11,12-环氧二十碳三烯酸对人脐静脉内皮细胞缺氧/复氧损伤的影响

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。

Effect of 11, 12-epoxyeicosatrienoic acids on hypoxia/reoxygenation injury in the human umbilical vein endothelial cells

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/reoxyge- nation 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% O_ 2 ,5% CO_ 2 and 93% N_ 2 ) for 3 hours, followed by reoxygenation (95% air and 5% CO_ 2 )for 1 hour. The evaluation of the endothelial cells were made by immunohistochemistry. The cell viability was monitored by MTT 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.