MAPK信号通路介导低剪切力诱导的人脐静脉内皮细胞氧化应激性损伤

摘要

目的：观察体外模拟低剪切力对血管内皮细胞产生的氧化应激性损伤并探讨其可能机制。方法用平行板流动腔装置于体外模拟低剪切力作用于体外培养的人脐静脉内皮细胞60 min后，用MitoSOX检测线粒体内活性氧簇（ROS）含量，用TUNEL及DAPI染色标记凋亡的细胞。低剪切力作用不同时间后，用Western blotting检测内皮型一氧化氮合酶（eNOS），P38，细胞外调节蛋白激酶（ERK），c-Jun及其磷酸化蛋白的表达水平，并分别用P38，ERK和c-Jun的抑制剂作用细胞后检查eNOS的负性调节位点Thr495的表达。结果体外模拟低剪切力可以明显诱导血管内皮细胞的氧化应激性损伤伤，呈时间依赖性激活eNOS、P38、ERK及c-Jun蛋白的磷酸化，但对总蛋白的合成无影响。ERK抑制剂可有效抑制eNOS-Thr495的活化并逆转LSS诱导的细胞内超氧化物歧化酶（SOD）减少。结论体外模拟低剪切力诱导的细胞氧化应激性损伤与促分裂素原活化蛋白激酶（MAPK）信号通路活化有关，ERK/eNOS的活化参与低剪切力调节的氧化应激。%Objective Atherosclerotic lesions occur preferentially in the arterial branches, bifurcations and curvatures where shear stress is low. We aimed to study the possible mechanisms involved in low shear stress (LSS)-induced oxidative damage in vascular endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) exposed for 60 min to simulated LSS using a parallel-plate flow chamber were examined for intracellular reactive oxygen species (ROS) and cell apoptosis with chemiluminescence assay and TUNEL staining, respectively. Western blotting was used to determine the levels of endothelial nitric oxide synthase (eNOS), P38, extracellular signal-regulate kinase (ERK) and c-Jun as well as their phosphorylation in cells with LSS exposure for different time lengths. To investigate the signaling pathway involved in LSS-induced oxidative damage, the cells were treated with P38, ERK and c-Jun inhibitors and examined for the expression of eNOS-Thr495 that negatively regulated eNOS. Results Exposure to LSS for 1 h resulted in markedly increased ROS accumulation and apoptosis in HUVECs. LSS exposure time-dependently enhanced the phosphorylation of eNOS, P38, ERK and c-Jun but did not significantly affect their total protein expressions. Inhibition of ERK with PD98059 deactivated eNOS-Thr495 and restored super oxide dismutase (SOD) activity, while inhibition of either p38 with SB202190 or c-Jun with SP600125 did no produce such effects. Conclusion LSS-induced oxidative damage is partly due to activated mitogen-activated protein kinases (MAPK), among which ERK contributes to decreased NO release in endothelial cells.