Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

DOI： 10.1016/j.bbrc.2024.150077

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The chemotherapeutic agent tegafur, a prodrug that prolongs the half-life of fluorouracil (5-FU), exerts antitumor effects against various cancers. Since tegafur is metabolized to 5-FU by CYP2A6 in the liver, the expression of CYP2A6 determines the effect of tegafur. Here, we report that the expression rhythm of Cyp2a5, a homolog of human CYP2A6, in female mice causes dosing time-dependent differences in tegafur metabolism. In the livers of female mice, CYP2A5 expression showed a circadian rhythm, peaking during the dark period. This rhythm is regulated by RORA, a core clock component, and abrogation of the CYP2A5 activity abolished the time-dependent difference in the rate of tegafur metabolism in female mice. Furthermore, administration of tegafur to mice transplanted with 4T1 breast cancer cells during the dark period suppressed increases in tumor size compared to female mice treated during the light period. Our findings reveal a novel relationship between 5-FU prodrugs and circadian clock machinery, potentially influencing antitumor effects, and contributing to the development of time-aware chemotherapy regimens for breast cancer.

DOI： 10.1016/j.bbrc.2024.149813

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Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light–dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

DOI： 10.3390/ijms25031841

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Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD.

DOI： 10.1016/j.trsl.2024.02.004

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The leading cause of death for patients with Duchenne muscular dystrophy (DMD), a progressive muscle disease, is heart failure. Prostaglandin (PG) D2, a physiologically active fatty acid, is synthesized from the precursor PGH2 by hematopoietic prostaglandin D synthase (HPGDS). Using a DMD animal model (mdx mice), we previously found that HPGDS expression is increased not only in injured muscle but also in the heart. Moreover, HPGDS inhibitors can slow the progression of muscle injury and cardiomyopathy. However, the location of HPGDS in the heart is still unknown. Thus, this study investigated HPGDS expression in autopsy myocardial samples from DMD patients. We confirmed the presence of fibrosis, a characteristic phenotype of DMD, in the autopsy myocardial sections. Additionally, HPGDS was expressed in mast cells, pericytes, and myeloid cells of the myocardial specimens but not in the myocardium. Compared with the non-DMD group, the DMD group showed increased HPGDS expression in mast cells and pericytes. Our findings confirm the possibility of using HPGDS inhibitor therapy to suppress PGD2 production to treat skeletal muscle disorders and cardiomyopathy. It thus provides significant insights for developing therapeutic drugs for DMD.

DOI： 10.3390/ijms25031846

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Pharmacology & Experimental Therapeutics (ASPET)  

Although vancomycin (VCM)-frequently used to treat drug-resistant bacterial infections-often induces acute kidney injury (AKI), discontinuation of the drug is the only effective treatment; therefore, analysis of effective avoidance methods is urgently needed. Here, we report the differences in the induction of AKI by VCM in 1/2-nephrectomized mice depending on the time of administration. Despite the lack of difference in the accumulation of VCM in the kidney between the light (ZT2) and dark (ZT14) phases, the expression of AKI markers due to VCM was observed only in the ZT2 treatment. Genomic analysis of the kidney suggested that the time of administration was involved in VCM-induced changes in monocyte and macrophage activity, and VCM had time-dependent effects on renal macrophage abundance, ATP activity, and interleukin (IL)-1β expression. Furthermore, the depletion of macrophages with clodronate abolished the induction of IL-1β and AKI marker expression by VCM administration at ZT2. This study provides evidence of the need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced AKI as well as the potential for macrophage-targeted AKI therapy. SIGNIFICANCE STATEMENT: There is a time of administration at which vancomycin (VCM)-induced renal injury is more and less likely to occur, and macrophages are involved in this difference. Therefore, there is a need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced acute kidney injury as well as the potential for macrophage-targeted acute kidney injury therapy.

DOI： 10.1124/jpet.123.001864

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Chronic kidney disease (CKD) induces an imbalance in the intestinal microbiota, affecting various physiological functions and leading to cardiovascular inflammation and fibrosis. However, the cardiotoxic impact of intestinal microbiota-derived uremic substances in advanced renal dysfunction remains unexplored. Therefore, we developed a 5/6 nephrectomy (5/6Nx) mouse model to investigate the intestinal microbiota and the effects of administering vancomycin (VCM) on the microbiota and the cardiac pathology associated with CKD. Despite VCM administration after the development of irreversible glomerulosclerosis and tubulointerstitial fibrosis, blood indoxyl sulfate and phenyl sulfate levels, which are intestinal bacteria-derived uremic substances, brain natriuretic peptide levels, and the fibrotic area in the heart were decreased. Moreover, VCM administration prevented 5/6Nx-induced weight loss and prolonged survival time. Our findings suggest that VCM-induced changes in the intestinal microbiota composition ameliorate heart failure and improve survival rates by reducing intestinal microbiota-derived cardiotoxic substances despite advanced renal dysfunction. This highlights the potential of using the intestinal microbiota as a target to prevent and treat cardiovascular conditions associated with CKD.

DOI： 10.1016/j.bbrc.2023.07.015

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Over 80% of patients affected by cancer develops cancer-related pain, one of the most feared consequences because of its intractable nature, particularly in the terminal stage of the disease. Recent evidence-based recommendations on integrative medicine for the management of cancer pain underline the role of natural products. The present systematic review and meta-analysis aims at appraising for the first time the efficacy of aromatherapy in cancer pain in clinical studies with different design according to the most updated Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 recommendations. The search retrieves 1002 total records. Twelve studies are included and six are eligible for meta-analysis. The present study demonstrates significant efficacy of the use of essential oils in the reduction of the intensity of pain associated with cancer (p < 0.00001), highlighting the need for earlier, more homogeneous, and appropriately designed clinical trials. Good certainty body of evidence is needed for effective and safe management of cancer-related pain using essential oils by establishment of a step-by-step preclinical-to-clinical pathway to provide a rational basis for clinical use in integrative oncology. PROSPERO registration: CRD42023393182.

DOI： 10.3390/ijms24087085

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Molecules  

Chronic pain is one of the most common causes of the need for clinical evaluation, acquiring more importance in the elderly with cognitive impairment. Reduced self-reporting capabilities cause unrelieved pain contributing to the development of agitation. Safe and effective pain treatment can afford the management of agitation without the serious increase in death risk associated with neuroleptics. To this aim, the essential oil of bergamot (BEO), proven by rigorous evidence to have strong preclinical anti-nociceptive and anti-allodynic properties, has been engineered (NanoBEO, patent EP 4003294) to allow randomized, double-blind, placebo-controlled trials (BRAINAID, NCT04321889). The present study: (1) assesses the analgesic effects of a single therapeutic dose of NanoBEO, as supplied by an airless dispenser for clinical translation, in models of inflammatory, neuropathic, and sensitization types of pain relevant to clinic; (2) provides a dose-response analysis of the efficacy of NanoBEO on scratching behavior, a typical behavioral disturbance occurring in dementia. A single therapeutic dose of NanoBEO confirms efficacy following thirty minutes pre-treatment with capsaicin and on the central sensitization phase induced by formalin. Moreover, it has an ID50 of 0.6312 mg and it is efficacious on static and dynamic mechanical allodynia. Altogether, the gathered results strengthen the potential of NanoBEO for clinical management of pain and agitation.

DOI： 10.3390/molecules27154987

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

Murine models are fundamental in the study of clinical conditions and the development of new drugs and treatments. Transgenic technology has started to offer advantages in oncology, encompassing all research fields related to the study of painful syndromes. Knockout mice or mice overexpressing genes encoding for proteins linked to pain development and maintenance can be produced and pain models can be applied to transgenic mice to model the most disabling neurological conditions. Due to the association of movement disorders with sensitivity and pain processing, our group focused for the first time on the role of the torsinA gene GAG deletion-responsible for DYT1 dystonia-in baseline sensitivity and neuropathic responses. The aim of the present report are to review the complex network that exists between the chaperonine-like protein torsinA and the baseline sensitivity pattern-which are fundamental in neuropathic pain-and to point at its possible role in neurodegenerative diseases.

DOI： 10.3390/ijms23158580

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The demand for natural products is steadily increasing, and pharmacotechnological engineering is needed to allow rigorous investigation of their efficacy and safety in clinical conditions representing still unmet needs. Among aged patients affected by dementia, up to 80% of residents in nursing homes suffer from chronic pain and 97% from fluctuant neuropsychiatric symptoms (NPS), of which the most challenging is agitation. It is, at least in part, due to undertreated pain and treated with antipsychotics almost doubling the risk of death. In the frame of a scoping review assessing the existence of essential oils undergoing engineering pharmacotechnological processes using solid lipid nanoparticle delivery systems for clinical translation in pain and/or neuropsychiatric symptoms of dementia following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR), here we identified that the sole essential oil engineered to overcome the criticisms of aromatherapy clinical trials in pain and dementia is the essential oil of bergamot (BEO). Therefore, we present the process leading to the actually ongoing randomized, double-blind, placebo-controlled NCT04321889 clinical trial to assess the efficacy and safety of intervention with bergamot in the management of agitation and pain in severe dementia to be followed also for the proof of concept of efficacy and safety of other essential oils.

DOI： 10.3390/pr10071340

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutics  

The essential oil of bergamot (BEO) has consistently proven antinociceptive and antiallodynic properties. Accordingly, the analgesic efficacy of the decolored essential oil (DEC), with higher levels of limonene, and the deterpenated (DET) fraction, with higher levels of linalool and linalyl acetate, was investigated using a formalin test after inhalation. The present study was aimed at characterizing the effects of BEO, its components with the highest pharmacological activity (represented by linalool, limonene, and linalyl acetate), and its DEC and DET fractions on the formalin test after transdermal administration relevant to clinical translation through topical application. To this aim, the schedule of intervention involved administration immediately after formalin injection or as a 5 min pretreatment followed by washout in ddY-strain mice. This study demonstrates, for the first time, the significant analgesic effect of all three constituents in the first and second phases, accounting for the efficacy of the essential oil in the formalin test. While all fractions revealed equal activity toward the phytocomplex in the early phase, the reduction in time of licking/biting during the late phase was more markedly induced by DEC. Moreover, pretreatment with BEO and its fractions followed by washout did not produce a significant reduction in licking/biting time in both phases of formalin-induced nociceptive response.

DOI： 10.3390/pharmaceutics14051006

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Background: Purpose of the present systematic review is to investigate preclinical evidence in favor of the working hypothesis of efficacy of cannabinoids in ocular pain treatment. Methods: Literature search includes the most relevant repositories for medical scientific literature from inception until November, 24 2021. Data collection and selection of retrieved records adhere to PRISMA criteria. Results: In agreement with a priori established protocol the search retrieved 2471 records leaving 479 results after duplicates removal. Eleven records result from title and abstract screening to meet the inclusion criteria; only 4 results are eligible for inclusion in the qualitative synthesis impeding meta-analysis. The qualitative analysis highlights the antinociceptive and anti-inflammatory efficacy of Δ8-tetrahydrocannabinol, cannabidiol and its derivative HU-308 and of new racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229. Moreover, CB2R agonists RO6871304 and RO6871085 and CB2R ligand HU910 provide evidence of anti-inflammatory efficacy. CB2 agonist HU308 reduces of 241% uveitis-induced leukocyte adhesion and changes lipidome profile. Methodological and design issues raise concern of risk of bias and the amount of studies is too small for generalization. Furthermore, the ocular pain model used can resemble only inflammatory but not neuropathic pain. Conclusions: The role of the endocannabinoid system in ocular pain is underinvestigated, since only two studies assessing the effects of cannabinoid receptors modulators on pain behavior and other two on pain-related inflammatory processes are found. Preclinical studies investigating the efficacy of cannabinoids in ocular inflammatory and neuropathic pain models are needed to pave the way for clinical translation.

DOI： 10.1016/j.biopha.2021.112505

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutics  

Bergamot essential oil (BEO) is endowed with consistent and reproducible antinociceptive and anti-allodynic properties when administered via an inhalation route. However, the effects of its main constituents and of its decolored (DEC) and deterpenated (DET) fractions, which are enriched in limonene or in linalool and linalyl acetate, respectively, on spontaneous motor activity related to anxiety and on formalin-induced licking/biting biphasic behavior have never been investigated before. Therefore, the present research aims to characterize the role of BEO components on an experimental pain model that is relevant to clinical translation. Under our present experimental conditions, a paper filter disc soaked with different volumes of the phytocomplex and of its fractions that was applied at the edge of the observation chamber allowed the effects on the spontaneous motor activity and on the formalin-induced nocifensive response in ddY-strain mice to be studied. The present research demonstrated the effects of the DEC fraction of BEO on motor activity and on formalin-induced licking/biting behavior for the first time, proving that limonene is implicated in reduced motor activity and that it is important for the analgesic effect.

DOI： 10.3390/pharmaceutics14020312

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Dysfunction of the circadian clock has been implicated in the pathogenesis of cardiovascular disease. The CLOCK protein is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in numerous physiological processes. However, here we report that chronic kidney disease (CKD)-induced cardiac inflammation and fibrosis are attenuated in Clk/Clk mice even though they have high blood pressure and increased serum angiotensin II levels. A search for the underlying cause of the attenuation of heart disorder in Clk/Clk mice with 5/6 nephrectomy (5/6Nx) led to identification of the monocytic expression of G protein-coupled receptor 68 (GPR68) as a risk factor of CKD-induced inflammation and fibrosis of heart. 5/6Nx induces the expression of GPR68 in circulating monocytes via altered CLOCK activation by increasing serum levels of retinol and its binding protein (RBP4). The high-GPR68-expressing monocytes have increased potential for producing inflammatory cytokines, and their cardiac infiltration under CKD conditions exacerbates inflammation and fibrosis of heart. Serum retinol and RBP4 levels in CKD patients are also sufficient to induce the expression of GPR68 in human monocytes. Our present study reveals an uncovered role of monocytic clock genes in CKD-induced heart failure.

DOI： 10.1038/s41467-021-23050-x

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Background: The demand for essential oils (EOs) has been steadily growing over the years. This is mirrored by a substantial increase in research concerned with EOs also in the field of inflammatory and neuropathic pain. The purpose of this present systematic review and meta-analysis is to investigate the preclinical evidence in favor of the working hypothesis of the analgesic properties of EOs, elucidating whether there is a consistent rational basis for translation into clinical settings. Methods: A literature search has been conducted on databases relevant for medical scientific literature, i.e., PubMed/MEDLINE, Scopus, and Web of Science from database inception until November 2, 2020, following the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) criteria for systematic reviews and meta-analyses. Results: The search was conducted in order to answer the following PICOS (participants/population, interventions, comparisons, outcomes, and study design) question: are EOs efficacious in reducing acute nociceptive pain and/or neuropathic pain in mice experimental models? The search retrieved 2,491 records, leaving 954 studies to screen after the removal of duplicates. The title and abstract of all 954 studies were screened, which left 127 records to evaluate in full text. Of these, 30 articles were eligible for inclusion. Conclusion: Most studies (27) assessed the analgesic properties of EOs on acute nociceptive pain models, e.g. the acetic acid writhings test, the formalin test, and the hot plate test. Unfortunately, efficacy in neuropathic pain models, which are a more suitable model for human conditions of chronic pain, had fewer results (only three studies). Moreover, some methodologies raised concerns in terms of the risk of bias. Therefore, EOs with proven efficacy in both types of pain were corroborated by methodologically consistent studies, like the EO of bergamot, which should be studied in clinical trials to enhance the translational impact of preclinical modeling on clinical pain research.

DOI： 10.3389/fphar.2021.640128

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Targeted protein degradation by proteolysis-targeting chimera (PROTAC) is one of the exciting modalities for drug discovery and biological discovery. It is important to select an appropriate linker, an E3 ligase ligand, and a target protein ligand in the development; however, it is necessary to synthesize a large number of PROTACs through trial and error. Herein, using a docking simulation of the ternary complex of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, H-PGDS, and cereblon, we have succeeded in developing PROTAC(H-PGDS)-7 (6), which showed potent and selective degradation activity (DC50 = 17.3 pM) and potent suppression of prostaglandin D2 production in KU812 cells. Additionally, in a Duchenne muscular dystrophy model using mdx mice with cardiac hypertrophy, compound 6 showed better inhibition of inflammatory cytokines than a potent H-PGDS inhibitor TFC-007. Thus, our results demonstrated that in silico simulation would be useful for the rational development of PROTACs.

DOI： 10.1021/acs.jmedchem.1c01206

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Neuropathic pain is characterized by mechanical allodynia and thermal hyperalgesia to heat, and it affects some 20% of European population. Patients suffering from several neurologic diseases experience neuropathic pain, often finding no relief in therapy. Transgenic mice expressing the gene encoding the human mutant (hMT) or the human wild-type (hWT) torsin A represent a preclinical model of DYT1 dystonia which is the most common form of early-onset inherited dystonia. Baseline thermal sensitivity and hyperalgesia to heat have never been studied in models of dystonia. Therefore, the aim of this research has been to characterize thermal sensitivity in baseline conditions and hyperalgesia to heat after the induction of neuropathic pain through the spinal nerve ligation (SNL) model in mice overexpressing human wild-type and mutated torsin A in comparison to non-transgenic C57BL/6 mice. According to our results, the paw withdrawal latency time to heat in the Hargreaves' test is significantly lower in the hMT mice (Kruskal-Wallis test = 6.933; p = 0.0312*; hMT vs. hWT p = 0.0317*). On the other hand, no significant differences in SNL-induced thermal hyperalgesia was found among the three strains (Friedman test = 4.933; p = 0.1019). Future studies are needed to better understand the role of torsin A in sensory processing of heat stimuli.

DOI： 10.3390/life11090985

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Dementia is one of the most common causes of disability worldwide characterized by memory loss, cognitive impairment, and behavioral and psychological symptoms (BPSD), including agitation. Treatment of the latter consists of the off-label use of harmful atypical antipsychotics, though a significant reduction is afforded by pain control. The use of an essential oil endowed with analgesic properties and devoid of toxicity would represent an important option for the management of agitation in dementia. Therefore, the aim of this study was to engineer a nanotechnology delivery system based on solid lipid nanoparticles loaded with bergamot essential oil (BEO) and devised in the pharmaceutical form of an odorless cream (NanoBEO) to confirm its analgesic efficacy for further development and application to control agitation in dementia. BEO has proven strong antinociceptive and anti-allodynic properties and, in its bergapten-free form, it is completely devoid of phototoxicity. NanoBEO has been studied in vivo confirming the previously reported analgesic activity of BEO to which is now added its anti-itching properties. Due to the nanotechnology delivery system, the stability of titrated BEO components is guaranteed. Finally, the latter invention, currently under patent consideration, is smell-devoid allowing efficacy and safety to be established in double-blind clinical trials; until now the latter studies have been impeded in aromatherapy by the strong odor of essential oils. A clinical trial NCT04321889 has been designed to provide information about the efficacy and safety of NanoBEO on agitation and pain in patients suffering from severe dementia.

DOI： 10.3390/pharmaceutics13030379

Language：Japanese   Publishing type：Research paper (scientific journal)  

Analgesic Effects of PREGABALIN OD TABLETS “NIPRO” in a Mouse Model of Neuropathic Pain

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Neuropathic pain is an intractable chronic pain condition that is mainly caused by allodynia. We had previously reported that intra-plantar administration of bergamot essential oil (BEO) containing an aromatic compound significantly suppressed partial sciatic nerve ligation (PSNL)-induced mechanical allodynia via opioid mu receptors in mice. However, it has also been reported that the inhalation of BEO reduced formalin-induced nociceptive responses. Therefore, we aimed to elucidate whether the analgesic action of BEO is mediated by olfactory stimulation through volatile components. In the current study, BEO was continuously administered with an osmotic pump during PSNL surgery, and the effects on mice behavior were examined pharmacologically using a double activity monitoring system, which can detect two-dimensional planar motion in a cage with an infrared beam sensor as well as active motion with a running wheel. Here, we report that the two-dimensional planar activity significantly increased in mice with PSNL in the light phase (from 8 o'clock to 20 o'clock) but not in the dark phase (from 20 o'clock to 8 o'clock) from the second day after surgery. However, this increase was not observed when BEO was continuously administered. The effect of BEO on the two-dimensional planar counts in mice with PSNL was antagonized by naloxone hydrochloride. Regarding the running wheel activity, the number of rotations decreased by PSNL in the dark phase from the 8th day after surgery. However, this was not apparent with BEO use. The effect of BEO on the number of rotations was also antagonized by naloxone hydrochloride. Furthermore, inhalation of BEO in PSNL mice did not affect mechanical allodynia or the two-dimensional planar motion or running wheel activities. These findings indicate that BEO exhibits an analgesic action, which is mediated by opioid receptors and not by the olfactory system.

DOI： 10.3389/fphar.2020.01310

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The essential oil obtained by the fresh fruit of Citrus bergamia Risso et Poiteau is used worldwide in aromatherapy to reduce pain, facilitate sleep induction, and/or minimize the effects of stress-induced anxiety. Preclinical pharmacological data demonstrate that bergamot essential oil (BEO) modulates specific neurotransmissions and shows an anxiolytic-relaxant effect not superimposable to that of the benzodiazepine diazepam, suggesting that neurotransmissions, other than GABAergic, could be involved. Several studies on essential oils indicate a role for serotonergic (5-HT) neurotransmission in anxiety. Interestingly, among serotonergic receptors, the 5-HT1A subtype seems to play a key role in the control of anxiety. Here, we report that modulation of the 5-HT1A receptor by selective agonist ((±)8-OH-DPAT) or antagonist (WAY-100635) may influence some of the anxiolytic-relaxant effects of BEO in Open Field and Elevated Plus Maze tests.

DOI： 10.3390/ijms21072597

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The expression levels of many cell-surface proteins vary with the time of day. Glycoprotein 2 (Gp2), specifically expressed on the apical surface of M cells in Peyer's patches, functions as a transcytotic receptor for mucosal antigens. We report that cAMP response element-binding protein (CREB) regulates the transcription of the Gp2 gene, thereby generating the circadian change in its expression in mouse Peyer's patches. The transcytotic receptor activity of Gp2 was increased during the dark phase when the Gp2 protein abundance increased. Rhythmic expression of clock gene mRNA was observed in mouse Peyer's patches, and expression levels of Gp2 mRNA also exhibited circadian oscillation, with peak levels during the early dark phase. The promoter region of the mouse Gp2 gene contains several cAMP response elements (CREs). Chromatin immunoprecipitation assays revealed that CREB bound to the CREs in the Gp2 gene in Peyer's patches. Forskolin, which promotes CREB phosphorylation, increased the transcription of the Gp2 gene in Peyer's patches. As phosphorylation of CREB protein was increased when Gp2 gene transcription was activated, CREB may regulate the rhythmic expression of Gp2 mRNA in Peyer's patches. These findings suggest that intestinal immunity is controlled by the circadian clock system.

DOI： 10.1111/gtc.12758

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The essential oil of bergamot (BEO) is one of the most common essential oils and is most familiar to the general public. The aims of this study were to investigate the effect of intraplantar (i.pl.) BEO on neuropathic allodynia induced by partial sciatic nerve ligation (PSNL) in mice and the opioid receptor subtypes involved in the antiallodynic effects of BEO. Our findings showed that a single dose of i.pl. administration of BEO significantly inhibited the PSNL-induced neuropathic pain using the von Frey test. The i.pl pretreatment with naloxone methiodide, a peripherally acting μ-opioid receptor preferring antagonist, β-funaltrexamine hydrochloride (β-FNA), a selective μ-opioid receptor antagonist, and β-endorphin antiserum significantly reversed the antiallodynic effect of BEO in the von Frey test, but not by naltrindole, the nonselective δ-opioid receptor antagonist and nor-binaltorphimine, the selective κ-opioid receptor antagonist. Furthermore, in the western blotting analysis, i.pl. administration of BEO resulted in a significant blockage of spinal extracellular signal-regulated protein kinase (ERK) activation induced by PSNL. Naloxone methiodide and β-FNA significantly reversed the blockage of spinal ERK activation induced by BEO. These results suggest that i.pl. injection of BEO-induced antiallodynic effect and blockage of spinal ERK activation may be triggered by activation of peripheral μ-opioid receptors.

DOI： 10.1016/j.neulet.2018.08.053

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One approach to increasing pharmacotherapy effects is administering drugs at times of day when they are most effective and/or best tolerated. Circadian variation in expression of pharmacokinetics- and pharmacodynamics-related genes was shown to contribute to dosing time-dependent differences in therapeutic effects of small molecule drugs. However, influence of dosing time of day on effects of high molecular weight formulations, such as drugs encapsulated in liposomes, has not been studied in detail. This study demonstrates that blood pressure rhythm affects dosing time-dependent variation in effects of high molecular weight formulations. Systolic blood pressure in sarcoma 180-bearing mice showed significant 24-h oscillation. Intratumoral accumulation of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA), an indicator of tumor vascular permeability, varied with dosing time of day, matching phases of blood pressure circadian rhythm. Furthermore, intratumoral accumulation of liposome-encapsulated oxaliplatin (Lipo-L-OHP) increased with increases in systolic blood pressure. Our findings suggest that circadian blood pressure oscillations may be an important factor to consider in dosing strategies for macromolecular drugs and liposomes in cancer therapy.

DOI： 10.1016/j.bbrc.2017.11.162

Language：English   Publishing type：Research paper (scientific journal)  

Clock genes encoding transcription factors that regulate circadian rhythms may inform chronomodulated chemotherapy, where time-dependent dose alterations might affect drug efficacy and reduce side effects. For example, inhibiting the essential cystine transporter xCT with sulfasalazine induces growth arrest in cancer cells. Although the anticancer effects of sulfasalazine have been studied extensively, its effects on transcriptional control of xCT expression have not been studied. Here, we show that sulfasalazine administration during the period of increased xCT expression improves its anticancer effects and that the Clock gene itself induces xCT expression and regulates its circadian rhythm. Our findings highlight the clinical potential of chronomodulated chemotherapy and the importance of xCT-mediated transcriptional regulation in the utility of such strategies. (C) 2017 AACR.

DOI： 10.1158/0008-5472.CAN-17-0720

Language：English   Publishing type：Research paper (scientific journal)  

The involvement of spinal release of histamine on nociceptive behaviors induced by spermine was examined in mice. Intrathecal spermine produced dose-dependent nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by spermine at 0.02 amol and 10 pmol were markedly suppressed by i.t. pretreatment with antiserum against histamine and were abolished in histidine decarboxylase-deficient mice. In histamine H-1 receptor-deficient mice, the nociceptive behaviors induced by spermine were completely abolished after treatment with 0.02 amol of spermine and significantly suppressed after treatment with 10 pmol of spermine. The i.t. pretreatment with takykinin NK1 receptor antagonists eliminated the nociceptive behaviors induced by 0.02 amol of spermine, but did not affect the nociceptive behaviors induced by 10 pmol of spermine. On the other hand, the nociceptive behaviors induced by spermine at both 0.02 amol and 10 pmol were suppressed by i.t. pretreatment with antagonists for the NMDA receptor polyamine-binding site. The present results suggest that the nociceptive behaviors induced by i.t. administration of spermine are mediated through the spinal release of histamine and are elicited via activation of NMDA receptors.

DOI： 10.1016/j.ejphar.2017.01.031

Language：English   Publishing type：Research paper (scientific journal)  

Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we showthat inhibition of G(0)/G(1) switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.ebiom.2016.10.008

Language：English   Publishing type：Research paper (scientific journal)  

Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found that Irp2 mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(19) tumors expressing the clock-mutant protein (CLOCK19) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

DOI： 10.1074/jbc.M115.713412

Language：English   Publishing type：Research paper (scientific journal)  

Chronic kidney disease (CKD) is associated with an increase in serum retinol; however, the underlying mechanisms of this disorder are poorly characterized. Here, we found that the alteration of hepatic metabolism induced the accumulation of serum retinol in 5/6 nephrectomy (5/6Nx) mice. The liver is the major organ responsible for retinol metabolism; accordingly, microarray analysis revealed that the hepatic expression of most CYP genes was changed in 5/6Nx mice. In addition, D-box-binding protein (DBP), which controls the expression of several CYP genes, was significantly decreased in these mice. Cyp3a11 and Cyp26a1, encoding key proteins in retinol metabolism, showed the greatest decrease in expression in 5/6Nx mice, a process mediated by the decreased expression of DBP. Furthermore, an increase of plasma transforming growth factor-1 (TGF-1) in 5/6Nx mice led to the decreased expression of the Dbp gene. Consistent with these findings, the alterations of retinol metabolism and renal dysfunction in 5/6Nx mice were ameliorated by administration of an anti-TGF-1 antibody. We also show that the accumulation of serum retinol induced renal apoptosis in 5/6Nx mice fed a normal diet, whereas renal dysfunction was reduced in mice fed a retinol-free diet. These findings indicate that constitutive Dbp expression plays an important role in mediating hepatic dysfunction under CKD. Thus, the aggravation of renal dysfunction in patients with CKD might be prevented by a recovery of hepatic function, potentially through therapies targeting DBP and retinol.

DOI： 10.1074/jbc.M115.696930

Language：English   Publishing type：Research paper (scientific journal)  

Clock gene regulates the circadian rhythm of various physiological functions. The expression of clock gene has been shown to be attenuated by certain drugs, resulting in a rhythm disorder. Mitomycin C (MMC) is often used in combination with ophthalmic surgery, especially in trabeculectomy, a glaucoma surgical procedure. The purpose of this study was to investigate the influence of MMC on clock gene expression in fibroblasts, the target cells of MMC. Following MMC treatment, Bmal1 mRNA levels was significantly decreased, whereas Dbp, Per1, and Rev-erb zeta mRNA levels were significantly increased in the mouse fibroblast cell line NIH3T3 cells. Microarray analysis was performed to explore of the gene(s) responsible for MMC-induced alteration of clock gene expression, and identified Nr3c1 gene encoding glucocorticoid receptor (GR) as a candidate. MMC suppressed the induction of Per1 mRNA by dexamethasone (DEX), ligand of GR, in NIH3T3 cells. MMC also modulated the DEX-driven circadian oscillations of Per2::Luciferase bioluminescence in mouse-derived ocular fibroblasts. Our results demonstrate a previously unknown effect of MMC in GR signaling and the circadian clock system. The present findings suggest that MMC combined with trabeculectomy could increase the risk for a local circadian rhythmdisorder at the ocular surface. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2015.09.086

Language：English   Publishing type：Research paper (scientific journal)  

Aquaporin 3 (AQP3) is located in the basal layer of the epidermis and regulates biological functions of skin such as water content and trans-epidermal water loss. A recent study showed that the biological function of skin exhibits a 24-hour rhythm, but the molecular mechanism of the variation remains poorly understood. Here we show that mice mutated in the core clock component CLOCK (Clk/Clk) show decreased stratum corneum hydration. An extensive search for the underlying cause led us to identify AQP3 as a new regulator to control the 24-hour variation in biological functions of skin. In mouse epidermis of wild-type mice, mAqp3 exhibits circadian rhythms; however, these are significantly decreased in Clk/Clk. Luciferase reporter gene analysis revealed that transcription of mAqp3 is activated by D-site-binding protein, a clock gene. A human homolog, hAQP3, also exhibited significant oscillation in human keratinocyte (HaCaT) cells synchronized with medium containing 50% serum, and this rhythm was regulated by the endogenous CLOCK/BMAL1 heterodimer. These data indicate that although the molecular mechanisms underlying the rhythmic expression of mAqp3 and hAQP3 are different, clock genes are involved in time-dependent skin hydration. Our current findings provide a molecular link between the circadian clock and AQP3 function in mouse dorsal skin and HaCaT cells.

DOI： 10.1038/jid.2014.13

Language：English   Publishing type：Research paper (scientific journal)  

The dopamine D3 receptor (DRD3) in the ventral striatum is thought to influence motivation and motor functions. Although the expression of DRD3 in the ventral striatum has been shown to exhibit 24-hour variations, the mechanisms underlying the variation remain obscure. Here, we demonstrated that molecular components of the circadian clock act as regulators that control the 24-hour variation in the expression of DRD3. The transcription of DRD3 was enhanced by the retinoic acid-related orphan receptor alpha (ROR alpha), and its activation was inhibited by the orphan receptor REV-ERB alpha, an endogenous antagonist of ROR alpha. The serum or dexamethasone-induced oscillation in the expression of DRD3 in cells was abrogated by the downregulation or overexpression of REV-ERB alpha, suggesting that REV-ERB alpha functions as a regulator of DRD3 oscillations in the cellular autonomous clock. Chromatin immunoprecipitation assays of the DRD3 promoter indicated that the binding of the REV-ERB alpha protein to the DRD3 promoter increased in the early dark phase. DRD3 protein expression varied with higher levels during the dark phase. Moreover, the effects of the DRD3 agonist 7-hydroxy-N, N-dipropyl-2-aminotetralin (7-OH-DPAT)-induced locomotor hypoactivity were significantly increased when DRD3 proteins were abundant. These results suggest that ROR alpha and REV-ERB alpha consist of a reciprocating mechanism wherein ROR alpha upregulates the expression of DRD3, whereas REV-ERB alpha periodically suppresses the expression at the time of day when REV-ERB alpha is abundant. Our present findings revealed that a molecular link between the circadian clock and the function of DRD3 in the ventral striatum acts as a modulator of the pharmacological actions of DRD3 agonists/antagonists.

DOI： 10.1124/mol.112.083535

Language：English   Publishing type：Research paper (scientific journal)  

Time-Dependent Interaction between Differentiated Embryo Chondrocyte-2 and CCAAT/Enhancer-Binding Protein alpha Underlies the Circadian Expression of CYP2D6 in Serum-Shocked HepG2 Cells
Differentiated embryo chondrocyte-2 (DEC2), also known as bHLHE41 or Sharp1, is a pleiotropic transcription repressor that controls the expression of genes involved in cellular differentiation, hypoxia responses, apoptosis, and circadian rhythm regulation. Although a previous study demonstrated that DEC2 participates in the circadian control of hepatic metabolism by regulating the expression of cytochrome P450, the molecular mechanism is not fully understood. We reported previously that brief exposure of HepG2 cells to 50% serum resulted in 24-h oscillation in the expression of CYP3A4 as well as circadian clock genes. In this study, we found that the expression of CYP2D6, a major drug-metabolizing enzyme in humans, also exhibited a significant oscillation in serum-shocked HepG2 cells. DEC2 interacted with CCAAT/enhancer-binding protein (C/EBP alpha), accompanied by formation of a complex with histone deacetylase-1, which suppressed the transcriptional activity of C/EBP alpha to induce the expression of CYP2D6. The oscillation in the protein levels of DEC2 in serum-shocked HepG2 cells was nearly antiphase to that in the mRNA levels of CYP2D6. Transfection of cells with small interfering RNA against DEC2 decreased the amplitude of CYP2D6 mRNA oscillation in serum-shocked cells. These results suggest that DEC2 periodically represses the promoter activity of CYP2D6, resulting in its circadian expression in serum-shocked cells. DEC2 seems to constitute a molecular link through which output components from the circadian clock are associated with the time-dependent expression of hepatic drug-metabolizing enzyme.

DOI： 10.1124/mol.111.076406

Language：English   Publishing type：Research paper (scientific journal)  

Background: Neuropathic pain is characterized by hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to NSAIDs or even opioids. Gabapentin, a GABA analogue, was originally developed to treat epilepsy. Accumulating clinical evidence supports the effectiveness of this drug for diverse neuropathic pain. In this study, we showed that the anti-allodynic effect of gabapentin was changed by the circadian oscillation in the expression of its target molecule, the calcium channel alpha 2 delta-1 subunit.
Results: Mice were underwent partial sciatic nerve ligation (PSL) to create a model of neuropathic pain. The paw withdrawal threshold (PWT) in PSL mice significantly decreased and fluctuated with a period length about 24 h. The PWT in PSL mice was dose-dependently increased by intraperitoneal injection of gabapentin, but the antiallodynic effects varied according to its dosing time. The protein levels of alpha 2 delta-1 subunit were up-regulated in the DRG of PSL mice, but the protein levels oscillated in a circadian time-dependent manner. The time-dependent oscillation of alpha 2 delta-1 subunit protein correlated with fluctuations in the maximal binding capacity of gabapentin. The anti-allodynic effect of gabapentin was attenuated at the times of the day when alpha 2 delta-1 subunit protein was abundant.
Conclusions: These findings suggest that the dosing time-dependent difference in the anti-allodynic effects of gabapentin is attributable to the circadian oscillation of alpha 2 delta-1 subunit expression in the DRG and indicate that the optimizing its dosing schedule helps to achieve rational pharmacotherapy for neuropathic pain.

DOI： 10.1186/1744-8069-6-83

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