外向钾通道在纳米SiO_2致内皮细胞毒性中的作用

Effect of outward potassium channels on nanometer silica induced toxicity in endothelial cells

目的探讨电压依赖性外向钾通道(voltage-dependent potassium channel,Kv)在纳米二氧化硅(SiO2)致血管内皮细胞毒性中的作用,以验证钾外流与纳米SiO2致细胞炎性反应的关系。方法以人脐静脉内皮细胞(HUVECs)正常培养为阴性对照组,单独加入20μg/ml纳米SiO2为阳性对照组,在20μg/ml纳米SiO2基础上添加不同剂量的Kv通道阻断剂氯化四乙胺(TEA-Cl)或4-氨基吡啶(4-AP)为实验组,检测各组细胞存活率、乳酸脱氢酶(LDH)活力、肿瘤坏死因子(TNF-α)以及白介素-6(IL-6)释放量。用全细胞膜片钳电生理测定正常HUVECs及染不同浓度纳米SiO2时Kv电流变化,验证纳米SiO2对血管内皮细胞Kv的影响。结果与阴性对照(正常细胞组)比较,纳米SiO2(阳性对照组)导致内皮细胞存活率明显降低,LDH活力、TNF-α和IL-6释放量明显增加。与阳性对照组比较,加入钾通道阻断剂TEA-Cl或4-AP后,细胞存活率明显升高,但高剂量阻断剂组反而降低。钾通道阻断剂也导致LDH活力降低,但在最高剂量时甚至达到阳性对照水平。只有高剂量TEA-Cl使TNF-α释放量明显减少,而4-AP自低到高剂量使TNF-α释放量逐渐降低,呈剂量-效应关系。TEA-Cl和4-AP均可明显降低纳米SiO2诱导IL-6释放量。膜片钳电生理实验中,内皮细胞Kv电流表现延迟整流特性,4-AP对该电流的抑制作用明显。与阴性对照(正常细胞组)比较,纳米SiO2导致外向钾电流明显增大,激活曲线左移,斜率因子降低(P<0.05),表明Kv活性增加,通道开放速率明显增高。结论纳米SiO2可引起细胞炎性反应,Kv开放引起的钾外流增加在该毒性效应中可能起着活化细胞、激活炎性体的早期信号作用。

Objective To study the effect of outward potassium channels on nanometer silica（nano-SiO2）induced vascular endothelial cell damage.Methods Human umbilical vein endothelial cells（HUVECs）were co-cultured by tetraethyl ammonium chloride（TEA-Cl）or 4-aminopyridine（4-AP）together with 20 μg/ml nano-SiO2 at the same time for 24 h.Taking the nano-SiO2 separately treated one as the control group,the cell survival rate,lactate dehydrogenase（LDH）activity,tumor necrosis factor（TNF-α）and interleukin（IL-6）production in culture supernatants were detected.Potassium channels were measured by whole cell patch clamp to verify what kind of channels were involved in the cell response to nano-SiO2.Results Nano-SiO2（positive control group）caused the survival rate of endothelial cells significantly lowered in comparison to the negative control and a significant increase in the LDH activity,TNF-α production and IL-6.Compared to the positive control group,cell survival rate increased significantly after adding potassiumion channel blockers of TEA-Cl and 4-AP,however,it reduced again in the high-dose blocker group.Channel blockers also resulted in LDH activity decrease at first and then increase at the high dose group even reaching to the level of positive control.Only high doses of TEA-Cl significantly reduced the TNF-α production,while 4-AP led to its release decreased gradually with a dose-dependent relationship.A significant reduction in IL-6 release was caused by adding both TEA-Cl and 4-AP.As the analysis after standardization showed that the effect of 4-AP was more significant than that of TEA-Cl.In the electrical physiological experiments with patch clamp,the outward potassium current showed a delayed rectifier characteristic,and the current inhibition by 4-AP was more significant.Compared with the control group,the outward K＋ current significantly increased,activation curve shifted toward to left and the slope factor became significant lower after adding nano-SiO2（P0.05）,indicating an increased activity and opening rate of the outward potassium channels.Conclusions The increase in potassium outflow caused by more frequent opening of the outward potassium channel might play a role in activating cells and inflammatory reaction as an early signal in the vascular endothelial cell damage by nano-SiO2.