Kv1.5蛋白在内毒素致血管内皮细胞损伤中的作用机制

Effect of Kv1.5 on Edotoxin-induced Endothelial Cell Injury

目的研究Kv1.5蛋白在内毒素致血管内皮细胞损伤中的作用机制。方法体外培养人脐静脉内皮细胞(HUVECs),给予脂多糖(LPS)刺激建立脓毒症模型,并分为:空白对照组、LPS组、LPS+Kv1.5蛋白通道特异性阻滞剂MT1(250nmol/L)组及LPS+MT2(500nmol/L)组,DAPI染色免疫荧光观察各组HUVECs凋亡情况,Western blot检测Caspase-3及Bcl-2蛋白的表达,RT-PCR检测Caspase-3及Bcl-2基因的表达。结果 DAPI染色免疫荧光显示,LPS组内皮细胞核固缩、凋亡小体形成,凋亡小体形成率由空白对照组的(4.2±0.7)%增加至(26.7±0.8)%(P<0.01),而250nmol/L、500nmol/L MT预处理后,凋亡小体形成率从LPS组(26.7±0.8)%分别下降至(12.4±1.0)%、(8.5±0.9)%(均P<0.01);Western blot检测结果显示,LPS组较空白对照组Bcl-2蛋白表达减少、Caspase-3蛋白表达增加(均P<0.01),给予500nmol/L MT预处理后,与LPS组比较,Bcl-2蛋白表达上调、Caspase-3蛋白表达下调(均P<0.01);RT-PCR检测结果显示,LPS组较空白对照组Bcl-2mRNA表达下调、Caspase-3mRNA表达上调(均P<0.01),给予500nmol/L MT预处理后,与LPS组比较,Bcl-2mRNA表达上调、Caspase-3mRNA表达下调(均P<0.01)。结论Kv1.5介导脓毒症相关内皮细胞损伤可能与线粒体凋亡途径有关。

Objective To study mechanism by which Kv1.5 protein promoted edotoxin-induced endothelial cell injury.Methods Human umbilical vein endothelial cells（HUVECs）were cultured in vitro and LPS was used to establish sepsis model.The cells were randomly divided into four goups：blank control group,LPS group,LPS＋mephetyl tetrazole（MT）1（250nmol/L）group and LPS＋ MT2（500nmol/L）group.DAPI staining was used to observe the apoptosis of HUVECs in each group.Western blotting was used to detect expression of Caspase-3and Bcl-2.The mRNA expression of Caspase-3and Bcl-2was detected by RT-PCR.Results DAPI staining showed that the rate of apoptotic body formation in LPS group increased from（4.2±0.7）%（blank control group）to（26.7±0.8）%（P0.01）,and after pretreatment with 250nmol/L,and 500nmol/L MT,the rate of apoptotic body formation decreased from（26.7±0.8）%to（12.4±1.0）% and（8.5±0.9）%,respectively（both P0.01）.Compared with control,the expression of Bcl-2protein was significantly decreased and Caspase-3in LPS group was increased（both P0.01）.Compared with LPS group,after pretreatment with 500nmol/L MT,the expression of Bcl-2protein was significanlty increased and Caspase-3was decreased（both P0.01）.The expression of Bcl-2 mRNA in LPS group was down-regulated and Caspase-3mRNA was up-regulated,as compared with control group（both P0.01）.Compared with LPS group,after pretreatment with 500nmol/L MT,the expression of Caspase-3mRNA was down-regulated and Bcl-2mRNA was up-regulated detected by RT-PCR（both P0.01）.Conclusion Kv1.5may mediate LPS-related HUVECs damage via the mitochondrial-ROS-vascular endothelial cell pathway.