左旋氨氯地平对血管内皮细胞氧化应激损伤的影响

Effect of L- amlodipine on H_2O_2- induced oxidattive stress in vascular endothelial cells

目的研究左旋氨氯地平对过氧化氢(H_2O_2)诱导人血管内皮细胞株EA.hy926氧化应激损伤的影响。方法实验随机分为5组,正常组,模型组(300μmol·L^(-1)H_2O_2)及左旋氨氯地平低、中、高剂量组(0.5,1,2μmol·L^(-1))。噻唑蓝法检测各组细胞活力;二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)探针检测各组细胞内活性氧水平;分光光度法检测各组丙二醛及超氧化物歧化酶含量;Annexin V-FITC流式双染检测各组细胞凋亡率;分光光度法检测各组天冬氨酸蛋白水解酶3(Caspase 3)活性;免疫印迹技术分析核因子-κB(NF-κB)通路的激活状况。结果与正常组比较,模型组中EA.hy926细胞活力下降(P<0.01),活性氧及丙二醛含量上升(P<0.01),超氧化物歧化酶含量下降(P<0.01),Caspase 3活性上升(P<0.01),NF-κB p65磷酸化水平降低(P<0.01);与模型组比较,0.5,1,2μmol·L^(-1)左旋氨氯地平能显著细胞活力(P<0.05),提高超氧化物歧化酶含量(P<0.05),降低活性氧及丙二醛含量(P<0.05),抑制Caspase 3活性及NF-κB p65磷酸活化水平(P<0.05)。结论左旋氨氯地平可抵抗H_2O_2诱导的人血管内皮细胞株EA.hy926细胞氧化应激作用,是通过抑制活性氧等物质的产生及细胞凋亡实现的。

Objective To explore the effect of L- amlodipine on hydrogen peroxide（ H2O2）- induced oxidattive stress in vascular endothelial cells EA. hy926. Methods The case was randomly divided into 5 groups,the normal group,model group（ 300 μmol·L^-1H2O2） and L-amlodipine groups in the low, middle and high dose（ 0. 5, 1, 2μmol·L^-1）. The viability of EA. hy926 cell was measured by MTT assay. Intracellular reactive oxygen species level was detected by DCFH- DA fluorescence probe. The content of malondialdehyde and superoxide dismutase was determined by kits. Annexin V- FITC flow cytometry dual staining was used to detect cell apoptosis. The activity of cysteinyl aspartate specific proteinase 3（ Caspase 3） was measured by spectrophotometry. The activation of nuclear factor kappa B（ NF- κB）signaling pathway was assayed by western blot. Results Compared with normal group, the cell viability in the model group was lower（ P〈0. 01）,the levels of malondialdehyde and oxygen species were higher（ P〈0. 01）, the content of superoxide dismutase was lower（ P〈0. 01）,the activity of Caspase 3 was higher（ P〈0. 01）,and thephosphorylation of NF- κB p65 was lower（ P〈0. 01）. Compared with model group,0. 5,1,2 μmol·L^-1of L- amlodipine could inhibit the change,the difference was significant（ P〈0. 05）. Conclusion These results suggested L- amlodipine was resistant to H2O2- induced oxidattive stress in EA. hy926 cell via inhibition of oxgen generation and cell apoptosis.