Biological effects of ultrashort electric pulses in a neuroblastoma cell line: the energy density role

Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz–1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.尽管目前已有大量关于电磁场（electromagnetic field, EMF）暴露的生物学效应的研究成果，但此类场与生物体间的相互作用机制仍存在争议。极低频（extremely low frequency, ELF）磁场可调控氧化还原稳态，我们此前的研究显示，持续24小时暴露于50 Hz–1 mT的极低频磁场，可产生促氧化效应，并影响SH-SY5Y细胞的表观基因组：通过其启动子的高甲基化降低miR-34b/c的表达水平。本研究旨在探究，在50 Hz、幅值为1 mT的磁场中暴露24小时的条件下，沉积能量密度（deposited energy density, ED）在诱导上述效应过程中所发挥的作用。为此，我们向SH-SY5Y细胞施加微秒至纳秒级时长的超短电脉冲，其沉积能量密度与此前的磁场暴露一致；此外，我们还探索了更高沉积能量密度的作用效果。我们开展了四项分析：①基因与microRNA表达水平；②细胞形态；③活性氧（reactive oxygen species, ROS）生成量；④细胞凋亡情况。结果发现，在极低沉积能量密度水平下，egr-1与c-fos的表达出现显著变化，但未观察到活性氧生成量、miR-34b/c表达水平的改变，也未出现细胞凋亡相关指标。为此，我们进一步探究了沉积能量密度递增的超短电脉冲对egr-1与c-fos表达的影响。当使用沉积能量密度更高的脉冲时，可引发细胞电穿孔及其他形态学变化（如细胞融合），egr-1与c-fos的表达变化也更为显著，但同样未观察到活性氧生成量、miR-34b/c表达水平的改变，亦未诱导细胞凋亡。上述结果表明，此前从未被研究过的极低水平电刺激即可引发转录水平的变化，同时证实了生物医学应用中所使用的电穿孔脉冲的安全性，并为该技术的进一步治疗应用开辟了可能。