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目的初步探讨银杏叶提取物GBE50对抗缺氧致人脐静脉内皮细胞(HUVECs)功能障碍的分子机制。方法通过消化法获得HUVECs,传代后干预分为N组(正常对照)、NG组(GBE50 25μg／ml干预4 h后不予缺氧处理)、H组(缺氧24 h)及HG组(GBE50 25μg／mL干预4 h后缺氧24 h)。应用反转录-聚合酶链反应半定量检测各组von Willebrand因子(vWF)、内皮素-1(ET-1)及过氧化物酶体增殖物激活受体γ(PPARγ)的mRNA表达水平,运用Western印迹法测定各组内皮型一氧化氮合酶(eNOS)及PPARγ的蛋白表达水平。结果①VWF的mRNA表达水平:N组为(0．55±0．05)％,H组为(0．84±0．14)％,H组vWF表达较N组明显增加(P<0．05);HG组为(0．58±0．24)％,与H组相比明显降低(P<0．05)。②ET-1的mRNA表达水平: N组为(0．14±0．03)％,H组为(0．31±0．03)％, H组ET-1表达较N组明显增加(P<0．05);HG为(0．26±0．02)％,与H组相比明显降低(P<0．05)。③eNOS蛋白表达水平:H组eNOS蛋白表达较N组明显增加(P<0．05),由(0．30±0．04)％升至(0．59±0．08)％;HG组降至(0．43±0．08)％,但与其他各组相比差异无统计学意义(P值均>0．05)。④PPARγ的mRNA表达水平:N组为(0．40±0．02)％,H组为(0．42±0．01)％,HG组为(0．42±0．01)％,各组之间的差异均无统计学意义(P值均>0．05)。⑤PPARγ的蛋白表达水平:N组为(0．10±0．02)％,H组为(0．13±0．05)％,HG组为(0．13+0．04)％,各组之间的差异均无统计学意义(P值均>0．05)。结论内皮细胞缺氧损伤时可导致ET-1、vWF及eNOS的表达增加,内皮细胞在缺氧前给予GBE50 25μg／mL后,可降低缺氧导致的ET-1和vWF的表达增加,部分降低因缺氧导致的eNOS蛋白表达的增高,提示缺氧及GBE50对上述因子的表达起作用。缺氧及GBE50对PPARγ的mRNA及蛋白水平均无影响,提示缺氧及GBE50不通过PPARγ途径起作用。
Objective To investigate the molecular mechanism of Ginkgo biloba extract GBE50 against hypoxia-induced dysfunction of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were obtained by digestion. After intervention, the interventions were divided into N group (normal control) and NG group (GBE50 25 μg/ml no hypoxia treatment after 4 h), H group (24 h hypoxia) and HG group (GBE50 Hypoxia 24 h after 25 μg/mL intervention for 4 h). The mRNA levels of von Willebrand factor (vWF), endothelin-1 (ET-1), and peroxisome proliferator-activated receptor gamma (PPARγ) were detected semiquantitatively by reverse transcription-polymerase chain reaction. The expression of endothelial nitric oxide synthase (eNOS) and PPARγ protein in each group was determined by Western blotting. Results 1The mRNA expression level of VWF was (0.55±0.05)% in N group and (0.84±0.14)% in H group. The expression of vWF in H group was significantly higher than that in N group (P<0.05). ); HG group (0.58 ± 0.24)%, compared with the H group was significantly lower (P <0.05). The mRNA expression level of 2ET-1 mRNA in N group was (0.14±0.03)%, and that in H group was (0.31±0.03)%. The expression of ET-1 in H group was significantly higher than that in N group (P< 0.05); HG was (0.26±0.02)%, which was significantly lower than that of H group (P<0.05). 3 Expression of eNOS protein: The expression of eNOS protein in group H was significantly higher than that in group N (P<0.05), from (0.30±0.04)% to (0.59±0.08)%; HG group decreased To (0.43 ± 0.08)%, but no significant difference compared with other groups (P values are> 0.05). The mRNA expression level of 4PPARγ was: (0.40±0.02)% in N group, (0.42±0.01)% in H group, and (0.42±0.01)% in HG group. There was no significant difference between the two groups (all P>0.05). 5PPARγ protein expression levels were: (0.10±0.02)% in N group, (0.13±0.05)% in H group, and (0.13+0.04)% in HG group. There was no significant difference between the two groups (all P>0.05). Conclusion Endothelial cell hypoxia can increase the expression of ET-1, vWF and eNOS. Endothelial cells administered GBE50 25 μg/mL before hypoxia can decrease the expression of ET-1 and vWF induced by hypoxia, and partly decrease the expression of ET-1 and vWF. The increase of eNOS protein expression caused by hypoxia suggests that hypoxia and GBE50 play an important role in the expression of these factors. Hypoxia and GBE50 had no effect on the mRNA and protein levels of PPARγ, suggesting that hypoxia and GBE50 do not act through the PPARγ pathway.