摘要
目的通过观察11,12-环氧二十碳三烯酸(11,12-EET)对缺氧/复氧人脐静脉内皮细胞损伤程度的影响,了解EET血管保护的可能途径,并初步探讨其作用机制。方法采用原代培养人脐静脉内皮细胞,随机分为对照组、缺氧/复氧组、11,12-EET对照组、11,12-EET缺氧/复氧组、细胞外信号调节的蛋白激酶(ERK1/2)抑制组和一氧化氮合酶抑制组。通过向培养瓶内通入混合气体(2%O2,5%CO2,93%N2)3h,复氧1h复制缺氧/复氧损伤模型。采用MTT法检测细胞存活率,比色法检测培养液中乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)及丙二醛(MDA)的变化,Westernblot方法检测内皮型一氧化氮合酶(eNOS)和ERK1/2的表达。结果11,12-EET在常氧条件下可对细胞造成轻度损伤,而在缺氧/复氧条件下能显著提高内皮细胞的存活率,降低LDH的漏出率,提高SOD的活性,降低MDA的含量,并且促进eNOS、磷酸化ERK1/2的表达。结论11,12-EET具有拮抗内皮细胞缺氧/复氧损伤作用,这与其提高缺氧/复氧内皮细胞SOD活性、清除氧自由基、减少缺氧/复氧对eNOS及磷酸化ERK1/2表达的抑制有关。
Objective To investigate the effects of 11, 12-epoxyeicosatrienoic acids （11, 12-EET） on the degree of hypoxia/reoxygenation injury in human umbilical vein endothelial cells （ HUVECs）, and reveal the possible pathway of EET on protection. Methods Primary cultured HUVECs were randomly divided into control group, hypoxia/reoxygenation group, 11, 12-EET control group, 11, 12- EET hypoxia/reoxygenation group, inhibition of extracellular signal-regulated kinase （ ERK1/2 ） group, and inhibition of nitric oxide synthase （NOS） group. Hypoxia/reoxygenation injury model in HUVECs was established by exposure to hypoxia （2% O2, 5% CO2 and 93% N2） for 3 hours, followed by reoxygenation （95% air and 5% CO2） for 1 hour. The evaluation of the endothelial cells were made by immunohistochemistry. The cell viability was monitored by MTF assay. Colorimetry method was used to assay the lactate dehydrogenase （LDH） , malondialdehyde （MDA） and activity of superoxide dismutase （SOD） in culture medium. Western blot was used to detect the expressions of endothelial nitric oxide synthase （eNOS） and phosphorylated ERK1/2 in HUVECs. Results 11, 12-EET caused minor injury in normal oxygen incubated HUVECs; however, in hypoxia/reoxygenation HUVECs, it raised the cell viability markedly, decreased the LDH release and MDA content, and increased the activity of SOD and the expressions of eNOS and phosphorylated ERK1/2. Conclusions 11, 12-EET may prevent against endothelial cell hypoxia/reoxygenation injury. The mechanism may be related to the increased activity of SOD, elimination of oxygen-derived free radicals, and reduction of eNOS and phosphorylated ERK1/2 lesion caused by hypoxia/reoxygenation.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2006年第6期803-807,I0007,共6页
Acta Academiae Medicinae Sinicae
基金
北京自然科学基金(7062007)~~
