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目的观察银杏叶提取物GBE50及其主成分总黄酮对缺氧致人脐静脉内皮细胞(HUVECs)活性氧自由基(ROS)生成及凋亡的保护作用。方法通过消化法获得HUVECs,传代后干预分为N组(正常对照)、H组(缺氧24 h)、HG组(缺氧前GBE50干预4 h)和HF组(缺氧前总黄酮干预4 h),其中GBE50和总黄酮各有12．5、25．0和50．0μg／mL 3个浓度。采用流式细胞仪测各组ROS水平及早期凋亡率(Annexin V),通过TUNEL测晚期凋亡率,探讨缺氧及GBE50和总黄酮对HUVECs凋亡及其ROS生成的影响。结果①H组ROS水平较N组明显升高(P<0．05),由(9．88±0．97)％升至(29．27±2．21)％;HG各浓度组与H组相比ROS水平均降低,但与N组及H组相比差异均无统计学意义(P值均>0．05);HF各浓度组与H组相比ROS水平均明显下降(P值均<0．05),其中HF组中总黄酮浓度为50．0μg／mL时ROS降到最低,为(7．42±1．81)％。②H组细胞Annexin V阳性率较N组明显增加(P<0．05),由(9．00±1．00)％升至(18．47±2．53)％:HG 25．0μg／mL时为(10．69±2．08)％,显著降低了HUVECs的Annexin V阳性率(P<0．05);HF各浓度组均能不同程度地降低Annexin V阳性率,但差异均无统计学意义(P值均>0．05),该保护效应在总黄酮浓度为50．0μg／mL时最明显,Annexin V阳性率为(14．25±2．22)％。③H组TUNEL阳性率较N组明显增加(P<0．05),由(1．28±0．65)％升至(8．59±1．10)％; HG 25．0μg／ml时降至(3．58±0．28)％,HF 50．0μg／ml为(3．06+0．40)％,较H组均明显下降(P值均<0．05)。结论缺氧可导致HUVECS ROS生成增加伴凋亡增多。而GBE50及总黄酮可降低缺氧所致的ROS生成增多及凋亡增加,对缺氧所致的内皮功能障碍有一定的保护作用。
Objective To observe the protective effect of ginkgo biloba extract GBE50 and its main component flavonoids on the production of reactive oxygen species (ROS) and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by hypoxia. Methods HUVECs were obtained by digestion. After intervention, the interventions were divided into N group (normal control), H group (24 h hypoxia), HG group (4 h before GBP50 intervention) and HF group (before hypoxia total flavonoid intervention 4 h), wherein GBE50 and total flavonoids have concentrations of 12.5, 25.0, and 50.0 μg/mL, respectively. Flow cytometry was used to measure the level of ROS and early apoptosis rate (Annexin V) in each group. The late apoptosis rate was measured by TUNEL to investigate the effects of hypoxia and GBE50 and total flavonoids on the apoptosis and ROS production of HUVECs. Results The level of ROS in the H group was significantly higher than that in the N group (P<0.05), from (9.88±0.97)% to (29.27±2.21)%; HG concentrations in each group and H group Compared with ROS, the mean levels of ROS decreased, but there was no significant difference between N group and H group (all P>0.05). The ROS levels in each HF group decreased significantly compared with H group (P values were <0.05), in which the total flavonoid concentration in the HF group was 50.0μg/mL, the ROS decreased to the lowest (7.42 ± 1.81)%. The positive rate of Annexin V in the 2H group was significantly higher than that in the N group (P<0.05), from (9.00±1.00)% to (18.47±2.53)%: HG 25.0 μg/mL At the time of (10.69±2.08)%, the Annexin V positive rate of HUVECs was significantly decreased (P<0.05); the concentrations of Annexin V in various concentrations of HF could reduce the positive rate of Annexin V to varying degrees, but the differences were not statistically significant. The significance of the study was significant (P>0.05). The protective effect was most obvious when the total flavonoid concentration was 50.0 μg/mL, and the positive rate of Annexin V was (14.25±2.22)%. The positive rate of TUNEL in 3H group was significantly higher than that in N group (P<0.05), from (1.28±0.65)% to (8.59±1.10)%; HG 25.0μg/ml decreased To (3.58±0.28)%, HF 50.0 μg/ml was (3.06+0.40)%, which was significantly lower than that in H group (P<0.05). Conclusion Hypoxia can lead to increased ROS production in HUVECS with increased apoptosis. The GBE50 and total flavonoids can reduce the increase of ROS generation and apoptosis caused by hypoxia, and have a protective effect on endothelial dysfunction caused by hypoxia.