| Background and Purpose: With the increasing development of technology and people’s living standards, personal mobile communication devices are widely used, radio frequency electromagnetic field radiation(RF-EMF) may bring all sorts of undesirable effects and public security risks, which is attracting more and more attention. Research shows that the nervous system is an important target in electromagnetic radiation exposure, and the imbalanced neuronal Ca2+ regulation is associated with a variety of nervous system damage. Therefore researching the relationship between radio frequency electromagnetic field radiation and calcium balance has become a focused direction of this research area in recent years. There are many reports about radio frequency electromagnetic radiation resulting in intracellular calcium regulation imbalance, but the complete mechanism of radio frequency electromagnetic radiation-induced calcium imbalance is still unclear. Researches on the mechanism of RF radiation induced intracellular calcium imbalance studied through the calcium channel of neuronal cell membrane are rarely reported. Therefore, this study will explore the influence of RF-EMF on neuronal calcium balance and the mechanism through the transient receptor potential C(TRPC) channels and the voltage-gated channels(VGCC) to provide more clues and references for studying the mechanism of electromagnetic radiation causing nerve damage.Methods: ① Primary cultured cortical neurons(PCCNs) and groups: primary cortical neurons are from 0-3 day old neonatal rat brain, culture 8 days after trypsin digestion for experiments. Experimental groups: sham exposure group, electromagnetic radiation exposure group and sham exposure + SKF-96365 treated group, electromagnetic radiation exposure + SKF-96365 treated group. SKF-96365 is the TRPC channel blockers.② Neurons electromagnetic radiation exposure model: Exposure system is s Xc-1800, the frequency is 1800 MHz, the specific absorption rate is 4 W / kg, 5 min open, 10 min close, continuous exposure.③ After RF-EMF exposure, neuronal death and vitality rate detection: the LDH and CCK-8 kit were used to detect 24 h PCCNs mortality and vitality rate at the end and after the end of exposure.④ Observation of the effects of RF-EMF exposure on neurite: β-III tubulin(Tuj-1) immunofluorescence staining and laser confocal microscopy testing the neurite growth conditions after RF-EMF exposure, and by adding TRPC channel blocker SKF-96365 as pretreatment before exposure, observe and compare the influence of RF-EMF exposure on neurite growth after blocking the TRPC channel.⑤ TRPC1, TRPC3 protein expression detection: detecting the protein expression levels of PCCNs after RF-EMF exposure in 12, 24, 48 h in TRPC1, TRPC3 channel with the Western blot. ⑥ Detection of [Ca2+]i changes: Fluo-4 AM staining, confocal microscopy measuring RF-EMF after exposure of 24 h, observe the changes of PCCNs store-operated calcium entry(SOCE) and K+ induced calcium entry.⑦ Whole-cell patch-clamp technique VGCC current detection: at the end of RF-EMF exposure of 24 h, apply different voltage and activate and open the channels, recording PCCNs VGCC current with whole cell mode.Result: ① At end and after the RF-EMF exposure of 24 h, the irradiated group PCCNs mortality and vitality test results and the corresponding control groups showed no significant difference(P> 0.05). It indicates the electromagnetic radiation dose not cause significant impact on neuronal survival and vitality in the experiment, which can be used as follow-up experimental studies of electromagnetic exposure model.② RF-EMF exposed 24 h, PCCNs in TRPC1 TRPC3 protein expression were decreased by 24% and 23%, and compared with the control group it was statistically significant( P <0.05). This indicates protein expression of TRPC channel is inhibited, which may cause cha nnel dysfunction.③ SKF-96365 treatment can leave exposed group PCCNs SOCE drop 24%, and RF-EMF exposed 24 h, PCCNs of SOCE drop 54%, compared with the control group, the differences of the two were both statistically significant(P <0.05). RF-EMF exposure + SKF-96365 combined treatment group’s SOCE decreased by 20%, the difference of the change with the simple RF-EMF exposure group was not statistically significant(P> 0.05). This indicates adding SKF-96365 did not effectively enhance effects of RF-EMF exposure caused SOCE decline, which shows RF-EMF causes indirect TRPC channels mediated calcium influx pathway damaged participating RF-EMF suppression of neuronal SOCE.④ RF-EMF exposing 48 h can cause PCCNs projections total length, number of primary projections and the number of branches declined by 20%, 12%, 31%, compared with the sham-exposed group being statistically significant(P <0.05). Sham exposed SKF-96365 treated group, the total length of the projection, the number of primary projections, the number of branches decreased by 10%, 12%, 24%, and shame-exposed group were statistically significant(P <0.05), while exposure SKF- 96365 combined treatment group, the total length of the projection, the projection number, change the number of branches and exposed groups showed no significant difference(P> 0.05). This indicates that RF-EMF exposure causes PCCNs TRPC channel damage by calcium imbalance, which can result i n inhibition of neurite growth.⑤ RF-EMF exposed for 24 h, different high threshold voltage induced activation VGCC current size and current density compared with the sham exposure group, the difference was not significant(P> 0.05). This indicates under this experimental model RF-EMF exposure is of no effect on activation of PCCNs VGCC.⑥ RF-EMF exposed for 24 h, K+ current induced by increased intracellular calcium PCCNs, compared with sham-exposed group it decreased by 26%, with a significant difference(P<0.05). After addition of TG, K+ induced neuron’s calcium influx increased and compared with the sham exposure group there is no significant difference(P> 0.05). This suggests that RF-EMF exposure had no effect on the function of VGCC mediated calcium influx, but affected the regulation of intracellular calcium flow path.Conclusion: In this experimental model of electromagnetic radiation exposure, RF-EMF continuously exposes for 24 h, resulting in TRPC1, TRPC3 protein decreased, leading to neuronal SOCE reduce and neuronal calcium dynamic characteristics change; in this experimental model it was not observed that RF-EMF’s 24 h exposure having impact on the high threshold activation VGCC current characteristics and the VGCC mediated calcium influx function, but it will affect the regulation of calcium influx by way of intracellular calcium causing change of the K+ induced neurons’ dynamic properties. We found that in the study of RF-EMF’s effect on the TRPC and VGCC Pathway, the changes were observed in the calcium PCCNs dynamic characteristics, although caused by different mechanisms, this may be one of the impacting mechanisms that causes the slowed growth of primary cortical neuronal. |
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