New Approach in Translational Medicine: Effects of Electrolyzed Reduced Water (ERW) on NF-κB/iNOS Pathway in U937 Cell Line under Altered Redox State

Abstract

It is known that increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can exert harmful effects, altering the cellular redox state. Electrolyzed Reduced Water (ERW) produced near the cathode during water electrolysis exhibits high pH, high concentration of dissolved hydrogen and an extremely negative redox potential. Several findings indicate that ERW had the ability of a scavenger free radical, which results from hydrogen molecules with a high reducing ability and may participate in the redox regulation of cellular function. We investigated the effect of ERW on H₂O₂-induced U937 damage by evaluating the modulation of redox cellular state. Western blotting and spectrophotometrical analysis showed that ERW inhibited oxidative stress by restoring the antioxidant capacity of superoxide dismutase, catalase and glutathione peroxidase. Consequently, ERW restores the ability of the glutathione reductase to supply the cell of an important endogenous antioxidant, such as GSH, reversing the inhibitory effect of H₂O₂ on redox balance of U937 cells. Therefore, this means a reduction of cytotoxicity induced by peroxynitrite via a downregulation of the NF-κB/iNOS pathway and could be used as an antioxidant for preventive and therapeutic application. In conclusion, ERW can protect the cellular redox balance, reducing the risk of several diseases with altered cellular homeostasis such as inflammation.

Keywords: antioxidant enzymes; electrolyzed reduced water; nitric oxide; superoxide anion.

Figure 1

Cytotoxic effect ERW-medium on U937 cells line. Cells were grown in an MQ-medium or MQ-NaOH medium or ERW-medium with/without H₂O₂ and subjected to an MTT assay to analyse cell cytotoxicity. Data is presented as means ± SD for triplicate experiments. * p < 0.05 vs. MQ-NaOH; ** p < 0.05 vs. MQ-NaOH + H₂O₂.

Figure 2

(A) Antioxidant activity of ERW water against oxidative stress measured by NBT test. Results were registered as stimulation index (SI). SI value of 1 was assigned to control cells; (B) effect of ERW water on GSH levels in U937 cells. * p < 0.05 vs. MQ-NaOH + H₂O₂. Data are presented as means ± SD for triplicate experiments.

Figure 3

Effect of an ERW medium on the protein expression (top) and activity (bottom) of antioxidant enzyme SOD (A); CAT (B); GPx (C); and GR (D). * p < 0.05 vs. MQ-NaOH + H₂O₂. Data is presented as means ± SD for triplicate experiments.

Figure 4

Effects of an ERW-medium on NF-κB, iNOS and 3-nitrotyrosine in U937 cells. Representative image of Western blot analysis for nuclear-p65 NF-κB (A); cytoplasmic-p65 NF-κB iNOS (B); ELISA for NF-κB p65 DNA binding activity (C); western blot analysis for iNOS (D); NO levels (E); and 3-nitrotyrosine protein expression (F). (A) Averaged band density of nuclear NF-κB from U937 cells normalized versus tubuline; (B) Averaged band density of cytoplasmic fraction of p65 NF-κB from U937 cells normalized versus β-actine; (C) effect of ERW on NF-κB p65 DNA binding activity; (D) effect of an ERW-medium on preventing H₂O₂-induced iNOS expression in U937 cells. Averaged band density of iNOS from U937 cells normalized versus β-actin. Cells were treated or not treated with H₂O₂ and/or an MQ-NaOH/ERW-medium; (E) effects of an ERW-medium after 4 h of incubation with H₂O₂ on NO production in U937 cells. NO levels were quantified by the accumulation of nitrite in the cell culture medium and are expressed as µmol NO/L⁻¹/10⁶ cells; (F) Effect of ERW on 3-nitrotyrosine formation. Values are mean ± SD of different experiments performed in triplicate. * p < 0.05 versus MQ-NaOH + H₂O₂ treated cells.