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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 investigate the role of voltage-dependent potassium channel (Kv) in the cytotoxicity of vascular endothelial cells induced by nanosilica (SiO2) to verify the relationship between potassium outflow and the inflammatory reaction induced by nano-SiO2. Methods Human umbilical vein endothelial cells (HUVECs) were cultured as negative control group. 20μg / ml nano-SiO2 was added as a positive control group, and 20μg / ml nano-SiO2 was added with different dosages of Kv channel blockers (TEA-Cl) or 4-aminopyridine (4-AP) were used as the experimental group. The cell viability, the activity of LDH, the level of tumor necrosis factor (TNF- -6) release amount. Whole cell patch clamp electrophysiological determination of normal HUVECs and different concentration of nano-SiO2 Kv current changes to verify the effect of nano-SiO2 on vascular endothelial cells Kv. Results Compared with the negative control group (normal cell group), nano-SiO2 (positive control group) resulted in a significant decrease of endothelial cell viability and LDH activity, TNF-α and IL-6 release. Compared with the positive control group, the cell viability was significantly increased by the addition of potassium channel blocker TEA-Cl or 4-AP, but the high dose blocker group decreased. Potassium channel blockers also led to a decrease in LDH viability but even reached positive control levels at the highest dose. Only high-dose TEA-Cl significantly reduced the release of TNF-α, whereas the release of TNF-α from low to high doses of 4-AP gradually decreased, showing a dose-response relationship. Both TEA-Cl and 4-AP significantly decreased the release of IL-6 by nano-SiO2. Patch electrophysiological experiments, Kv current endothelial cells showed delayed rectification, 4-AP on the current inhibition. Compared with the negative control group (normal cell group), nano-SiO2 resulted in a significant increase of outward potassium current, a shift of the activation curve to the left and a decrease of the slope factor (P <0.05), indicating that the Kv activity was increased and the channel opening rate was significantly increased. Conclusions Nano-SiO2 can cause cellular inflammatory reaction. The increase of potassium efflux induced by Kv opening may play an important role in activating the cells and activating the early signaling of inflammasome.