Biochemical and Biophysical Research Communications
Hydrogen-rich medium protects mouse embryonic fibroblasts from oxidative stress by activating LKB1-AMPK-FoxO1 signal pathway
Introduction
Oxidative stress refers to a pathophysiological state that arises by virtue of the strong cellular oxidizing potential of reactive oxygen species (ROS); importantly, persistent oxidative stress can damage cellular molecules, including DNA, protein, and lipids [1]. For these reasons, oxidative stress has been accepted as one of the major etiologies underlying many common diseases, including metabolic dysregulation, neurological diseases and cancer, as well as ageing [2]. A number of phytochemicals, including polyphenols, have been the focus of recent research interest owing to their antioxidant properties in oxidative-stress–related diseases. Despite the considerable body of literature demonstrating the antioxidant effects of phytochemicals in vitro and in animal models, most clinical trials of dietary antioxidants have failed to effectively prevent oxidative-stress–related diseases [3]. Indeed, some well-known antioxidant flavonoids actually exert pro-oxidant properties in the presence of a transition metal [4]. Similarly, ascorbic acid, beta-carotene, and lycopene show both antioxidant and pro-oxidant effects, depending on the dose [5]. Therefore, identification of an effective antioxidant without side effects is a high priority.
In 2007, Ohsawa et al. reported the landmark observation that molecular hydrogen (H2) exerts therapeutic antioxidant effects by selectively scavenging hydroxyl and peroxynitrite radicals, and protects the brain against ischemia-reperfusion injury [6]. Subsequent studies have supported the therapeutic potential of H2 against a variety of human diseases and in animal models [7], and the absence of toxicity, even at high concentrations, and rapid cellular diffusion of H2 have drawn considerable research attention to H2 as a therapeutic agent [8], [9]. Nevertheless, little is known about the reactive oxygen species (ROS)-scavenging properties of H2. Beyond this, it is clear that the pleiotropic therapeutic effects of H2 cannot be accounted for solely on the basis of a radical-scavenging mechanism. Indeed, numerous reports have demonstrated that the therapeutic effects of H2 can reflect modulation of the activity of various protein intermediaries, including the Src family tyrosine kinase Lyn; the mitogen-activated protein kinases (MAPKs), ERK (extracellular signal-regulated kinase), p38 and c-Jun N-terminal kinase (JNK); the MAPK kinase kinase, ASK1; the serine/threonine kinase AKT; the rho family GTPase, GTP-Rac1; the transcription factors NF-κB and STAT3; and the hormone ghrelin [7]. However, effects of H2 on AMPK, a major regulator of cellular and whole-body energy homeostasis that has been considered a therapeutic target for metabolic diseases [10], [11], has remained almost unknown. So far, there has been only one report describing the effect of H2 on AMPK [12], but it is practically unknown how H2 modulates AMPK signal pathway.
In this study, we explored the underlying mechanisms that H2 activates AMPK signal pathway, ultimately affecting the expression of genes involved in ROS detoxification. To study the influence of molecular H2, we generated hydrogen-rich media by directly dissolving H2 gas in the media. Here, we report that H2 acts as a signaling molecule, demonstrating that hydrogen-rich medium activates the LKB1-AMPK signal pathway without ATP depletion, leading to induction of FoxO1-dependent transcription of genes encoding antioxidants. Hydrogen-rich media also effectively reduced ROS levels in mouse embryonic fibroblasts (MEFs) treated with hydrogen peroxide (H2O2) and protected these cells from apoptosis in an AMPK-dependent manner.
Section snippets
Preparation of hydrogen-rich media
Hydrogen-rich medium was prepared by dissolving H2 gas (Hankook Special Gases Co., Korea) into serum-free Dulbecco's Modified Eagle Medium (DMEM) at a pressure of 0.1 MPa (MPa) in a Pyrex bottle for 30 min. At this point, H2 levels in media, measured using an ENH-1000 dissolved hydrogen meter (Trustlex Co., Osaka, Japan), were saturated at a concentration of 0.6 mM. Thereafter, fetal bovine serum was added to achieve a final concentration of 10%.
Antibodies and reagents
Anti-phospho-AMPKα (Thr172) and anti-caspase-3
Hydrogen-rich medium activates AMPK through the kinases, LKB1 and CaMKKβ
The commonly used routes of hydrogen administration in medical research include inhalation of 1–4% hydrogen gas, intraperitoneal injection/intravenous infusion of hydrogen-rich saline, and oral intake of hydrogen-saturated water. Here, to test the effects of H2, we generated hydrogen-rich media by directly dissolving H2 gas into cell culture media until its levels were saturated at a concentration of 0.6 mM (Fig. 1A). The molecular H2 level in hydrogen-rich media/phosphate-buffered saline was
Discussion
Since the initial discovery of its antioxidant properties, molecular H2 has been investigated in a wide range of animal models and human diseases for potential therapeutic effects. To date, molecular H2 has been reported to have beneficial effects in all organs encompassing 31 disease categories [7]. However, such pleiotropic effects of H2 cannot be explained solely by its radical-scavenging properties; instead, emerging evidence suggests that H2 may also act as a gaseous signaling molecule
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by grants from the Korean Health Technology R&D Project (AhR project; HI14C2700), and the National Research Foundation of Korea (NRF), funded by the Korea government (MEST; No 2011-0030072).
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These authors equally contributed to the present work.