Effects of Schumann resonance on the proliferation and migration of normal human epidermal keratinocytes and the expression of DEFB1 and SIRT1

When the skin is damaged and its barrier function is disrupted, the proliferation and migration of epidermal keratinocytes are vital for repairing the damaged area. The Schumann resonance at 7.8 Hz has been reported to protect rat cardiomyocytes against oxidative stress and inhibit the proliferation of B16 mouse melanoma cells. However, its effect on the skin is unknown.

atmospheric pollutants such as PM 2.5, antimicrobial peptides produced by epidermal keratinocytes, such as β-defensin (DEFB1) and cathelicidin, also protect the skin against bacteria, viruses, and other pathogens, improving the skin barrier function.When the skin is damaged and its barrier function is disrupted, the proliferation and migration of epidermal keratinocytes are vital for repairing the damaged area, but substances derived from Staphylococcus aureus in the resident skin flora may be aggravating factors for inflammation. 3Newly synthesized sirtuin 1 (SIRT1)-activating factors (piper amide derivatives (E)-3-(2,4-dichlorophenyl)-Nphenylacrylamide, NED416) upregulate SIRT1 expression and promote cell migration in NHEKs and fibroblasts, 4 indicating that SIRT1 may be a repair factor of damaged skin.
In this study, we investigated whether the application of electromagnetic waves at 7.8 Hz, a Schumann resonance, affects the proliferation and migration of NHEKs, as well as its effect on the expression of the antimicrobial peptide gene DEFB1 and SIRT1, which is involved in cell proliferation.

| Cell proliferation assay
One hundred microliters of NHEKs at a concentration of 4 × 10 4 cells/mL was seeded into individual wells of a 96-well plate (n = 6).
After culturing for 24, 48, or 72 h, the plates were placed on top of a 7.8-Hz ultra-low-frequency pulse generator (CF-FM783-BA, Wal front, operating current 1 mV) (Figure 1) emitting block pulses of 7.8-Hz electromagnetic waves for 10 min.Plates were further incubated for 96 h, at which point the culture solution was removed and wells were washed with phosphate-buffered saline (PBS, Sigma-Aldrich Japan LLC).One hundred microliters of 50 μg/mL neutral red (NR, Nacalai Tesque Inc.) was added to each well, and the plates were incubated for 3 h.The NR solution was removed, and the cells were fixed with 200 μL of 1% formaldehyde.The fixation solution was removed, and 100 μL of 1% acetic acid/50% 2-propanol was added to each well to visualize the NR that was taken up intracellularly.
Absorbance was measured at 540 nm using a microplate reader (Immuno Mini, NJ-2300, Nalge Nunc International Co.).Cell proliferation was evaluated by calculating the NR uptake in cells treated with 7.8-Hz electromagnetic waves relative to the mean NR uptake in untreated control cells.

| Cell migration assay
Two milliliters of NHEKs at a concentration of 12 × 10 4 cells/mL was seeded into 35-mm dishes (n = 3).After the cells reached confluence, they were cultured for a further 24 h to ensure all of the cells were in G 0 phase.A 200-μL pipette tip was used to draw a cross on each dish, detaching the cells in the scratch, and the culture medium was changed.A mycoplasma-derived synthetic diacyl lipopeptide (fibroblast-stimulating lipopeptide, FSL-1, Adipogen Life Sciences Inc.) 5 was added at a concentration of 0.1 μg/ mL, and the dishes were cultured for 24 h.Immediately after the scratch was made and 24 h later, the dishes were placed on top of a 7.8-Hz ultra-low-frequency pulse generator emitting 7.8-Hz electromagnetic waves for 10 min.After 24 h, the area around the scratch was examined under a microscope to assay cell migration into the wound.

| DEFB1 gene expression analysis
One milliliter of NHEKs at a concentration of 4 × 10 4 cells/mL was seeded into individual wells in a 24-well plate (n = 5-9).Cells were stimulated with 0.1 μg/mL FSL-1, and the plates were cultured for 24 or 48 h.The plates were placed on top of a 7.8-Hz ultra-lowfrequency pulse generator emitting 7.8-Hz electromagnetic waves for 10 min.After 48 h of culture, the culture medium was removed and the cells were washed with PBS, at which point they were lysed using 1 mL of 1% sodium dodecyl sulfate (Nippon Gene Co.).We added 1 μL of 1% KOH (Nacalai Tesque Inc.) and 20 μL of 20 mg/mL proteinase K solution (Thermo Fisher Scientific Inc., Waltham MA, USA) for every 180 μL of cell suspension.The mixture was incubated at 37°C for 15 min, and RNA was extracted using the RNA Clean XP (Beckman Coulter Inc.).
The RNA was subjected to reverse transcription with a Super Script™ IV VILO™ Master Mix (Thermo Fisher Scientific Inc.) for 10 min at 25°C, 10 min at 50°C, and 5 min at 85°C to obtain cDNA.

| SIRT1 gene expression analysis
Five milliliters of NHEKs at a concentration of 3 × 10 4 cells/mL was seeded in T25 flasks (n = 3-5).After culturing for 24, 48, or 72 h, the flasks were placed on top of a 7.8-Hz ultra-low-frequency pulse generator emitting 7.8-Hz electromagnetic waves for 10 min.After 72 h of culture, the cells were recovered, RNA was extracted, and reverse transcription was performed as described for DEFB1 gene expression analysis.PCR was conducted using Taqman® Gene Expression Assays (SIRT1 Hs01009006_m1, Thermo Fisher Scientific Inc.) as described for DEFB1 gene expression analysis.

| Statistical analysis
Measured values are expressed as means and standard deviations.
The statistical software EZR version 1.41 was used for statistical analysis, 6 and t-test and Dunnett's test were used for multiple comparisons with untreated controls.p < 0.05 was considered statistically significant.

| RE SULTS
Cell proliferation was increased 2.8-fold by the application of 7.8-Hz electromagnetic waves (Figure 2).Cell migration was enhanced by FSL-1 stimulation and was further increased by the application of 7.8 Hz electromagnetic waves (Figure 3).

| DISCUSS ION
In this study, we investigated the effect of the application of electromagnetic waves at an ultra-low frequency (7.8 Hz) to NHEKs.The application of 7.8 Hz electromagnetic waves increased the proliferation and migration of NHEKs.We observed a 2.8-fold increase in the rate of NHEK proliferation as a result of 7.8-Hz wave pulse application.A study on the addition of recombinant human epidermal growth factor to the HaCaT human keratinocyte cell line found that it increased cell proliferation 1.2-to 1.3-fold, irrespective of the presence or absence of serum. 7Therefore, the increase in proliferation we observed was extremely high.We also found that ultra-low-frequency electromagnetic waves increased the expression of DEFB1, which is an antimicrobial peptide gene, and SIRT1, which is involved in cellular senescence.These data suggest that 7.8 Hz electromagnetic waves can activate to repair a skin damage and a skin barrier dysfunction via inducing the expression of DEFB1 and SIRT1.
Previous studies have examined the effects of the application of electromagnetic fields at frequencies other than 7.8 Hz.In human bone marrow mesenchymal stem cells, 50-Hz electromagnetic fields suppress proliferation and increase SIRT1 expression, 8 and they increase the amount of melanin and tyrosinase activity in human melanocytes. 9Another study found that the application of electromagnetic fields at 1 Hz enhances the migration of HaCaT cells, whereas 80-Hz fields decrease migration. 10Thus, there is a F I G U R E 2 Effect of the application of 7.8-Hz electromagnetic waves on cell proliferation.The neutral red (NR) uptake during the application of 7.8-Hz electromagnetic waves increased 2.8-fold compared with the mean NR uptake of the untreated control.n = 6, values are presented as mean ± SD. ***p < 0.001, significant difference compared with control.
possibility that different electromagnetic wavelengths may exert different actins even on the skin.
We found that stimulation with FSL-1, which has the same Nterminal sequence as an S. aureus-derived diacyl lipopeptide, 3 promoted cell migration and upregulated the antimicrobial peptide gene DEFB1.The application of 7.8-Hz electromagnetic waves increased cell migration by a greater amount than FSL-1 stimulation and also tended to increase DEFB1 gene expression.β-defensin is suppressed in patients with atopic dermatitis. 11The application of 7.8-Hz electromagnetic waves to NHEKs may stimulate the expression of βdefensin in a manner equivalent to that of FLS-1 and may promote the function of antimicrobial peptides.From this point of view, 7.8-Hz electromagnetic waves may bring a good effect to a patients with atopic dermatitis.
As wound healing is delayed in SIRT1-knockout mice, 12 our data suggest that the application of 7.8-Hz electromagnetic waves may be useful to skin ulcer via activating SIRT1 expression.
There are some limitations in the present study.First, we used human cultured cells to examine the effect of 7.8-Hz electromagnetic waves and all of the findings are from in vitro experiments.
Second, we only used one cell strain of NHEKs and there may be an individual difference in the effect of 7.
8-Hz electromagnetic waves to the cells.More additional studies to use different cell strains, molecular analysis and clinical study should be performed to conclude the effect of 7.8-Hz electromagnetic waves to skin barrier function.In conclusion, our results suggest that the application of 7.8-Hz electromagnetic waves may contribute to the recovery of skin barrier function by upregulating the expression of several genes including DEFB1 and SIRT1, orchestrating to maintain skin homeostasis.

Further
studies are required to ascertain the mechanism of action of the application of 7.8-Hz electromagnetic waves to the skin, with the aim of enabling the clinical administration of 7.8-Hz electromagnetic waves for disorders such as atopic dermatitis, intractable cutaneous ulcer and for skin damage due to laser exposure or similar causes in aesthetic dermatology.

F I G U R E 3
Effects of the application of 7.8-Hz electromagnetic waves and FSL-1 stimulation on cell migration.FSL-1 stimulation and the application of 7.8-Hz electromagnetic waves both increased cell migration.(A) Untreated control, (B) FSL-1 stimulation, (C) application of 7.8-Hz electromagnetic waves, -: 0.6 mm.FSL-1: fibroblast-stimulating lipopeptide 1. F I G U R E 4 Effects of the application of 7.8-Hz electromagnetic waves and FSL-1 stimulation on DEFB1 gene expression.The DEFB1 gene expression level was increased 2.8-fold by FSL-1 stimulation (FSL-1) and 2.4-fold by the application of 7.8-Hz electromagnetic waves (7.8 Hz).n = 5-9, values are presented as mean ± SD. *p < 0.05, significant difference compared to the control.†p < 0.1, significant tendency compared to the control.DEFB1, β-defensin; FSL-1, fibroblast-stimulating lipopeptide 1. F I G U R E 5 Effect of the application of 7.8-Hz electromagnetic waves on SIRT1 gene expression.The SIRT1 expression level was increased 4.9-fold after the application of 7.8-Hz electromagnetic waves.n = 3-5, values are presented as mean ± SD. *p < 0.05, significant difference compared to the control.SIRT1: sirtuin 1.