Fig 7. The schematic diagram of MES₅₅₀₀ mechanism.

Therapeutic use of electric current has been practiced for various kinds of inflammation, and the mechanism was thought to be its analgesic effect or by blood flow improvement [38]. It is well known that in an electrolytic reaction, electrical current generates H₂O₂ from water [39], and as shown in our study, this reaction might also occur in electrical stimulation used for therapeutic purposes. While many reports indicate that reactive oxygen species (ROS), such as H₂O₂, exerts pro-inflammatory effects through activation of NF-κB, more recent studies have shown that ROS are not only responsible for inducing inflammation, but also have potent anti-inflammatory properties [40, 41]. Increase in intracellular concentration of H₂O₂ have been demonstrated to diminish TLR4-induced activation of NF-κB and production of pro-inflammatory cytokines in neutrophils, epithelial cells and other cell populations [42]. Aside from T cells and primary splenocytes, MES₅₅₀₀ was also able to reduce the inflammatory cytokine expression in other cells such as MG6 (microglia), SHSY5Y (neuroblastoma lineage) (S5 Fig) and HeLa cells (S7 Fig). MES₅₅₀₀ did not have any effect at the basal state of these cells, indicating the safety of the treatment, and it modulated the excess reaction after inflammatory reagent stimulation. Moreover, MES₅₅₀₀ reduced the induction of IL-1β upon stimulation of these cells with lipopolysaccharide (LPS), an immune activator that binds to Toll-like receptor (TLR) 4 (S5A–S5C Fig). These data consolidate our findings that MES₅₅₀₀ has immune suppressor effect.