蛇毒三肽pENW对血管内皮的保护作用及机制探讨

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。

The Protective Effect of Snake Venom Tripeptide pENW on Endothelial Cells and Its Mechanisms

Objective： To investigate the protective action of tripeptide pENW on vascular endothelial ceils in vitro and to reveal its protective mechanisms, to provide the basis for pENW being developed as a novel antithrombotic agent. Methods： H2 O2 （ 800 μmol· L^ - 1） or LPS （ 1 mg·L^ - 1） was used to emulate the oxidative injury or inflammatory injury to endothelial cells and Griess Reagent was used to measure nitric oxide（NO） content. LPS（ 100 μg·L-1） was used to stimulate NO production from vascular endothelial cells. In order to observe the protective effect of pENW, the test groups were divided into normal control group （ treated with phosphate buffered solution）, model group （ established by the treatment of H2 02 800 μmol· L^ - 1or LPS 1 mg·L^-1）, positive control group （treated with edaravone 10-5 mol·L^-1 or aspirin 10-4 mol·L^-1 before model established） and various concentrations of pENW treated groups （treated with pENW 10^- 4, 10^-5, 10^-6and 10^-7 mol·L^-1 before model established, respectively ）. In order to find out the acting mechanisms of pENW, the test groups were divided into control group （treated with phosphate buffered solution）, model group （treated with LPS 100 μg·L^-1）, positive control group （treated with aspirin 10-4 mol·L^-1 before LPS 100 μg·L^-1 added ） and various concentrations of pENW treated groups （treated with pENW 10^-4, 10^-5, 10^-6 and 10^-7 mol·L^-1 before LPS 100 μg·L^-1 added, respectively）. Results： pENW in a concentration-dependent manner inhibited H202 and LPS induced endothelial injury. The cell viability rates were increased to （97. 6±40. 4） % and （64. 7 ±8. 0） % in pENW 10^-4 mol·L^-1 at the presence of H2O2 or LPS groups, respectively. Under the physiological conditions, NO production in pENW （10^-4 mol·L^-1） group was increased to （48. 68± 10. 81） μmol· L^ - 1, but that of control group was （15.50±2. 82） μmol· L^ - 1 （P 〈0. 01）. pENW inhibited LPS induced magnified production of NO in a concentration-dependent manner. Conclusions： pENW protected endothelial injury, which was related to the modulation on NO production.