其mRNA[分别为（0．0994±0．0231）、（0．0382±0．0073）、（0．0065±0．0010）]表达，P〈0．01。结论 GSP可能通过抑制细胞内氧化应激而影响AGEs诱导的内皮细胞TF生成，改善血管促凝血活性。

Objective To investigate the inhibitory effect of grape seed polyphenol （GSP） on expression of tissue factor （TF） protein and mRNA induced by advanced glycation end products （AGEs） in human umbilical vein endothelial cells（HUVEC）. Methods AGE-BSA was prepared by incubating bovine serum albumin （BSA）with glucose for 90 days at 37℃. After pre-incubation of GSP at the concentrations of 5, 15, 25 μg/ml for 4 hours, the cultured HUVEC was stimulated with 200 μg/ml AGE-BAS. Thereafter, surface TF protein expression was determined by flow cytometer. TF mRNA levels were assayed by reverse-transcription polyrnerase chain reaction （RT-PCR） . Intracellular oxidative stress was detected by laser scanning confocal microscopy （LSCM）. Results In HUVEC without any treatment intracellular reactive oxygen species （ROS） was slightly generated （0. 83±0. 23）,and TF protein and mRNA were expressed at lower levels [（4. 42± 0. 69）% for TF protein, 0. 0020 ±0. 0008 for TF mRNA3. Whereas, the stimulation of HUVEC with AGE-BSA significantly enhanced intracellular ROS production and subsequently increased the expressions of TF protein and mRNA[3. 12±0. 31 for ROS, （42. 28±5.80） % for TF protein, 0. 2964±0. 0403 for TF mRNA, compared to control, all P〈0. 013, while unmodified BSA was without effect. Interestingly, the pre-incubation of 5, 15, 25 g/ml GSP, in a concentration-dependent manner, markedly prevented AGE- induced up-regulation of TF protein and mRNA levels[（28. 45 ± 2. 19）%, （17. 49 ± 3. 84）%, （8. 54±1. 57） % for TF protein, 0. 0994±0. 0231, 0. 0382±0. 0073, 0. 0065±0. 0010 for TF mRNA respectively, compared with AGE-BSA group, all P〈0. 013. Meanwhile, LSCM further confirmed that GSP distinctly suppressed the formation of intracellular ROS （2.36±0. 34, 1.88±0.43, 1.23±0.20, respectively, compared with AGE-BSA group, all P〈0.01）. Conclusions These results demonstrate that GSP can inhibit AGEs-induced expressions of TF protein and mRNA through its antioxidant properties, which contributes to improvement of blood coagulant activity in the vessel walls. Hence, GSP may have therapeutic potential in the treatment of patients with diabetic vascular complications.