Short ELF-EMF Exposure Targets SIRT1/Nrf2/HO-1 Signaling in THP-1 Cells

Abstract

Extremely low frequency electromagnetic fields (ELF-EMFs) have been known to modulate inflammatory responses by targeting signal transduction pathways and influencing cellular redox balance through the generation of oxidants and antioxidants. Here, we studied the molecular mechanism underlying the anti-oxidative effect of ELF-EMF in THP-1 cells, particularly with respect to antioxidant enzymes, such as heme oxygenase-1 (HO-1), regulated transcriptionally through nuclear factor E2-related factor 2 (Nrf2) activation. Cells treated with lipopolysaccharides (LPS) were exposed to a 50 Hz, 1 mT extremely low frequency electromagnetic fields for 1 h, 6 h and, 24 h. Our results indicate that ELF-EMF induced HO-1 mRNA and protein expression in LPS-treated THP-1 cells, with peak expression at 6 h, accompanied with a concomitant migration to the nucleus of a truncated HO-1 protein form. The immunostaining analysis further verified a nuclear enrichment of HO-1. Moreover, ELF-EMF inhibited the protein expressions of the sirtuin1 (SIRT1) and nuclear factor kappa B (NF-kB) pathways, confirming their anti-inflammatory/antioxidative role. Pretreatment with LY294002 (Akt inhibitor) and PD980559 (ERK inhibitor) inhibited LPS-induced Nrf2 nuclear translocation and HO-1 protein expression in ELF-EMF-exposed cells. Taken together, our results suggest that short ELF-EMF exposure exerts a protective role in THP-1 cells treated with an inflammatory/oxidative insult such as LPS, via the regulation of Nrf-2/HO-1 and SIRT1 /NF-kB pathways associated with intracellular glutathione (GSH) accumulation.

Keywords: THP-1 cells; extremely low frequency electromagnetic fields (ELF-EMF); heme oxygenase-1 (HO-1); nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2); silent information regulator 1 (SIRT1).

Figure 1

Extremely low frequency electromagnetic field (ELF-EMF) effects on nuclear translocation of heme oxygenase-1 (HO-1) in THP-1 cell line. (A) Representative image of western blot analysis of HO-1 protein in cytoplasmic and nuclear fractions extracted from THP-1 cells cultured with lipopolysaccharides (LPS) and ELF-EMF-exposed or sham-exposed (top). Averaged band density of HO-1 normalized vs. β-actin for cytoplasm and vs. Lamin B for nucleus (bottom). Data are expressed as mean ± SD of three independent experiments performed in triplicate. * p < 0.05 ELF-EMF-exposed vs. sham-exposed cells. (B) Fluorescence expression at nuclear level and co-localization analyses. Immunolabeling with TO-PRO (blue fluorescence) and heme oxygenase-1 (red fluorescence) in THP-1 cells at basal condition at T0 (g), in LPS-treated cells exposed to ELF-EMF at 1 h (b), 6 h (d), and 24 h (f) or sham-exposed (a,c,e). Bars represent HO-1 co-expression reported as Pearson’s correlation coefficient. * p < 0.05 ELF-EMF-exposed vs. sham-exposed cells. The Supplementary materials illustrates the single fluorescence channels used. (C) The mRNA expression of HO-1 in THP-1 cells exposed to ELF-EMF and compared to sham-exposed cells. HO-1 expression levels (2^−∆∆Ct) are reported as mean ± standard error (SE). * p < 0.05 ELF-EMF vs. sham assumed as 1.

Figure 2

ELF-EMF effects on nuclear factor erythroid 2-related factor 2 (Nrf2) and sirtuin 1 (SIRT1)/ nuclear factor kappa B (NF-kB) pathway.(A) Representative western blots (top) and data analysis (bottom) of Nrf2 analyzed in cytoplasmic and nuclear extracts from THP-1 cells treated with LPS and ELF-EMF-exposed or sham-exposed. β-actin and lamin B were used as the internal standards for cytoplasmatic and nuclear fraction, respectively. (B) Representative western blots (top) and densitometric analysis (bottom) of Sirt-1, cytoplasmatic NF-kBp65, and nuclear phosphorylated NF-kBp65 protein level in THP-1 cells treated with LPS and ELF-EMF-exposed or sham-exposed. Data are normalized vs. β-actin for Sirt-1 and to respective unphosphorylated protein for p-65 subunit. Data are expressed as mean ± SD of three independent experiments performed in triplicate. * p < 0.05 ELF-EMF-exposed vs. sham-exposed cells.

Figure 3

Effect of ELF-EMF exposure on Akt and ERK pathways. (A) Analysis of the phosphorylation levels of Akt and ERK in THP-1 cells treated with LPS and ELF-EMF-exposed or sham-exposed by western blotting. (B) Representative image of immunoblotting for Nrf-2 and HO-1 in LPS-treated and ELF-EMF-exposed or sham-exposed THP-1 cells for 6 h, pre-treated or not with selective inhibitors of Akt (Ly294002, 1 μmol/L) and ERK (PD980559, 1 μmol/L). Data are reported as the relative expression of nuclear Nrf-2 vs. lamin B and whole cell lysate HO-1 vs. β-actin. Data are expressed as mean ± SD of three independent experiments performed in triplicate. * p < 0.05 ELF-EMF-exposed vs. sham-exposed cells.

Figure 4

Effect of ELF-EMF exposure on intracellular reactive oxygen species (ROS) formation and glutathione-redox status. (A) Intracellular ROS was quantified by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) probe measuring fluorescence intensity. Data are expressed as the fold increase of ROS production in ELF-EMF-exposed cells relative to the sham-exposed ones. (B) Cells were lysed and the intracellular concentration of glutathione (GSH)/oxidized glutathione (GSSG) in all groups was quantified following enzymatic recycling assay. (C) Glutathione peroxidase and (D) Glutathione reductase activity was normalized with respect to the total protein content of cell lysates. One unit of enzyme activity was defined as 1 µmol of NADPH oxidized/min. Data are expressed as mean ± SD of three independent experiments performed in triplicate. * p < 0.05 ELF-EMF-exposed vs. sham-exposed cells. GPx: glutathione peroxidase; GR: glutathione reductase.