Role Of Voltage-dependent Outward K~+Channels In The Nanometer Silica-induced Toxic Effect In Human Vascular Endothelial Cells

With the development of nanomaterial, the exposure to nanometer particles has beenincreasing. It was common in the production and use of the material. In addtion, suchparticles were produced in the thermal processing of industries and environmental pollution,which made workers and general populations expose to them. Certainly its potential healtheffect becomes an important research area. It had been proved that nanoparticles could enterinto the blood system directly and distribute the whole body, leading to the cardiovasculardisease. It was known that the inflammasome （a protein complex） mediated inflammatoryreactions, and that intracellular K⁺outflow, namely reduced intracellular K⁺concentration,was one of the preconditions for inflammasome activation. Therefore, in this study, we willexplore the issues in order to provide some scientific basis for understanding of K⁺channelsin nanoparticle toxic effect.Objective: To understand the role of voltage-dependent outward potassium channels （Kv） onthe nanometer silica-induced toxic effect in HUVECs, and to verify the relationship betweenpotassium efflux and inflammatory response.Method: Human umbilical vein endothelial cells （HUVECs） were incubated in vitro as a testsystem. Cells cultured normally were used as the negative control group.20g/ml ofnanometer silica （nano-SiO₂） added to the system was taken as the positive control group. Onthe basis of nano-SiO₂particles, different doses of K⁺channel blockers margatoxin （MGTX,final concentration of0.1,1,10,100nM） were added as the blocker group to observe the cellresponse under the condition of channel blocked or not. Cell survival rate, lactatedehydrogenase （LDH） leakage rate, tumor necrosis factor （TNF-α） and interleukin （IL-6）production in culture supernatants were determined as parameters of the response. By usingof whole cell patch clamp, outward potassium channels （Kv） were measured in normalHUVECs, then verified by adding K⁺channel blockers （TEA,4-AP, MGTX）, and weobtained the median inhibitory dose （ID50） by fitting curves of each blockers. Taking the normal cells as the control group, the maximum current amplitude of Kv was recorded afteradding the nano-SiO₂of different concentration （20g/ml,40g/ml）, and we obtained thelowest effect dose. Meanwhile, the current-voltage curves and channel dynamics wereanalyzed. After adding nano-SiO₂, the outward K⁺channels subtype Kv1.3expression on thecell membrane was preliminarily observed by using immunofluorescence technique.Results: After adding MGTX, the cells survival rate increased along with blocker dose, butthe difference was not statistically significant （P>0.05）. LDH leakage rate at first decreasedthen increased, but was still lower than the positive control group （P<0.05）. Under the effectof MGTX, the TNF-production induced by nano-SiO₂decreased significantly compared tothe positive control group. A significant reduction in IL-6release was caused by addingMGTX of low concentration （0.1nM）. As the analysis after standardization showing, theMGTX’s effect was great, but the effect of4-AP was more significant than that of TEA andMGTX, and4-AP had the best correlation with various inflammatory markers. In theelectrical physiological experiments with patch clamp, the outward potassium current on thecell membrane showed the delayed rectifier and voltage-dependent characteristic, and thecurrent was inhibited of different degree by the TEA,4-AP and MGTX potassium channelblockers. The current was almost entirely blocked by5mM4-AP, and had no change afterwashing.100nM MGTX also could completely block the current, and after washing only asmall part of currents was recovered, which meant that the current recorded was partly Kv1.3current. Compared with the control group, the Kv current maximum amplitude approximatelyincreased by63.9%after added nano-SiO₂particles with20g/ml, but the difference was notstatistically significant （P>0.05）. While the current amplitude increased by120.2%with40g/ml nano-SiO₂, and there was the significant difference （P=0.0385）. Under the differentvoltage stimulation, the outward Kv current gradually increased with statistical significancecompared with the control group （P<0.05）. From the steady-state activation curve fitting, theactivation curve shifted toward to left with the V1/2changing from11.961.17mV to4.251.55mV, and the slope factor k by13.541.16significantly reduced to11.451.49compared with the control group （P<0.05）. In the immunofluorescence experiments, cellsbecame irregular in shape, and the cells’ membranes were ruptured, which showed theapparent cytotoxic changes. But the fluorescence intensity of the intact cell membrane wasstrengthen, which noted that the Kv1.3’s expression had increased.Conclusion: MGTX could inhibit the toxic reaction in human vascular endothelial cellsinduced by nano-SiO₂. Furthermore, the outward K⁺channel blocker4-AP had more significant effect. There are the outward Kv channels in HUVECs, which showed the delayedrectifier characteristics. Nano-SiO₂could induce cells toxic effect, and the activity of theoutward Kv channel was enhanced, the opening rate was heightened and current intensitywas increased. The outward K⁺channel might play an important role in the vascularendothelial cells damage induced by nano-SiO₂, which might serve as an early signal.