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1. Elucidation of molecular mechanisms


1-1 Electric potential therapeutic device and its working hypothesis

 Therapeutic devices generate high-voltage electric fields function by guiding a high-voltage alternating current (boosted from a 100-V power supply to a maximum of 9,000 V) to an insulated electrode that then generates an electric field between itself and a counter electrode. A human body is placed between the electrodes within the electric field. This induces a very small current (several tens of microamperes) throughout the body. The effects associated with this process are reportedly “remission of headache, shoulder stiffness, insomnia, and chronic constipation.” This process has been approved and certified by the Ministry of Health, Labour and Welfare of Japan. A working hypothesis related to the mode of action of this therapeutic device obtained by an evaluation committee of experts in medicine, science, and engineering based on a literature survey is as follows:


【Working hypothesis】 Because it has been confirmed that body hair sway and stimulate the skin and that the skin temperature in humans increases on the application of an electric field, the action of the electric field, which seems to influence the positive effects, appears to be as follows:
“ The action of the electric field stimulates the tactile sensation of the skin and sensory receptors that sense pressure, thereby acting on blood circulation and the body’s regulatory functions.”




1-2 Introduction

 The mechanisms related to health benefits conferred by a therapeutic device that generates high-voltage electric fields is not fully understood. To address this, a strategic approach from the perspective of integrative molecular medicine was devised. The focus of the present study was to discover molecules in humans that can be endogenously activated by a therapeutic device that generates a high-voltage electric field. Based on these findings, molecular-level evidence that supports the pharmacological effects of this device will be accumulated by analyzing interactions between the active molecules and their target proteins. This chapter discusses new insight into the molecular mechanisms; however, some technical terms and names of chemical substances could not be altered. Therefore, even medical/pharmaceutical experts may find the research results presented here somewhat difficult to understand unless they are aware of the latest developments in molecular pharmacology. For those interested in more detailed research information, a list of relevant articles is available at the end of this chapter.




1-3 Focusing on human metabolites

 We start by discussing a breakthrough made by our research. Exposure to a high-voltage electric field generated by the therapeutic device (30 min) significantly increased 9-hydroxyoctadecadienoic acid (9-HODE), 13-HODE, and 13-hydroperoxyoctadecadienoic acid (13-HpODE) levels (Nakagawa-Yagi Y et al, 2016). These data were obtained from human plasma lipidomics experiments conducted using plasma samples obtained from 35 participants. In contrast, levels of HODE-related diol-metabolites, epoxide-metabolites, ketone-metabolites, and/or prostaglandins remained unchanged (Nakagawa-Yagi Y et al, 2016). In medical and pharmaceutical fields, phenomena in which nonspecific changes are induced by exogenous stimuli are believed to increase the possibility of causing undesirable effects. Therefore, the specific induction of 13-HODE, 13-HpODE, and 9-HODE by the therapeutic device is considered interesting.




1-4 Endogenous lipid-derived signal molecules that act on thermosensors

 Next, we explain the reason why we used a lipidomics approach. Comprehensive investigation of this process is critical as it helps eliminate prejudices. We therefore conducted comprehensive metabolomics for screening purposes (Nakagawa-Yagi Y et al, 2014). Significant changes were observed in levels of several fatty acids and fatty acid amides under conditions in which no changes occurred in levels of metabolites related to citric acid and ornithine cycles (Nakagawa-Yagi Y et al, 2014). Screening of these 161 metabolites led us to focus on lipid-derived metabolites (Nakagawa-Yagi Y et al, 2014). In recent years, there has been a rapid increase in the number of molecular medicine research papers that have reported that certain endogenous lipid-derived metabolites serve as signal molecules (Patti GJ et al, 2012; Piomelli D et al, 2014). In addition, a research approach that elucidates interactions between signal molecules, which function as the key, and receptor proteins, which function as keyholes, is essential to clarify the molecular mechanisms related to an unknown pharmacological action (Itoh et al, 2008: Nieto-Posadas A et al, 2012; Norn C et al, 2015). What roles do 13-HODE and 9-HODE play in the human body? We came across an interesting article that to answer this question (Patwardan AM et al, 2010). The authors reported a new finding related to transient receptor potential vanilloid 1 (TRPV1) protein, a nociceptor that is sensitive to noxious stimuli. TRPV1 is a member of the TRP channel family with a 6-transmembrane basic structure and is considered a thermosensor protein that acts as a nonselective cation channel (Tominaga M et al, 1998; Szolcsanyi J et al, 2012). Surprisingly, 13-HODE and 9-HODE were identified by Patwardhan et al. as signal molecules generated in the skin in response to ~43°C thermal stimuli. Their report suggested that 13-HODE and 9-HODE serve as mediators of TRPV1, acting as a biological defense system that detects noxious thermal stimuli. At present, TRPV1 is considered to function as a polymodal nociceptor that is mainly expressed in unmyelinated C fibers of primary sensory nerves (Julius D, 2013). Meanwhile, lipid-derived signal molecules, including 13-HODE and 9-HODE, have been found in human skin epidermis and dermis at reasonable concentrations (Kendall AC et al, 2015). These studies have demonstrated that a mechanism in which such lipid-derived signal molecules that are increased in the skin tissue act on TRPV1 in the peripheral sensory neuron terminals is highly likely.




1-5 Endogenous lipid-derived signal molecules that act on mechanosensors

 A research published in 2010 pointed out the role of transient receptor potential vanilloid 2 (TRPV2) as a mechanostretch sensor in the muscle of the gastrointestinal tract (Mihara H et al, 2010). In that article, lysophosphatidylcholine (lysoPC) was demonstrated to have pharmacological activity as a TRPV2 receptor agonist in vitro and was confirmed to promote the movement of contents within the intestinal tract in vivo. However, in what process is lysoPC produced in the human body? Glycerophosphatidylcholine breakdown products, for example, can be summarized as follows. Choline and phosphatidic acid are produced when phospholipase D is activated. Meanwhile, phosphorylcholine and diacylglycerol are formed when phospholipase C is activated. At the screening stage, choline and phosphorylcholine have been identified to remain unchanged upon exposure to a high-voltage electric field generated by the therapeutic device (Nakagawa-Yagi Y et al, 2014). However, no data are available on lysoPC generated by activated phospholipase A₂ (PLA₂). By chance, we came across an interesting article that the electric field induces PLA₂ activity (Thuren T et al, 1987). With regard to PLA₂, attention has previously been focused on inflammation evoked in association with arachidonic acid (FA-20:4) release, but subtypes acting to attenuate inflammation, such as group IID secretory PLA₂, have recently been found (Miki Y et al, 2013). Research on new types of PLA₂ involved in a central player in the anti-inflammatory effect to chronic inflammation observed in various intractable diseases is an area of attention in contemporary biomedicine. As such, we decided to investigate the effect of a high-voltage electric field generated by the therapeutic device on lysoPC levels. The results of lipidomics analysis of plasma samples collected from 50 participants showed significant increases in lysoPC-22:4 levels (Nakagawa-Yagi Y et al, 2017). In contrast, lysophosphatidic acid (lysoPA) levels were not affected. Furthermore, in silico docking simulation revealed that lysoPC-22:4 has a high affinity for a pocket in the TRPV2 receptor (Nakagawa-Yagi Y et al, 2017). LysoPC-22:4 molecules have been suggested to promote the movement of gastrointestinal contents to the anal side via TRPV2 receptor binding. This is likely to be at least one molecular mechanism that explains the remission of chronic constipation by exposure to a high-voltage electric field generated by the therapeutic device. Furthermore, TRPV2 has been known to act as a mechanosensor in the intercalated discs of the cardiac muscle (Kataoka Y et al, 2014) and as a sensor of cell extension at the tip of neurites (Shibasaki K et al, 2010). Accumulation of new findings related to TRPV2 will help draw attention to the field of unknown research.




1-6 Endogenously active molecules related to sleep

 A previous study found that peroxisome proliferator-activated receptor-alpha (PPAR-α) can be a drug target for ameliorating a type of sleep disorder in which sleep/wakeful phases are delayed (Shirai H et al, 2007). They reported that the activity time in a delayed sleep phase syndrome model was shifted by 3 h after administering the PPAR-α agonist bezafibrate for 14 days. Interestingly, oleoylethanolamide (OEA), an endogenous signal molecule with PPAR-α agonist-like action, was found to be significantly increased in the blood after exposure to a high-voltage electric field generated by the therapeutic device (Guzman M et al, 2004; Nakagawa-Yagi Y et al, 2014). Moreover, a crystal structure showing the molecular docking of OEA with the human PPAR-α protein indicated a binding mode similar to that of the fibrate analog AZ242 (Nakagawa-Yagi Y et al, 2014). Therefore, it is reasonable to speculate that the delayed sleep phase can be normalized by shifting the biological rhythm using the stimulatory effect of OEA on PPAR-α. Another study showed that significant increases in OEA levels in cerebrospinal fluid samples taken from 20 healthy volunteers after 24 h of sleep deprivation (Koethe D et al, 2009). Furthermore, uridine and prostaglandin D₂ are examples of endogenous sleep-promoting substances that increase under sleep deprivation conditions (Inoue S et al, 1984). In our study, exposure to a high-voltage electric field generated by the therapeutic device significantly increased uridine diphosphate levels but did not change the prostaglandin D₂ level (Nakagawa-Yagi Y et al, 2014; Nakagawa-Yagi Y et al, 2016). In future, it will be necessary to investigate whether uridine diphosphate has a sleep-promoting effect similar to uridine.




1-7 Endogenously active molecules that alleviate pain

 An analgesic mechanism induced by OEA via a non-PPAR-α pathway is conceivable (Suardiaz M et al, 2007; Fehrenbacher JC et al, 2009; Nakagawa-Yagi Y et al, 2014). Another possibility is an analgesic mechanism induced by beta-endorphin secreted via G protein-coupled receptor 40 on skin keratinocytes (Fell GL et al, 2014; Nakagawa-Yagi Y et al, 2015). In addition, an analgesic mechanism induced by uridine diphosphate via a purinergic receptors is conceivable (Okada M et al, 2002). On the other hand, an analgesic mechanism induced by local skin hypersensitivity-defunctionalization system is also conceivable. In this case, however, it is expected to take some time before the effect appears because it occurs through the defunctionalization of the nociceptor. A typical example of this analgesic mechanism is shown below. Qutenza®, a patch containing 8% 8-methyl-N-vanillyl-trans-6-nonenamide (capsaicin), is clinically applied in the USA and EU to relieve pain in cases of post-therapeutic neuralgia (Anand P et al, 2011; Vay L et al, 2012). More research should be conducted as other molecular mechanisms may exist.




1-8 Endogenously active molecules related to blood flow

 When a certain signal molecule acts on the peripheral sensory neuron terminals, neurohormones are released by a mechanism called axon reflex, and increases in this phenomenon in blood flow have been revealed. Thus, we investigated the effects of exposure to a high-voltage electric field generated by the therapeutic device and found that substance P in the plasma significantly increased at the 30-min time point after the exposure (Nakagawa-Yagi Y et al, 2016). Under these conditions, no significant changes were observed in the levels of calcitonin gene-related peptide, vasoactive intestinal peptide, bradykinin, or motilin (Nakagawa-Yagi Y et al, 2016). The release of substance P from sensory nerve terminal causes the activation of nitric oxide synthase in vascular endothelial cells and guanylate cyclase in smooth muscle cells. Vascular relaxation, which increases blood flow, is induced by an increase in intracellular cyclic guanosine monophosphate levels. Given the cause of most blood flow increases, it is pharmacologically important to consider the changes in levels of intracellular secondary messengers such as cyclic guanosine monophosphate. In fact, the 1998 Nobel Prize in Physiology and Medicine was awarded to Furchgott, Ignarro, and Murad for their studies on vascular relaxation phenomena via the nitric oxide synthase–guanylate cyclase–cyclic guanosine monophosphate system. With regard to vascular relaxation by endogenous lipid-derived signal molecules, linoleic acid (FA-18:2) and its metabolites (13-HODE and 13-HpODE) have been reported to induce relaxation of the coronary artery (Pomposiello SI et al, 1998). Moreover, OEA has been found to induce vascular endothelium-dependent relaxation of mesenteric arteries, which play a major role in blood circulation in the digestive tract (AlSuleimani YM et al, 2013). In particular, attention must be paid to the fact that the half-maximal effective concentration of the concentration dependency curve for the relaxation effect on mesenteric arteries in that study is close to the OEA concentration in human blood [46.8 nM] (Psychogios N et al, 2011). This suggests that the improvement in blood flow by OEA in mesenteric arteries also occurs in humans. Future studies should focus on the importance of signal molecule levels in blood and the levels necessary for pharmacological effects.


References

AlSuleimani YM et al (2013) Mechanisms of vasorelaxation induced by oleoylethanolamide in the rat small mesenteric artery. European Journal of Pharmacology 702: 1-11.
Anand P et al (2011) Topical capsaicin for pain management : therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch. British Journal of Anaesthesia 107: 491-502.
Fehrenbacher JC et al (2009) Rapid pain modulation with nuclear receptor ligands. Brain Research Review 60: 114-124.
Fell GL et al (2014) Skinβ-endorphin mediates addiction to UV light. Cell 157: 1527-1534.
Guzman M et al (2004) Oleoylethanolamide stimulates lipolysis by activating the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-α). Journal of Biological Chemistry 279: 27849-27854.
Inoue S et al (1984) Differential sleep-promoting effects of five sleep substances nocturnally infused in unrestrained rats. Proceedings of the National Academy of Sciences USA 81:6240-6244.
Julius D (2013) TRP channels and pain. Annual Review of Cell and Developmental Biology 29: 355-384.
Itoh T et al (2008) Structural basis for the activation of PPARγ by oxidized fatty acids. Nature Structure Molecular Biology 15: 924-931.
Kataoka Y et al (2014) TRPV2 is critical for the maintenance of cardiac structure and function in mice. Nature Communications5: 3932.
Kendall AC et al (2015) Distribution of bioactive lipid mediators in human skin. Journal of Investigative Dermatology 135: 1510-1520.
Koethe D et al (2009) Sleep deprivation increases oleoylethanolamide in human cerebrospinal fluid. Journal of Neural Transmission 116: 301-305.
Mihara H et al (2010) Involvement of TRPV2 activation in intestinal movement through nitric oxide production in mice. Journal of Neuroscience 30: 16536-16544.
Miki Y et al (2013) Lymphoid tissue phospholipase A₂ group IID resolved contact hypersensitivity by driving antiinflamatory lipid mediators. Journal of Experimental Medicine 210: 1217-1234.
Nieto-Posadas A et al (2012) Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site. Nature Chemical Biology 8: 78-85.
Norn C et al (2015) Mutation-guided unbiased modeling of the fat sensor GPR119 for high-yield agonist screening. Structure 23: 2377-2386.
Okada M et al (2002) Analgesic effects of intrathecal administration of P2Y nucleotide receptor agonists UTP and UDP in normal and neuropathic pain model rats. Journal of Pharmacology and Experimental Therapeutics 303: 66-73.
Patti GJ et al (2012) Metabolomics implicates altered sphingolipids in chronic pain of neuropathic origin. Nature Chemical Biology 8: 232-234.
Patwardhan AM et al (2010) Heat generates oxidized linoleic acid metabolites that activate TRPV1 and produce pain in rodents. Journal of Clinical Investigation 120: 1617-1626.
Piomelli D et al (2014) Peripheral gating of pain signals by endogenous lipid mediators.Nature Neuuroscience 17: 164-174.
Pomposiello SI et al (1998) Linoleic acid induces relaxation and hyperpolarization of the pig coronary artery. Hypertension 31: 615-620.
Psychogios N et al (2011) The human serum metabolome. PLoS ONE 6(2): e16957.
Shibasaki K et al (2010) TRPV2 enhances axon outgrowth through its activation by membrane stretch in developing sensory and motor neurons. Journal of Neuroscience 30(13): 4601-4612.
Shirai H et al (2007) PPAR is a potential therapeutic target of drugs to treat circadian rhythm sleep disorders. Biochemical and Biophysical Research Communications 357: 679-682.
Suardiaz M et al (2007) Analgesic properties of oleoylethanolamide (OEA) in visceral and inflammatory pain. Pain 133: 99-110.
Szolcsanyi J et al (2012) Multisteric TRPV1 nocisensor: a target for analgesics. Trends in Pharmacological Sciences 33: 646-655.
Thuren T et al (1987) Triggering of the activity of phospholipase A₂ by an electric field. Biochemistry 26: 4907-4910.
Tominaga M et al (1998) The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21: 531-543.
Vay L et al (2012) The thermo-TRP ion channel family: properties and therapeutic implications. British Journal of Pharmacology165: 787-801.




List of relevant articles

1)Nakagawa-Yagi, Y, Hara H, Fujimori T, Yamaguchi T, Midorikawa A, Hara A; Non-targeted human plasma metabolomics reveals the changes in oleoylethanolamide, a lipid-derived signaling molecule, by acute exposure of electric field.
Integrative Molecular Medicine, Vol.1, No.2:29-37(2014)
doi:10.15761/IMM.1000108
2)Nakagawa-Yagi Y, Hara H, Yoshida Y, Midorikawa A, Hara A; Discovery of a novel effect of electric field exposure on human plasma beta-endorphin and interleukin-12 levels: Insight into mechanisms of pain alleviation and defense against infection by electric field therapy.
Integrative Molecular Medicine, Vol.2, No.3:200-204(2015)
doi:10.15761/IMM.1000139
3)Nakagawa-Yagi, Y, Hara H, Nakagawa, F, Sato, M, Hara A; Acute exposure to an electric field induces changes in human plasma 9-HODE, 13-HODE, and immunoreactive substance P levels: Insight into the molecular mechanisms of electric field therapy.
Integrative Molecular Medicine, Vol.3, No.2:600-605(2016)
doi:10.15761/IMM.1000210
4)Nakagawa-Yagi Y, Hara H, Tuboi H, Abe J, Hara A; Effect of 3-hydroxybutyrate, an endogenous histone deacetylase inhibitor, on FOXO3A mRNA expression in human epithelial colorectal Caco-2 cells: Insight into the epigenetic mechanisms of electric field therapy.
Integrative Molecular Medicine, Vol.3, No.5:764-768(2016)
doi:10.15761/IMM.1000241
5)Nakagawa-Yagi Y, Hara H, Nakanishi H, Tasaka T, Hara A; Acute exposure to an electric field induces changes in human plasma lysophosphatidylcholine (lysoPC)-22:4 levels: Molecular insight into the docking of lysoPC-22:4 interaction with TRPV2.
Integrative Molecular Medicine, Vol.4, No.2:1-7(2017)
doi:10.15761/IMM.1000274
6)Nakagawa-Yagi Y, Hara H, Nakanishi H, Kanai C, Hara A; Molecular insight into the docking of lysophosphatidylethanolamine (lysoPE)-22:6 interaction with GPR119: Acute exposure to an electric field induces changes in human plasma lysoPE-22:6 and lysoPE-20:4 levels.
Integrative Molecular Medicine, Vol.4, No.5:1-7(2017)
doi:10.15761/IMM.1000305 Note: Integrative Molecular Medicine (country of publication: United Kingdom) is a peer-reviewed open journal.


2. Evaluation studies in humans

 Some studies that have evaluated high-voltage electric field therapy are briefly introduced below:


2-1 Article title:Effects of electrical Healthtron on curing of non-communicable diseases: Case study of Banlad hospital Petchaburi province (in Thai).
<First author and affiliation> Nawarat S(Banlad Hospital・Petchaburi・Thailand)
<Article published in> Region 4 Medical Journal 18, (2):139-149(1999)
<Evaluation in humans>
 This study evaluated the effect of exposure to a high-voltage electric field generated by the therapeutic device (30 min/day, 30 times) based on the indices “signs of improvement,” “no signs of change,” and “signs of worsening” in 74 healthcare workers with myalgia (67 individuals), stress (35 individuals), insomnia (30 individuals), allergies (16 individuals), hypertension (12 individuals), and diabetes mellitus (11 individuals) at Banlad Hospital in Petchaburi, Thailand. The results demonstrated a significant improvement in insomnia and myalgia after exposure to a high-voltage electric field.


2-2 Article title:High-voltage electrostatic therapy for chronic sleep disorder in aged patients (in Chinese).
<First author and affiliation> Zhang L (Chinese PLA General Hospital, Beijing, China)
<Article published in> Academic Journal of PLA Postgraduate Medical School 33, (7): 730-732 (2012) <Evaluation in humans>
 Seventy elderly patients (all aged >65 years) with sleep disorders were randomly allocated to the high-voltage electric field therapeutic device exposure group (35 patients, once daily for 25–30 min, 10–15 times in total) or the cognitive behavioral therapy group (32 patients, 3 weeks). The patients completed the Pittsburg Sleep Quality Index (PSQI) questionnaire before and after treatment to evaluate treatment-induced changes. In the high-voltage electric field exposure group, significant improvements were observed in scores of all PSQI subscales (sleep quality, latency, duration, efficiency, disturbance, medications, and daytime dysfunction). On the other hand, in the cognitive behavioral therapy group, significant improvements were observed only in PSQI subscale scores related to sleep quality, latency, and duration but not in those related to sleep efficiency, disturbance, and medications or daytime dysfunction. Both the total score and some subscale scores were significantly better in the high-voltage electric field exposure group than in the cognitive behavioral therapy group.


2-3 Article title:Effect of high-voltage electrostatic therapy on sleep disorder in older adults (in Chinese).
<First author and affiliation> Zhang L (Nan Lou of Chinese PLA General Hospital, Beijing, China)
<Article published in> Chinese Journal of Rehabilitation Theory and Practice 18, (3): 286-288 (2012)
<Evaluation in humans>
 Exposure to a high-voltage electric field generated by a therapeutic device (once daily for 20–30 min, 10–15 times in total) was evaluated using the Pittsburg Sleep Quality Index (PSQI) questionnaire in 30 elderly individuals with sleep disorders (age range, 66–92 years). Significant improvements in the PSQI total score and all subscale scores (sleep quality, latency, duration, efficiency, disturbance, medications, and daytime dysfunction) were observed after the intervention using the therapeutic device. A beneficial effect of high-voltage electric field exposure on insomnia was observed.


2-4 Article title:Electric field exposure improves subjective symptoms related to sleeplessness in college students: A pilot study of electric field therapy for sleep disorder.
<First author and affiliation> Takashi Otsuki (Morinomiya University of Medical Sciences)
<Article published in> CImmunology, Endocrine & Metabolic Agents in Medicinal Chemistry17, (1):37-48(2017) doi:10.2174/1871522217666170815163329
<Evaluation in humans>
 The effect of exposure to a high-voltage electric field generated by the therapeutic device (once daily for 30 min, 5 days total) was evaluated using the OSA Sleep Inventory in 19 college students with sleep disorders. Significant improvements in scores related to sleep length, sleepiness upon rising, and refreshed feeling upon waking were induced by intervention using the therapeutic device. A beneficial effect of high-voltage electric field exposure on sleep was observed after the 5-day treatment.


2-5 Article title:A pilot study on electric field therapy for chronic pain with no obvious underlying diseases.
<First author and affiliation> Toshikazu Shinba (Shizuoka Saiseikai General Hospital)
<Article published in> Journal of Japanese Society for Integrative Medicine 5, (1): 68-72 (2012)
<Evaluation in humans>
 Subjects who felt pain on a daily basis with no known underlying diseases underwent high-voltage electric field therapy using the therapeutic device (once daily for 20 min, 4 times every few days). A significant improvement in the visual analog scale scores related to pain was observed.


2-6 Article title:The effects of electric field therapeutic device (Healthtron) on stiffness in the neck and shoulder area-Changes in subjective symptoms, blood circulation, and the autonomic nervous system-
<First author and affiliation> Fujio Ito (Ito Orthopaedic and Internal Medicine Clinic)
<Article published in> The Journal of the Japanese Society of Balneology and Physical Medicine 68, (2): 110-121 (2005)
<Evaluation in humans>
 The visual analog scale score was followed over time in 30 patients with shoulder stiffness (12 patients received the general treatment, 18 patients received the general treatment + high-voltage electric field therapy). In the general treatment group, significant improvements in visual analog scale scores were observed from day 14. In the general treatment + high-voltage electric field therapy, significant improvements in visual analog scale scores were observed from day 7 in the left shoulder. In addition, significantly increased blood flow in the trapezius muscle in patients exposed to the high-voltage electric field alone were observed by using a near-infrared spectroscopy.


 It is important to note that the studies that have evaluated the therapeutic device were conducted not only in medical institutions in Japan but also in hospitals in the Kingdom of Thailand and the People’s Republic of China to clarify health benefits.

 

3. A part of the research paper in 2018

  Nakagawa-Yagi Y et al, Integrative Molecular Medicine Vol.5, No.3: Page 1-6 (2018)
Acute electric field downregulates human plasma immunoreactive interleukin-6 and -1β levels: Molecular mechanisms underlying inflammation alleviation through electric field therapy


Introduction

 A therapeutic device exposing the human body to high-voltage electric potential (HELP) has been approved by the Ministry of Health, Labour and Welfare in Japan [1-19]. High-voltage electric field (EF) therapy is reported to be an effective treatment for shoulder stiffness, headache, insomnia, and chronic constipation [1-20]. Although EF therapy was discovered more than 80 years ago, the molecular mechanisms associated with its health benefits remain elusive. Altogether, the results of these studies suggest that HELP exposure may be an alternative therapy for several conditions. Our previous attempts to identify biomarker induced by HELP exposure using plasma metabolomics and lipidomics have led to the detection of lipid-derived signaling molecules such as 3-hydroxybutyrate (3-HBA), cis-8,11,14-eicosatrienoic acid, 9-hydroxyoctadecadienoic acid (9-HODE),
13-hydroxyoctadecadienoic acid (13-HODE), oleoylethanolamide (OEA), lysophosphatidylethanolamine (lysoPE)-20:4, and lysoPE-22:6 [21-25]. Endogenous lipid-derived signaling molecules have been suggested as candidate molecules, representing the interface between symptoms and electroceutical target proteins [21-26]. A recent study conducted by Smani et al. reported that pretreatment with lysophosphatidylcholine (lysoPC) induced decreases in the levels of pro-inflammatory cytokines in the murine model of peritoneal sepsis caused by Acinetobacter baumannii [27]. In our previous study, we observed HELP exposure-induced upregulation of lysoPC-22:4 levels in the plasma of healthy individuals [28]. Therefore, we hypothesized that the increased plasma lysoPC-22:4 levels following EF exposure may be linked to changes in pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) In the present study, we report that the immunoreactive levels of IL-1β and IL-6 may be downregulated by HELP treatment (9 kV/electrode + 9 kV/electrode, 30 min). In addition, we conducted a binding study to explore the interactions between lysoPC-22:4 and a homology model of transient receptor potential melastatin 8 (TRPM8) using the template structure (PDB ID 6BPQ).



Results

Effect of HELP exposure on immunoreactive cytokines in the plasma of healthy individuals


 We examined the effect of HELP exposure (9 kV/electrode + 9 kV/electrode) for 30 min on immunoreactive cytokines in the plasma of healthy individuals at multiple time points (Figure 1). The levels of immunoreactive IL-1β were significantly downregulated at the 0time and 30-min time point after HELP exposure compared with those observed before the exposure (IL-1β-After 0time: 0.66-fold, p=0.00005; IL-1β-After 30-min: 0.83-fold, p=0.025). Moreover, the levels of immunoreactive IL-6 were significantly downregulated at the 30-min time point after HELP exposure compared with those observed before the exposure (IL-6: 0.68-fold, p=0.039). Under these conditions, HELP exposure did not affect the levels of immunoreactive IL-10, IL-18, TNF-α, or TGF-β (Figure 1c-f).
Effect of HELP exposure on immunoreactive hormones in the plasma of healthy individuals
We examined the effect of HELP exposure (9 kV/electrode + 9 kV/electrode) for 30 min on immunoreactive hormones in the plasma of healthy individuals to determine the specificity (Table 1). HELP exposure did not affect the levels of immunoreactive adrenaline, DHEAS, histamine, insulin, neuropeptide Y, serotonin, or somatostatin.



Docking simulation of lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6 with a homology model of TRPM8

 Acute EF exposure induces a notable increase in the levels of lysoPC-22:4 in the plasma of healthy subjects [28]. LysoPC-16:0 activates TRPM8 in CHO cells expressing TRPM8 [30]. Therefore, we examined the in silico docking of lysoPC-22:4 in the active site of TRPM8 using the AutoDock Vina software. We set the number of output poses to 20, with a total of 100 candidate conformations. LysoPC-22:4 showed good binding energy (-10.8 kcal/mol) (Table 2). LysoPC-22:4 formed hydrogen bonds with Tyr-745, Glu-782, and Tyr-1005 (Figure 1a). The results indicate that lysoPC-22:4 binds to the TRPM8 channel. Under these conditions, icilin (a well-known TRPM8 agonist) showed a strong interaction energy of -11.4 kcal/mol (Figure 1b, Table 2). In addition, we examined the in silico docking of lysoPE-20:4 and lysoPE-22:6 to the active site of TRPM8. A similar docking score was obtained using lysoPE-20:4 instead of lysoPC-22:4 (Table 2). LysoPE-22:6 showed a strong interaction energy of -11.4 kcal/mol (Table 2). LysoPE-20:4 interacted with Glu-782, Asn-799, Asp-802, Arg-842, and Tyr-1005, and lysoPE-22:6 interacted with Asn-799 and Arg-842 (Figure 1c and 1d, Table 2). Subsequently, we examined the in silico docking of 13-HODE, 9-HODE, and OEA to the active site of TRPM8 to determine the specificity. The binding energies were -9.1, -9.3, and -9.8 kcal/mol for 13-HODE, 9-HODE, and OEA, respectively (Table 2).



Discussion

In this study, we showed that the levels of IL-1β and IL-6 are sensitive to acute EF exposure in healthy humans. Notably, the absence of a pro-inflammatory cytokine TNF-α response indicates that the IL-1β and IL-6 responses are not adverse nonspecific actions of the immune function in humans. Our previous studies have shown that the IL-6 levels in the plasma of healthy individuals are ineffective after 15 min of acute EF exposure (Nakagawa-Yagi Y et al, 2015). An acute EF exposure of 30 min may be necessary to develop a significant inhibitory effect on the IL-6 levels in the plasma. Further studies are warranted to identify the optimal condition for the downregulation of pro-inflammatory cytokines such as IL-6 and IL-1β induced by EF exposure. The molecular mechanisms of changes in the plasma levels of IL-6 and IL-1β following HELP exposure are complex and may be interpreted in several ways. The endogenous lipid-derived metabolite 3-hydroxybutyrate (3-HBA) has been suggested to function as an endogenous inhibitor of the NLRP3 inflammasome (Goldberg EL et al, 2017). Using nontargeted human metabolomics, we recently demonstrated that the increase in plasma 3-HBA levels is elicited by EF exposure (Nakagawa-Yagi Y et al, 2016). Interestingly, Youm et al. reported that 3-HBA inhibits the secretion of IL-1β in LPS-stimulated human monocytes without significantly affecting the production of TNF-α (Youm Y-H et al, 2015). In addition, there is evidence that IL-6 is a known downstream target of IL-1β, consistently increased in the blood of patients with NLRP3 inflammasome-mediated conditions (Ridker PM et al, 2016). Thus, it is reasonable to speculate that EF exposure may inhibit the NLRP3 inflammasome through the upregulation of 3-HBA.


Furthermore, we previously showed an acute EF exposure (9 kV/electrode + 9 kV/electrode, 30 min)-induced increase in the levels of lysoPC-22:4 (approximately 1.47-fold) in other lipid-derived signaling molecules (Nakagawa-Yagi Y et al, 2017). Considering the role of the TRP channel family in changes in the plasma levels of lysoPC-22:4, Andersson et al. reported an increase in [Ca2⁺]i induced by lysoPC-16:0 in CHO cells transfected with TRPM8 (Andersson DA et al, 2007). Unfortunately, lysoPC-22:4 is not commercially available as a pure chemical reagent for pharmacological experiments. Thus, at present, it is not possible to investigate the effect of lysoPC-22:4 on the intracellular levels of calcium in CHO-K1 or HEK293T cells stably expressing hTRPM8. An increasing number of reports on virtual simulation are available in the literature (Nakagawa-Yagi Y et al, 2012; Nakagawa-Yagi Y et al, 2017). Studies involving in silico molecular docking have been conducted to support the pharmacological results. However, the crystal structure of hTRPM8 has not yet been determined. Thus, we focused on the homology modeling of TRPM8. In the present study, the docking simulation showed that lysoPC-22:4 has good binding affinity (-10.8 kcal/mol). The docking scores were compared with several well-known transient receptor potential vanilloid 1 (TRPV1) agonists such as 13-HODE, 9-HODE, and OEA to determine the relative affinity further (Ahern GP, 2003). These results suggest that TRPV1 agonists exhibit weaker affinity than icilin, lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6 with a homology model of TRPM8. In key interacting residues, a previous study using the TRPM8 homology model (PDB ID: 1QGR) with icilin binding pockets reported hydrogen bonding to Tyr-745 (Pedretti A et al, 2009). Another study on menthol, a well-known TRPM8 agonist, showed that mutating arginine at position 842 in S4 of TRPM8 to alanine decreases the affinity for menthol (Janssens A et al, 2011; Patwardhan AM et al, 2010). Once the crystal structure of TRPM8 is determined, it may be interesting to identify the binding pocket of lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6 in hTRPM8. However, lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6 also activate the G protein-coupled receptor 119, raising the possibility that these receptors may serve as targets for lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6 during EF exposure (Nakagawa-Yagi Y et al, 2017).


 Considerable evidence for the modulation of cytokines has been obtained from animal models of arthritis and neuropathic pain (Pan RY et al, 2000; Naito Y et al, 2009; Sacerdote P et al, 2013; Venkatesha SH et al, 2015). In particular, Naito et al. reported that static EF exposure inhibits the increased expression of IL-1β, but not of TNFα in arthitic hind paws (Naito Y et al, 2009). Interestingly, the sensitivity of cytokines in that study was comparable to that observed in the present study. On the other hand, Khalil et al. reported that TRPM8 in macrophages modulates colitis through a balance-shift in the production of pro-inflammatory and anti-inflammatory cytokines (Khalil M et al, 2016). Moreover, Ramachandran et al. reported that activation of TRPM8 by icilin attenuates trinitrobenzenesulfonic acid- or dextran sodium sulfate-induced colonic inflammation in in vitro models (Ramachandran R et al, 2013). Thus, it is reasonable to speculate that EF exposure may alleviate inflammation through the binding of TRPM8 by lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6. However, it is unclear whether changes in the levels of IL-1β and IL-6 may be attributed to neurons, macrophages, melanocytes, or keratinocytes. Although the underlying mechanisms of anti-inflammation by EF exposure remain to be elucidated, the role of lysoPC-22:4, lysoPE-20:4, or lysoPE-22:6 as endogenous agonist of TRPM8 may be potential mechanisms. Thus, it is conceivable that the decrease in IL-1β and IL-6 levels is, at least in part, responsible for the improvement observed in leprosy patients with neuroinflammation undergoing EF exposure (Shiga K et al, 1967; Nakamura K et al, 1970). Moreover, Proudfoot et al. reported that activation of TRPM8 elicits analgesia in chronic neuropathic pain models (Proudfoot CJ et al, 2006). There is also evidence that activation of TRPM8 exerts an analgesic effect on acute and inflammatory pain (Liu B et al, 2013). Interestingly, Vanmolkot and de Hoon reported increased blood C-reactive protein (CRP) levels in young adult patients with migraine (Vanmolkot FH et al, 2007). Using lipidomic analysis of serum samples, Ren et al. recently reported that the levels of lysoPE-22:6 are decreased in migraine patients (Ren C et al, 2018). Thus, it is reasonable to speculate that EF exposure alleviates headache such as migraine via upregulation of lysoPE-22:6. It may be interesting to evaluate the possible effect of EF exposure on migraine in future studies.


 Chronic inflammation in aging has also been proposed as a strong risk factor for morbidity and mortality in elderly individuals (Franceschi C et al, 2014). Of note, centenarians cope with chronic subclinical inflammation through an anti-inflammatory response termed “anti-inflammaging” (Minciullo et al, 2016). It may be interesting to evaluate the possible effect of repetitive HELP exposure on human longevity in future studies.


Considerable evidence regarding an association between IL-6 and sleep quality has been obtained from studies involving aging females and a meta-analysis of cohort studies (Fridman EM et al, 2005; Hong S et al, 2005; Okun ML et al, 2007). Interestingly, Irwin et al. reported that sleep disturbance is associated with high levels of CRP and IL-6, but not TNF-α (Irwin MR et al, 2016). In contrast, Milrad et al. reported that poor sleep quality is associated with greater circulating levels of TNF-α, IL-1β, and IL-6 (Milrad SF et al, 2017). In the present study, repeated EF treatment was not performed. Therefore, it is reasonable to speculate that EF therapy alleviates insomnia, at least in part, through the downregulation of IL-1β and IL-6 (Nawarat S et al, 1999; Zhang L et al, 2012; Ohtsuki T et al, 2017). Further basic reseasrch studies are warranted to elucidate the alleviative effect of endogenous lipid-derived signaling molecules such as lysoPC-22:4, lysoPE-20:4, or lysoPE-22:6 on sleep disturbance.

In conclusion, acute HELP exposure induced marked inhibitory effects on the plasma levels of IL-1β and IL-6 in healthy individuals. in silico molecular docking of lysoPC-22:4, lysoPE-20:4, and lysoPE-22:6 was observed for TRPM8. Our findings provide insight into the molecular mechanisms involved in the alleviation of headache and insomnia induced by the HELP device. These mechanisms may also be important for defense against inflammaging.





References

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 PDF of academic paper is here(English)
 

4. A part of the research paper in 2019

 
Nakagawa-Yagi Y et al, Integrative Molecular Medicine Vol.6, No.5: Page 1-8 (2019)

Targeted lipidomics reveals changes in N-acyl serines by acute exposure to an electric field: Molecular insights into the docking of N-18:1 serine interaction with TRPV1 or PPAR-α



Introduction

 High-voltage electric field (EF) therapy may be an effective treatment for shoulder stiffness, headache, insomnia, and chronic constipation [1-19]. Although EF therapy was discovered about 90 years ago, the molecular mechanisms associated with its health benefits remain elusive. A therapeutic device that exposes the human body to high-voltage electric potential (HELP) was approved by the Ministry of Health, Labour and Welfare in Japan [1-19]. A review of the literature suggests that HELP exposure may be an alternative therapy for several conditions. Our previous attempts at finding HELP exposure-induced plasma biomarkers, using liquid chromatography (LC)-time-of-flight mass spectrometry, led to the detection of lipids such as palmitic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, cis-8,11,14-eicosatrienoic acid (DGLA), cis-5,8,11,14,17-eicosapentaenoic acid, and cis-4,7,10,13,16,19-docosahexaenoic acid [12]. Recent methods for quantifying lipid mediators have taken advantage of selected reaction monitoring (SRM) analysis [17-18]. Using SRM, we showed enhanced levels of HELP exposure-induced lipids, including lysophosphatidylcholine-22:4, lysophosphatidylethanolamine-20:4, and lysophosphatidylethanolamine-22:6 in the plasma of healthy individuals [17-18]. Therefore, we hypothesized that changes in plasma lyso-form of phosphatidylethanolamine levels after EF exposure may be linked to changes in N-acyl serines. The stimulatory effects of N-acyl serines, such as N-18:1 serine and N-16:0 serine on cell number in osteoblastic MC3T3 E1 cells, reported recently [20], prompted an investigation into detection of plasma N-acyl SERs using SRM in healthy subjects subjected to a single HELP stimulation. In this study, we demonstrated that N-18:1 serine and N-16:0 serine were upregulated by HELP exposure (9 kV/electrode + 9 kV/electrode, 30 min). In addition, we conducted an in silico docking simulation to explore the interactions between N-18:1 serine or N-16:0 serine and the active site of TRPV1, or PPAR-α


Results

Effect of HELP exposure on N-acyl serine in the plasma of healthy individuals

 We analyzed the effect of HELP exposure (9 kV/electrode + 9 kV/electrode) for 30 min on N-acyl serines (Figure 1). Plasma levels of N-16:0 serine and N-18:1 serine were significantly upregulated at the time-0 after HELP exposure compared with levels before exposure (N-16:0 serine: 1.42-fold, p=0.0305; N-18:1 serine: 1.49-fold, p=0.0315). Under these conditions, HELP exposure did not affect the levels of N-18:0 serine, N-18:2 serine, N-20:4 serine, and N-22:6 serine (Figure 1).



Effect of HELP exposure on N-acyl ethanolamine ethanolamines in the plasma of healthy individuals

 Because N-acyl serines are structurally similar to N-acyl ethanolamines, we next analyzed the effect of HELP exposure (9 kV/electrode + 9 kV/electrode) for 30 min on N-acyl ethanolamines (Figure 2). Plasma levels of N-16:0 ethanolamine, N-18:1 ethanolamine, and N-18:2 ethanolamine were significantly upregulated at the 30-min time point after HELP exposure compared with those observed before the exposure (N-16:0 ethanolamine: 1.24-fold, p=0.0300; N-18:1 ethanolamine: 1.27-fold, p=0.0285; N-18:2 ethanolamine: 1.34-fold, p=0.0170). Under these conditions, HELP exposure did not affect the level of N-18:0 ethanolamine (Figure 2).



Docking simulation of N-18:1 serine, N-16:0 serine, N-18:1 ethanolamine, N-16:0 ethanolamine, or N-18:2 ethanolamine with a homology model of TRPV1

 N-18:1 ethanolamine and N-18:2 ethanolamine evoke an increase in intracellular calcium in HEK293 cells expressing hTRPV1 [24]. Therefore, we hypothesized that increased plasma N-18:1 serine and N-16:0 serine levels after HELP exposure may be linked to its activation as an endogenous agonist of TRPV1. We examined the in silico docking of N-18:1 serine, N-16:0 serine, or a well-known TRPV1 agonist, capsaicin, in the active site of TRPV1 using AutoDock Vina software [25-27]. We set the number of outputs poses to 20, with a total of 100 candidate conformations. Capsaicin showed a strong interaction energy of -7.915 kcal/mol (Table 1). Capsaicin formed hydrogen bonds with Thr-550, Ser-512, and Glu-570 (Figure 3a, Table 1). Under these conditions, N-18:1 serine showed good binding energy of -6.359 kcal/mol (Table 1). N-18:1 serine formed hydrogen bonds with Thr-550, and Ala-666 (Table 1, Figure 3b). A similar docking score was obtained using 9-HODE instead of N-18:1 serine (Table 1). 9-HODE, an endogenous agonist of TRPV1, formed hydrogen bonds with Thr-550, and Ala-666 (Table 1, Figure 3c). In addition, N-16:0 serine showed good binding energy of -6.077 kcal/mol (Table 1). N-16:0 serine formed hydrogen bonds with Thr-550, and Ala-666 (Table 1, Figure 3d). Subsequently, we examined the in silico docking of N-acyl ethanolamines to the active site of TRPV1 to determine specificity. The binding energies were -6.421, -6.227, and -5.767 kcal/mol for N-18:2 ethanolamine, N-18:1 ethanolamine, and N-16:0 ethanolamine, respectively (Table 1).



Docking simulation of N-18:1 serine, N-16:0 serine, N-18:1 ethanolamine, N-16:0 ethanolamine, or N-18:2 ethanolamine with a model of PPAR-α

 N-18: ethanolamine is known to induce lipolysis as a putative endogenous activator of PPAR-α [13, 28-29]. We examined the in silico docking of N-18:1 serine, N-16:0 serine, or a well-known PPAR-α agonist, bezafibrate, in the active site of PPAR-α using AutoDock Vina software [30]. We set the number of outputs poses to 20, with a total of 100 candidate conformations. Bezafibrate showed a strong interaction energy of -9.323 kcal/mol (Table 2). Bezafibrate formed hydrogen bonds with Ser-280, Tyr-314, His-440, and Tyr-464 (Figure 4a, Table 2). Under these conditions, N-18:1 serine showed good binding energy of -7.366 kcal/mol (Table 2). N-18:1 serine formed hydrogen bonds with Ser-280, Tyr-314, and His-440 (Figure 4b, Table 2). In addition, N-16:0serine showed good binding energy of -7.161 kcal/mol (Table 2). N-16:0 serine formed hydrogen bonds with Ser-280, Tyr-314, and Tyr-464 (Figure 4c, Table 2). A similar docking score was obtained using N-18:1 ethanolamine instead of N-16:0 serine (Table 2). N-18:1 ethanolamine formed hydrogen bonds with Ser-280, Tyr-314, and His-440 (Figure 4d, Table 2). Subsequently, we examined the in silico docking of N-16:0 ethanolamine and N-18:2 ethanolamine to the active site of PPAR-α to determine specificity. The binding energies were -6.570 and -6.979, respectively (Table 2).



Effect of N-18:1 serine on FABP1 mRNA expression in human hepatocellular carcinoma HepG2 cells

 Because FABP1 and peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) are PPAR-α-responsive gene [12, 31], we next confirmed the effect of N-18:1 serine treatment on FABP1 mRNA using qRT-PCR (Figure 5a). In human HepG2, N-18:1 serine produced a dose-dependent increase in FABP1 mRNA expression (N-18:1 serine-10 µM: 1.48-fold, p=0.0164; N-18:1 serine-30 µM: 1.98-fold, p=0.0112; N-18:1 serine-100 µM: 2.02-fold, p=0.0009). Next, we determined if bezafibrate, a well-known PPAR-α agonist, mimicked the effect of N-18:1 serine in human HepG2 cells. Bezafibrate significantly enhanced FABP1 mRNA expression (bezafibrate-10 µM: 2.86-fold, p=0.0119). We also examined the effect of N-18:1 serine on ACOX1 mRNA expression in human HepG2 cells. N-18:1 serine increased ACOX1 mRNA expression (Figure 5b). We further evaluated whether the PPAR-α antagonist GW6471 attenuated the effect of N-18:1 serine-stimulated FABP1 expression. N-18:1 serine (30 µM)-stimulated FABP1 response was almost completely abolished by 30 µM GW6471 (Figure 5c).



Discussion

 In this study, we showed that the levels of N-18:1 serine and N-16:0 serine were sensitive to acute EF exposure in healthy humans. The blood concentration of N-18:1 serine in human healthy controls was consistent with that obtained previously by quantitative analysis (Braghithy S et al, 2019). In our previous screening using non-targeted metabolomics, we found that EF exposure (9 kV/electrode + 9 kV/electrode)-induced 1.24-fold increase in N-18:1 ethanolamine (Nakagawa-Yagi Y et al, 2014). In the present study, using SRM analysis, EF exposure induced a 1.27-fold increase in the same lipid moiety. Little is known about the biosynthesis of N-acyl serines, though some possible N-acyl ethanolamines biosynthetic pathways have been proposed (Ogura Y et al, 2016). Interestingly, a family of phospholipase A/acyltransferase (PLAAT) that contributes to N-acyl ethanolamines formation has been discovered (Uyama T et al, 2017). However, the detailed mechanisms of EF-induced changes in N-18:1 serine, N-16:0 serine, N-18:1 ethanolamine, N-16:0 ethanolamine, and N-18:2 ethanolamine remain to be elucidated.


 The molecular targets of N-18:1 serine and N-16:0 serine are complex and may be interpreted in several ways. In the present study, docking simulation with TRPV1 showed that N-18:1 serine and N-16:0 serine have good binding affinity values. The results indicate that N-18:1 serine and N-16:0 serine binds to the TRPV1 channel. Ahern reported that N-18:1 ethanolamine can activate TRPV1 channels following treatment of the protein kinase C activator (Ahern GP, 2003). Therefore, interaction of the Tyr-511 residue does not have a critical role in the active site of TRPV1. In contrast to N-18:1 ethanolamine, the formation of a hydrogen bond with Thr-550 was detected with N-18:1 serine, N-16:0 serine, and the endogenous TRPV1 agonist, 9-HODE (Patwardhan AM et al, 2010). Another study, using cryo-electron microscopy structures of TRPV1 with capsaicin-binding pockets, reported hydrogen bonding to Thr-550 (Yang F et al, 2015; Yang F et al, 2017). These findings indicate that N-18:1 serine or N-16:0 serine may act as an endogenous agonist for TRPV1. Raboune et al. reported that N-22:6 serine has agonist activity in TRPV1-transfected HEK cells (Raboune S et al, 2014). Evaluation of the agonistic effect of N-18:1 serine or N-16:0 serine in functional assays may be warranted in future studies. The TRPV1 channel is a known pharmacological target to relive pain in post-therapeutic neuralgia (Vay L et al, 2012). In particular, Anand et al. reported that treatment with Qutenza, a patch containing 8% capsaicin, produced an analgesic effect through defunctionalization of the nociceptor (Anand P et al, 2011). Borbiro et al. recently reported that activating TRPV1 channels elicits analgesia by inhibiting mechanosensitive Piezo channel activity (Borbiro I et al, 2015). Thus, it is reasonable to speculate that EF exposure might alleviate pain through the binding of TRPV1 by N-18:1 serine, or N-16:0 serine. On the other hand, Ito et al. recently reported involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy (Ito N et al, 2013). Although repetitive EF treatment was not performed in the present study, it may be interesting to evaluate the possible effect of HELP exposure on skeletal muscle atrophy in aging.


 Eberlein et al. reported that an emollient containing N-16:0 ethanolamine improved atopic dermatitis symptoms in a prospective cohort study (Eberlein B et al, 2008), while Esposito et al. reported that N-16:0 ethanolamine treatment improved colon inflammation in an ulcerative colitis model through toll-like receptor 4 (TLR4)-dependent PPAR-α activation (Esposito G et al, 2014). In the present study, the docking simulation with PPAR-α showed that N-acyl serines have good binding affinity values for N-18:1 serine and N-16:0 serine. In particular, N-18:1 serine binding to PPAR-α was stabilized through the formation of hydrogen bonds with Ser-280, Tyr-314, and His-440. In key interacting residues, a previous study using the PPAR-α model (PDB ID:1i7g) with N-18:1 ethanolamine binding pockets reported hydrogen bonding to Ser-280, Tyr-314, and Phe-273 (Nakagawa-Yagi Y et al, 2014). Because FABP1 and ACOX1 are PPAR-α-responsive genes (Nakagawa-Yagi Y et al, 2014; Landrier JF et al, 2004), we examined their expression in this study. We previously reported that N-18:1 ethanolamine induced 2.8-fold increase in FABP6 gene expression using the Affymetrix GeneChip human genome U133 Plus 2.0 array (Nakagawa-Yagi Y et al, 2014). Our present study results showed that N-18:1 serine (30 µM) induced an approximately 2-fold increase in FABP1 mRNA expression in human HepG2 cells, which was inhibited by the PPAR-α antagonist GW6471. Thus, HELP exposure may induce FABP1 mRNA expression, at least in part, through activating PPAR-α by N-18:1 serine.


 There is evidence that PPAR-α agonists have unique pharmacological effects in vivo. Brandstät et al. reported that fibrates, such as bezafibrate, clofibrate, or fenofibrate, extends the lifespan of adult Caenorhabditis elegans (Brandstät S et al, 2013). Zeitler et al. reported that fenofibrate (200 mg/day) induced a quiescent state in patients with multiple benign symmetric lipomatosis (MSL) characterized by a rapid progression (Zeitler H et al, 2008). On the other hand, Shirai et al. reported that bezafibrate alleviated circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) (Shirai H et al, 2007). In bone mass and osteoclast formation, Stunes et al. reported that fenofibrate maintains bone mass in ovariectomized rats (Stunes AK et al, 2011), while Patel et al. reported that fenofibrate treatment induces decrease of osteoclast formation in cultured derived from mouse marrow (Patel JJ et al, 2014). Interestingly, Smoum et al. observed that treatment with N-18:1 serine stimulated the proliferation of osteoblastic MC3T3 E1 cells and primary cavarial osteoblast in vitro (Smoum R et al, 2010) and further, N-18:1 serine rescued ovariectomy-induced bone loss in vivo (Smoum R et al, 2010). On the other hand, Hashimoto reported activation of callus formation by EF exposure in a mongrel rabbit in vivo model (Hashimoto T et al, 1975). In addition, there is evidence that static EF exposure stimulates the differentiation of human cultured osteoblastic cells (Su CY et al, 2017). Although the underlying mechanisms of bone formation by EF exposure remain to be elucidated, evidence points to N-18:1 serine’s role as an endogenous signaling molecule. Thus, it is conceivable that the increase in N-18:1 serine levels in proportion to the duration of HELP treatment is, at least in part, responsible for slightly high bone density in males aged 70 to 90 years old (Harakawa S et al, 2014). In future, the possible effect of EF therapy on MSL, DSPS, and bone mass should be evaluated.


 Interestingly, Scuderi et al. reported that N-16:0 ethanolamine treatment exerts neuroprotective effects in a rat model of Alzheimer’s disease through PPAR-α involvement (Scuderi C et al, 2014). In addition, Campolongo et al. reported that post-training administration of N-18:1 ethanolamine in rats enhanced memory consolidation in a Morris water maze performance (Campolongo P et al, 2009). These memory-enhancing effects were mimicked by the PPAR-α agonist, GW7647, and was abolished in a mutant animal lacking PPAR-α (Campolongo P et al, 2009). Thus, it is reasonable to speculate that EF therapy facilitates memory consolidation via the activation of the PPAR-α signaling pathway by N-18:1 serine, N-16:0 serine, N-16:0 ethanolamine, N-18:2 ethanolamine, and N-18:1 ethanolamine. In future, it will be of interest to evaluate the possible effects of EF therapy on aging-related memory function.


 In conclusion, acute HELP exposure induced marked effects on plasma N-18:1 serine and N-16:0 serine levels in healthy subjects. In silico molecular docking of N-18:1 serine and N-16:0 serine was observed in models of TRPV1 and PPAR-α. In human HepG2 cells, N-18:1serine-stimulated FABP1 mRNA expression was sensitive to the PPAR-α antagonist, GW6471. Our findings provide insight into the molecular mechanisms behind the health benefits induced by HELP.




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