Effects Of 50 Hz Electromagnetic Fields Exposure On The Proliferation And Differentiation Of Embryonic Neural Stem Cells And Its Underlying Mechanisms

Backgrounds:50 Hz electromagnetic fields(50 Hz-EMF), belonging to the extremely low frequency electromagnetic fields(ELF-EMF), is ubiquitous in the environment. It has raised public concerns about the pontential effects on human health and its biological effects are still controversial. The central nervous system is one of the most sensitive target organs/systems responding to EMF exposure. The embryonic brain development, a critical stage during the development and maturation of central nervous system, is known to be much more sensitive to EMF exposure. The process of embryonic neural stem cells(e NSCs), including its self-renew, directional differentiation, migration and so on, directly affects the normal embryonic brain development. Previous studies have found that 50 Hz-EMF exposure could promote the proliferation and differentiation of NSCs, enhance neurogenesis, and even result in the improvement in learning and memory in adult mice. However, the effects of 50 Hz-EMF exposure on embryonic brain development remain controversial and its mechanisms are not clear. Therefore, in the present study, in order to establish the cell model in vitro, the e NSCs were successfully cultured. Then the effects of 50 Hz-EMF exposure on the proliferation and differentiation of e NSCs were investigated, and the mechanism is explored on e NSCs by detecting the TRPC channels and the transcription factors of basic helix-loop-helix(b HLH) family directly regulating the proliferation and differentiation of e NSCs.Methods:(1) e NSC were derived from the fetal telencephalon at the embryonic day 13.5(E13.5) of mice. The cultured e NSCs were exposed to 50 Hz-EMF with two kinds of exposure modes in recent studies. The the specific exposure conditions were as follows: i) Mode 1: 0.5, 1 and 2 m T; 5 min on/10 min off; 1 day, 2 days and 3 days; ii) Mode 2: 1 m T; 4 hours per day(4 h/d); 1 day, 2 days, and 3 days.(2) The proliferation, maintenance, differentiation, apoptosis of e NSCs and the neurite outgrowth of e NSCs-derived neurons were measured by CCK-8 assay, Ed U incorporation, neurosphere formation assay, flow cytometer analysis, real time PCR, TUNEL, and immunofluorescence cytochemistry, respectively.(3) To further verify the influence of 50 Hz-EMF exposure on the cell behavior of e NSCs at the molecular levels, the expression of b HLH transcription factors, which directly regulate the cell behavior of e NSCs, were measured by real time PCR, Western blot and immunofluorescence cytochemistry.(4) The expression of transient receptor potential channels(TRPCs) were detected by real time PCR and Western blot in differentiating e NSCs. The concentration of intracellular calcium was measured via Fura 2-AM(a calcium fluorescent indicator).(5) To explore the molecular mechanism of TRPC1 in mediating the effects of 50 Hz-EMF exposure on the neuronal differentiation of e NSCs, the TRPC1 was down-regulated through the TRPC1-si RNA treatment and then the m RNA expression of b HLH transcription factors, the concentration of intracellular calcium, the ratio of neuronal differentiation and the neurite outgrowth of e NSCs-derived neurons were measured.(6) The data analysis was carried out with SPSS 20.0(a stasitical software).Results:(1) Under the condition of exposure mode 1(0.5-2 m T, 5 min on/10 min off), the cell viability, the numbers of Ed U positive cells, the distribution of cell cycle, the m RNA expression of cell cycle-related genes(P53, P21 and GADD45), the diameters of neurospheres were not changed in proliferating e NSCs. The number of either differentiated neurons(Tuj1 positive cells) or astrocytes(GFAP positive cells) was not altered in differentiating e NSC. However, we observed the alterations in the transcript levels of differentiation-related genes(Down-regulation: Sox2. Up-regulation: Math1, Math3, Ngn1 and Tuj1).(2) Under the condition of exposure mode 2(1 m T, 4 h/d), the cell viability, the numbers of Ed U positive cells, the numbers of both primary and secondary neurospheres were significantly increased after 50 Hz-EMF exposurefor 3 d. T he m RNA expression of genes regulating e NSCs proliferation(Sox2, Hes1 and Hes5) was also up-regulated. When the cells were induced to differentiation, we found that both the numbers of e NSCs derived neurons(Tuj1 positive cells) and the Tuj1 m RNA expression were increased, while neither the numbers of e NSCs derived astrocytes(GFAP positive cells) nor the GFAP m RNA expression were not changed. Meanwhile, the m RNA and protein expression of proneural genes(Neuro D and Ngn1) were markedly increased after the 3 d50 H z-EMF exposure. After 50 Hz-EMF exposure for 2 d a nd 3 d, t he expression of TRPC1 m RNA and protein in differentiating e NSCs were up-regulated. The concentration of intracellular calcium was also increased after the 3 d 50 Hz-EMF exposure. After down-regulating the protein expression of TRPC1 by TRPC1-si RNA treatment, the cells were exposed to 50 Hz-EMF for 3 d, and we found that TRPC1-si RNA treatment prevent the 50 Hz-EMF induced promotive effects on the neuronal differentiation from e NSCs, the neurite outgrowth of differentiated neurons, the m RNA expression of Neuro D and Ngn1 and the concentration of intracellular calcium.Conclusion:(1) The effects of 50 H z-EMF exposure on e NSCs depend on the specific exposure mode. Intermittent exposure(0.5-2 m T, 5 min on/10 min off) does not change e NSCs proliferation and directional differentiation. Relatively continuous exposure(1 m T, 4 h/d) enhances e NSCs proliferation, promotes neuronal differentiation and the neurite outgrowth of e NSCs derived neurons.(2) 50 Hz-EMF exposure(1 m T, 4 h/d) increases the expression of transcription factors in b HLH family, which can directly regulate the proliferation and differentiation of e NSCs.(3) 50 Hz-EMF exposure(1 m T, 4 h/d) increases the expression of TRPC1 and the concentration of intracellular calcium.(4) D o wn-regulation of the expression of TRPC1 could prevent the neuronal differentiation of e NSCs induced by 50 Hz-EMF, suggesting that 50 Hz-EMF exposure promotes the neuronal differentiation of e NSCs by up-regulating TRPC1 expression.