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目的:体外实验评价pENW对血管内皮细胞氧化应激损伤和炎性损伤的保护作用,并对其作用机制进行探讨,以提供pENW可开发成为抗血栓药物的依据。方法:原代培养的血管内皮细胞以消化合并刮取法取自牛胸主动脉。H2O2(800μmol.L-1)和LPS(1 mg.L-1)分别用于模拟氧化应激损伤和炎性损伤,经典的G riess Reagent法用于检测一氧化氮的含量。LPS(100μg.L-1)用于刺激血管内皮细胞分泌一氧化氮。在考察pENW对血管内皮细胞的保护作用时,设空白对照组(给予等体积的磷酸盐缓冲溶液)、模型组(分别给予H2O2800μmol.L-1或LPS 1 mg.L-1进行造模)、阳性药给药组(在H2O2造模前给予依达拉奉10-5mol.L-1或在LPS造模前给予阿司匹林10-4mol.L-1)和不同剂量pENW给药组(造模前分别给予pENW 10-4,10-5,10-6和10-7mol.L-1)。在探讨病理条件下pENW对血管内皮细胞释放一氧化氮的调节作用时,设空白对照组(给予等体积的磷酸盐缓冲溶液)、模型组(给予LPS 100μg.L-1)、阳性对照组(造模前给予阿司匹林10-4mol.L-1)和不同剂量pENW给药组(造模前分别给予pENW 10-4,10-5,10-6和10-7mol.L-1),而在进行正常生理条件下pENW调节血管内皮细胞一氧化氮释放作用的研究时,除不设模型组外,其余各组亦不给LPS。结果:pENW能剂量依赖性地减轻H2O2和LPS引起的血管内皮细胞损伤。在pENW10-4mol.L-1+H2O2和pENW 10-4mol.L-1+LPS组,细胞存活率分别升高至(97.6±40.4)%和(64.7±8.0)%;10-4mol.L-1的pENW使血管内皮细胞在正常生理条件下合成的一氧化氮由空白组的(15.50±2.82)升高至(48.68±10.81)μmol.L-1(P<0.01);但pENW能抑制LPS(100μg.L-1)引起的一氧化氮大量升高,且抑制作用呈剂量依赖性。结论:pENW对血管内皮细胞损伤具有保护作用,其作用与调节内皮细胞一氧化氮的释放有关。
OBJECTIVE: To evaluate the protective effect of pENW on oxidative stress injury and inflammatory injury of vascular endothelial cells in vitro and to explore its mechanism of action in order to provide a basis for the development of pENW as an antithrombotic drug. Methods: Primary cultured vascular endothelial cells were harvested from the bovine thoracic aorta by digestion and scraping. H2O2 (800 μmol.L-1) and LPS (1 mg.L-1) were used to simulate oxidative stress injury and inflammatory injury, respectively. The classical Griess Reagent method was used to detect the content of nitric oxide. LPS (100 μg.L-1) was used to stimulate vascular endothelial cells to secrete nitric oxide. When examining the protective effect of pENW on vascular endothelial cells, a blank control group (with an equal volume of phosphate buffer solution) and a model group (H2O2 800 μmol.L-1 or LPS 1 mg.L-1, respectively) were used for modeling. Positive drug administration group (administration of edaravone 10-5 mol.L-1 before H2O2 injection or aspirin 10-4 mol.L-1 before LPS injection) and different doses of pENW administration group (before modeling pENW 10-4, 10-5, 10-6 and 10-7 mol.L-1 were given respectively. To investigate the effect of pENW on the release of nitric oxide from vascular endothelial cells under pathological conditions, a blank control group (administer an equal volume of phosphate buffer solution), a model group (LPS 100 μg.L-1), and a positive control group ( Aspirin 10-4 mol.L-1 and different doses of pENW were administered before modeling (pENW 10-4, 10-5, 10-6, and 10-7 mol.L-1 were given before modeling). Under normal physiological conditions, pENW regulates the release of nitric oxide from vascular endothelial cells. In addition to the model group, LPS was not administered to other groups. RESULTS: pENW dose-dependently reduced the damage of vascular endothelial cells induced by H2O2 and LPS. In the pENW10-4mol.L-1+H2O2 and pENW 10-4mol.L-1+LPS groups, cell viability increased to (97.6±40.4)% and (64.7±8.0)%, respectively; 10-4 mol.L- 1 of pENW increased the production of nitric oxide in vascular endothelial cells under normal physiological conditions from (15.50±2.82) to (48.68±10.81) μmol.L-1 (P<0.01); however, pENW inhibited LPS. (100μg.L-1) caused a large increase in nitric oxide, and the inhibition was dose-dependent. Conclusion: pENW has a protective effect on vascular endothelial cell injury, and its action is related to regulating the release of nitric oxide from endothelial cells.