Kv1.5蛋白对内毒素致血管内皮细胞氧化应激损伤的影响

Effects of Kv1.5 Protein on LPS-induced Oxidative Stress Injury in Endothelial Cells

目的研究Kv1.5蛋白对内毒素(LPS)所致血管内皮细胞氧化应激损伤的影响。方法体外培养人脐静脉内皮细胞(HUVECs),给予5μg/mL LPS刺激建立脓毒症细胞模型,并分为:空白对照组、LPS组、LPS+MT1组(以Kv1.5特异性通道阻滞剂MT 250nmol/L预处理30min)及LPS+MT2组(MT浓度为500nmol/L)。采用MTT法检测内皮细胞存活率;ELISA法检测细胞上清液中内皮细胞E-选择素(E-selectin)、细胞间粘附分子(ICAM-1)浓度变化;激光共聚焦显微镜观察内皮细胞内ROS水平;比色法检测脂质过氧化物丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性。结果与空白对照组比较,LPS组内皮细胞存活率明显下降[(32.54±1.87)%vs.(98.64±0.92)%,P<0.01],而LPS+MT1组及LPS+MT2组内皮细胞存活率分别为(77.39±1.15)%、(93.22±1.15)%,均较LPS组上升(均P<0.01);LPS组内皮细胞E-selectin、ICAM-1分泌增加(均P<0.01),予以250、500nmol/L MT预处理后,E-selectin、ICAM-1分泌减少(均P<0.01);激光共聚焦显微镜观察内皮细胞内ROS水平发现,LPS组细胞内ROS水平升高为空白对照组的(3.39±0.42)倍(P<0.05),予以250、500nmol/L MT预处理后,ROS水平分别为空白对照组的(1.88±0.36)倍、(1.46±0.47)倍,与LPS组比较,差异有统计学意义(均P<0.05);且LPS组较空白对照组MDA水平上升、SOD活性降低(均P<0.01),而MT预处理可降低MDA含量、增加SOD活性(均P<0.01)。结论 Kv1.5蛋白与内毒素诱导的血管内皮细胞损伤密切相关;MT通过阻断Kv1.5通道可减轻内皮细胞脂质过氧化损伤。

Objective To investigate effects of Kv1.5 protein on LPS-induced peroxidation injury in endothelial cells.Methods Human umbilical vein endothelial cells（HUVECs）were cultured in vitro.Sepsis model was established by stimulating HUVECs with LPS（5μ/mL）.The experiment was divided into four groups：Control group,LPS group,LPS＋ MT1 group（250 nmol/L of MT was used for pretreatment for 30 min to block Kv1.5 channels）and LPS＋MT2（500 nmol/L MT）.Then the survival rate of endothelial cells was detected by MTT assay.ELISA was used to detect the changes of E-selectin and ICAM-1 in the supernatant,and to measure MDA and SOD activity.Results Compared with the control group,the survival rate of endothelial cells in LPS group was significantly decreased[（32.54±1.87）%vs.（98.64±0.92）%,P〈0.01],while the survival rate of LPS＋MT1 group and LPS＋ MT2 group was（77.39±1.15）% and（93.22±1.15）%,which was significantly higher than that of LPS group（P〈0.01）.At the same time,the secretion of E-selectin and ICAM-1 in endothelial cells of LPS group increased（P〈0.01）,and decreased after pretreatment with 250 and 500 nmol/L MT（P〈0.01）.By observation with laser confocal microscopy,intracellular ROS level in endothelial cells in LPS group were increased to（3.39±0.42）times as much as that of the blank control group（P〈0.05）,and after pretreatment with 250 and 500 nmol/L MT,ROS level was（1.88±0.36）times and（1.46±0.47）times as much as that of the blank control group,respectively,which was significantly different from that of LPS group（P〈0.05）.Compared with the blank control group,MDA level was significantly increased and SOD activity decreased in LPS group（P〈0.01）,while MT pretreatment significantly reduced MDA content and increased SOD activity（P〈0.01）.Conclusion Kv1.5 protein is closely related to LPS-induced injury of vascular endothelial cells.MT can reduce lipid peroxidation injury of endothelial cells by blocking Kv1.5 channel.