Stimulation of the Nonneuronal Cholinergic System by Highly Diluted Acetylcholine in Keratinocytes

Abstract

The physiological effects of acetylcholine on keratinocytes depend on the presence of nicotinic and muscarinic receptors. The role of nonneuronal acetylcholine in keratinocytes could have important clinical implications for patients with various skin disorders such as nonhealing wounds. In order to evaluate the efficacy of highly diluted acetylcholine solutions obtained by sequential kinetic activation, we aimed to investigate the effects of these solutions on normal human keratinocytes. Two different concentrations (10 fg/mL and 1 pg/mL) and formulations (kinetically activated and nonkinetically activated) of acetylcholine were used to verify keratinocyte viability, proliferation, and migration and the intracellular pathways involved using MTT, crystal violet, wound healing, and Western blot compared to 147 ng/mL acetylcholine. The activated formulations (1 pg/mL and 10 fg/mL) revealed a significant capacity to increase migration, cell viability, and cell proliferation compared to 147 ng/mL acetylcholine, and these effects were more evident after a single administration. Sequential kinetic activation resulted in a statistically significant decrease in reactive oxygen species production accompanied by an increase in mitochondrial membrane potential and a decrease in oxygen consumption compared to 147 ng/mL acetylcholine. The M1 muscarinic receptor was involved in these effects. Finally, the involvement of ERK/mitogen-activated protein kinases (MAPK) and KI67 confirmed the effectiveness of the single treatment on cell proliferation. The intracellular pathways of calcium were investigated as well. Our results indicate for the first time that highly diluted and kinetically activated acetylcholine seems to play an active role in an in vitro model of wound healing. Moreover, the administration of acetylcholine within the physiological range may not only be effective but is also likely to be safe.

Highly Diluted Acetylcholine Promotes Wound Repair in an In Vivo Model

“… 19 , 20 Highly diluted forms used have been obtained through a preparation method called “sequential kinetic activation” (SKA) 20 , 21 ; this method was also applied to prepare a new Ach formulation used to induce healing in a wound in vitro model. 11 In this study low doses of sequentially kinetically activated Ach seem to play an active role in an in vitro model of wound healing, suggesting that Ach administered in a physiological range may not only be effective but also likely to be safe. The use of SKA technique is common in different fields, including pharmaceutical technology, to obtain a high therapeutic potential at very low dosages.…”

“…Several important functions in KC, including control of cell viability, proliferation, adhesion, migration, and differentiation, have been attributed to the non-neuronal cholinergic system, particularly muscarinic receptors. 1 , 11 , 12 Simultaneous stimulation of nicotinic and muscarinic receptors by acetylcholine (Ach) seems to be necessary to synchronize and balance ionic and metabolic events within the cell during epidermal turnover. 13 , 14 Some evidence has been found about the therapeutic capability of cholinomimetics or blockers in skin diseases.…”

“…The use of SKA technique is common in different fields, including pharmaceutical technology, to obtain a high therapeutic potential at very low dosages. 11 Thus, the aim of this study is to evaluate the role of low doses of Ach SKA versus nonactivated diluted formulation in in vivo wound healing model.…”

Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model

“…Hence, perhaps it contributes to reduced scarring of the cornea. Our findings are promising, as it has been reported by Uberti et al that kinetically energized ultra‐low doses of ACh show remarkably great wound healing properties both in vitro in human keratinocytes and in vivo in mice. Even though our results cannot be directly compared with Uberti's findings, throughout our studies on ACh in corneal wound healing, we have observed that the lower dose of ACh exerts stronger wound healing and anti‐fibrotic effects.…”

Femtograms of Interferon-γ Suffice to Modulate the Behavior of Jurkat Cells: A New Light in Immunomodulation

“…Similarly, the use of low doses of cytokines, neurotransmitters, and hormones has recently been shown to control and harmonize cellular functions in order to restore the original homeostatic physiological condition. In a recent study, Uberti et al demonstrate that the activated formulations of acetylcholine increase keratinocyte viability, proliferation, and migration and decrease the production of reactive oxygen species and oxygen consumption [ 22 ]. This study also confirms previous results [ 14 ] about the relevance of the mechanical activation of the solutions to potentiate the pharmacological action of low-dose compounds.…”