The involvement of senescent cells in the lungs of idiopathic pulmonary fibrosis has been strongly suggested. In our previous study, we confirmed cellular senescence of macrophages and expression of p16^INK4a, a key marker of cellular senescence, in the lungs from bleomycin-induced pulmonary fibrosis model mice. However, the role of macrophage senescence in fibrosis has not been clarified. In the present study, we investigated the role of macrophage senescence in lung fibrosis using macrophage-specific p16 knockout (p16 cKO) mice. While bleomycin treatment increased p16 mRNA expression in whole lung tissue from the control mice, such an effect was not observed in the p16 cKO mice. We also examined expression of p16 mRNA in macrophages isolated from the lung and confirmed that the levels were below the detection limit in p16 cKO mice. However, Sirius Red staining revealed no difference in collagen accumulation by bleomycin treatment between lungs from control and p16 cKO mice. In addition, there was no difference in the soluble collagen concentrations, cell numbers or protein concentrations in bronchoalveolar lavage fluid, which are indicators of pulmonary fibrosis. These results suggest that macrophage senescence may have little effect on lung fibrosis.

NP-1815-PX is a selective P2X4 receptor antagonist. Administration of a P2X4 receptor antagonist to asthma model mice improves the asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effect of these antagonists on tracheal smooth muscles (TSMs) has not been investigated. This study examined the effect of NP-1815-PX on guinea pig TSM contraction. In epithelium-intact TSMs, NP-1815-PX strongly suppressed ATP-induced contraction, which was suppressed by indomethacin or ONO-8130 (an EP₁ receptor antagonist). NP-1815-PX strongly suppressed U46619 (a TP receptor agonist)- and PGF_2α-induced epithelium-removed TSM contractions, which were largely inhibited by SQ 29,548 (a TP receptor antagonist). Additionally, NP-1815-PX strongly suppressed the U46619-induced increase in intracellular Ca²⁺ concentration in TP receptor-expressing cells. In contrast, NP-1815-PX did not substantially inhibit TSM contractions induced by carbachol, histamine, neurokinin A, and 50 mM KCl. These findings indicate that: 1) NP-1815-PX inhibits guinea pig TSM contractions mediated through the TP receptor in addition to P2X4 receptor whose stimulation induces EP₁ receptor-related mechanisms, and 2) NP-1815-PX may be a useful therapeutic for asthma.

Although the exact pathogenesis of idiopathic pulmonary fibrosis (IPF) is still unknown, the transdifferentiation of fibroblasts into myofibroblasts and the production of extracellular matrix components such as collagen, triggered by alveolar epithelial cell injury, are important mechanisms of IPF development. In the lungs of IPF patients, apoptosis is less likely to be induced in fibroblasts than in alveolar epithelial cells, and this process is involved in the pathogenesis of IPF. We used a library containing approved drugs to screen for drugs that preferentially reduce cell viability in LL29 cells (lung fibroblasts from an IPF patient) compared with A549 cells (human alveolar epithelial cell line). After screening, we selected eperisone, a central muscle relaxant used in clinical practice. Eperisone showed little toxicity in A549 cells and preferentially reduced the percentage of viable LL29 cells, while pirfenidone and nintedanib did not have this effect. In an in vivo study, eperisone inhibited bleomycin (BLM)-induced pulmonary fibrosis, respiratory dysfunction, and fibroblast activation. In contrast, pirfenidone and nintedanib were less effective than eperisone in inhibiting BLM-induced pulmonary fibrosis under this experimental condition. Finally, we showed that eperisone did not induce adverse effects in the liver and gastrointestinal tract in the BLM-induced pulmonary fibrosis model. Considering these results, we propose that eperisone may be safer and more therapeutically beneficial for IPF patients than current therapies.

Cigarette smoking in asthmatics is known to cause severe symptoms, pulmonary dysfunction, neutrophilic inflammation, and decreased glucocorticoid sensitivity. The differential effects of dexamethasone (Dex) and roflumilast (Rof) on airway inflammation, lung function and airway responsiveness in ovalbumin-induced asthmatic mice with or without concurrent cigarette smoke (CS) exposure were examined.

　In the CS-exposed asthmatic mice, the airway neutrophils and lung compliance were increased, and the central airways resistance (Rn) and airway hyperreactivity (AHR) were decreased when compared with the asthmatic mice. Dex inhibited the airway eosinophils, airway resistance (Raw) and AHR in the asthmatic mice. In the CS-exposed asthmatic mice, Dex reduced the airway eosinophils but exacerbated the airway neutrophils, lung tissue resistance and AHR. Rof decreased the Rn in the asthmatic mice, but did not affect in the CS-exposed asthmatic mice.

　Based on these results, in asthmatic mice with CS exposure, Dex was effective in reducing eosinophilic inflammation but exacerbated the neutrophilic inflammation, lung tissue resistance and AHR. Rof was ineffective in improving inflammation and lung function in asthmatic mice with CS exposure.

Virus infection may affect the epigenetic regulations in host cells, including post-translational histone modifications. Ubiquitination of histone H2B, has been reported to be involved in transcription activation. However, it remains unknown the role of histone ubiquitination in the pathology of virus infection. CNOT4 that is a component of the CCR4-NOT complex has a ubiquitin transferase activity at the RING domain (L16). Here, we found that CNOT4 was responsible for histone H2B ubiquitination in the host cells, which was linked to H3K4 methylation. In addition, upon influenza virus infection CNOT4 interacted to virus protein, resulting in the loss of H2B ubiquitination and H3K4 methylation. Moreover, the cells with a mutation in L16 of CNOT4, have increased virus replication. These results suggest that the CNOT4 is involved in the virus replication through histone H2B ubiquitination.

Affibody is a class of smallest antibodies consisted of 58 amino acids, of which 13 amino acids are variable. Phage display is currently used for the production of affibody, but this method needs huge library construction for each antigen. In this study, we tried to develop the affibody against receptor binding domain (RBD) of SARS-CoV-2 spike protein in silico. SARS-CoV-2 virus binds to human ACE2 via spike protein, which is, therefore, a promising target for preventing SARS-CoV-2 infection. We analyzed three-dimensional coordinates of RBD structure and set antibody binding site on the interface surface between RBD and ACE2. We then designed many affibody structures based on the electrostatic potential of the binding site on RBD, and performed in silico docking prediction of constructed affibodies and RBD. Thereafter, two affibody molecules that showed the highest and second highest docking score as well as two molecules with much lower scores were expressed by wheat germ cell-free protein synthesis method. Binding affinity between expressed affibody molecules and RBD was examined with AlphaScreen assay, and two high score affibody molecules showed significantly higher luminescent signals due to binding than control low score molecules. In contrast, all four molecules showed no binding signals to negative control DHFR. The present study shows the successful non-animal-derived, cell-free and in silico generation of artificial antibody, targeting precise protein surface of SARS-CoV-2 RBD.

Mast cells are important sentinel cells in the first line of host defense against viral and bacterial entry. SARS-CoV-2 can activate mast cells present in the respiratory tract in the initial stage of the disease. In the present study, we attempted to dissect the mechanisms underlying the interaction of the function of mast cells, especially focusing on the mast cell-derived chymase, with the cell entry of SRAS-CoV-2 by using a well-established mast cell line, rat basophil leukemia (RBL-2H3) cells. RBL-2H3 cells were infected by pseudovirons and the viral entry was evaluated. The interactions between SRAS-CoV-2 spike and mast cell-derived chymase were observed and predicated binding-site were determined by using spike-truncating variants. During the viral infection, the interaction of chymase with SRAS-CoV-2 may have both detrimental and positive impacts. Whether SRAS-CoV-2 spike is a potential substrate of chymase, and if its cleavage modulates biological properties of spike-bearing virus would be focused on in future planned study.

Delayed wound healing is a major problem in patients with diabetes melitus, which significantly impairs their quality of life. Prostaglandin (PG) D₂ is a major inflammatory lipid mediator synthesized by hematopoietic PGD₂ synthase (HPGDS) from PGH₂, a common precursor of all of PGs. In the present study, we investigated the role of PGD₂ in cutaneous wound healing in streptozotocin (STZ)-induced diabetic mice. C57BL/6 mice were injected with 50 mg/kg of STZ intraperitoneally daily for 5 days. Four weeks after the injection of STZ, a full thickness wound was created with an 8-mm diameter biopsy punch on the dorsal of mice showing the hyperglycemia (>300 mg/dL). Wound healing was significantly decelerated in diabetic mice compared with non-diabetic mice. The mRNAs of HPGDS, Cyclooxygenase (COX) 1, COX2, DP1 and DP2 receptors in mouse skin were measured by quantitative PCR. The skin of diabetic mice had significantly increased mRNAs of HPGDS and DP2 receptors as compared with the skin of non-diabetic mice. In addition, there was no significant change in the amount of DP1 receptors mRNA and COX1 mRNA, but the amount of COX2 mRNA tended to increase. In addition, immunohistochemical analysis revealed that HPGDS was upregulated in epidermal Langerhans cells of diabetic mice. These results suggest  that in hyperglycemic skin, production of PGD₂ is increased in Langerhans cell and may be involved in delayed inflammation via DP2 receptors.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by the development of skin fibrosis. No topical treatment for the skin manifestations of SSc has been developed. We previously reported that celecoxib, a selective inhibitor of COX-2, and/or its derivative DM-celecoxib suppressed cardiac and renal fibrosis. Therefore, the effect of celecoxib on the SSc fibrosis was investigated. In in-vivo study, bleomycin was injected s.c. into a single location of the hair less mouse Hos:HR1 every 3 days for 10 days to induce scleroderma. For the treatment of celecoxib, celecoxib was dissolved into acetone and topically applied, whereas control mice received acetone only. Ten days after the first injection, skin section was obtained and histological analysis were performed. To clarify the mechanism of celecoxib action, normal human skin fibroblast cell line NB1RGB was stimulated with TGF-β to induced fibroblast-myofibroblast transformation and the effects of celecoxib on the expression of α-SMA was analyzed. We found that topical application of celecoxib significantly decreased skin thickness and the expression of α-SMA in bleomycin-induced scleroderma model mice. Consistent with these results, celecoxib inhibited the expression of α-SMA induced by TGF-β in NB1RGB cells. Further studies are now conducting to clarify the action of celecoxib.

Hochuekkito (HET), a Kampo medicine, is used to treat post-operative and post-illness general malaise and decreased motivation. HET is known to regulate immunity and inflammation. However, the mechanism underlying the anti-anxiety effect of HET is unknown. In this study, we revealed the effect of HET on lipopolysaccharide (LPS) -induced anxiety-like behavior　and examined the mechanism underlying LPS induced anxiety-like behaviors. Following the administration of LPS (300 µg/kg, i.p.), mice demonstrated inflammation-induced anxiety-like behaviors in hole-board and light-dark box tests. Systemic administration of LPS increased the serum levels of interleukin-6. Repeated administration of HET (1.0 g/kg, p.o.) once a day for 2 weeks before LPS treatment ameliorated anxiety-like behavior and reduced LPS-induced serum levels of interleukin-6 in 5 h after LPS treatment. Additionally, HET decreased LPS-induced secretion of interleukin-6 in human macrophage cell line (THP-1) and mouse macrophage cell line (RAW264.7) as in mouse models. Therefore, our findings suggest that HET may be useful in treating inflammation-induced anxiety-like behavior.

We recently reported the potential impacts of perilla seed oil (PO), a rich source of a-linolenic acid (ALA, C18:3, n-3), on cognitive function in healthy elderly Japanese individuals. Here, supplements containing either PO alone or PO with nobiletin-rich air-dried immature ponkan powder were examined for their effects on brain functions in 49 healthy elderly Japanese individuals. PO group or the PO + ponkan powder (POPP) group for a 12-month randomized, double-blind, parallel-armed study. Taking a total of fifteen capsules daily for twelve months was not associated with any clinically significant side effects. From baseline to 12-months intervention, taking POPP significantly increased the cognitive index scores obtained by the three evaluation methods more than taking PO alone supplementation. The increased cognitive function observed in the POPP group was accompanied by increases in the levels of a-linolenic acid and docosahexaenoic acid in the erythrocyte plasma membranes and serum levels of brain-derived neurotropic factor (BDNF) and biological antioxidant potential. Our results demonstrate that a 12-months intervention with PO plus nobiletin-rich ADPP supplementations enhances serum BDNF and the antioxidant potential and may improve the age-related cognitive decline in healthy elderly individuals by enhancing the erythrocyte w-3 fatty acid levels.

Anredera cordifolia (AC) is a perennial plant of the Basellaceae family, and is attracting attention as a health food because it is easy to cultivate and has high nutritional value. So far, it has been reported that AC has an improving effect on lipid abnormalities and hypertension in mice and rats. However, it is not known how long-term administration of AC extract affects the central nervous function. In this study, we investigated the effects of an oral intake of AC extract on learning and memory using the senescence accelerated mouse-prone 8 (SAMP8) mouse.

SAMP8 mice (15 weeks old) were divided into two groups; a tap water intake group (CN: n = 10) and an AC extract water intake group (AC: n = 9) for 34 weeks under the free-feeding condition. Learning and memory function was assessed using Novel object recognition (NOR) task (age of 23 weeks) and the Morris water maze (MWM) task (aged 30 weeks). Following the completion of behavioral tests, blood biochemistry parameters and hippocampal levels of brain-derived neurotropic factor, postsynaptic density protein 95, NR2A, and p-cAMP-response element binding (CREB)/CREB ratio were measured. The AC group spent more time exploring the novel object in the NOR task, and showed better acquisition and retention in the MWM task than the CN group. In addition, AC elevated the levels of the aforementioned neuronal plasticity-related proteins, and did not affect blood biochemistry parameters. These results suggest that the AC extract may improve learning and memory in SAMP8 mice without causing any noticeable side effects on the body.

Parkinson's disease (PD) is a progressive neurodegenerative disease that exhibits motor dysfunction due to deficit of dopaminergic neurons in the substantia nigra. Oxidative stress is a factor involved in the onset and progression of PD. We have previously shown that Actinidia arguta has an antioxidative activity. In this study, we evaluated the effect of prophylactic administration of Actinidia arguta (Sarunashi) in MPTP induced PD mice model. Male C57BL/6J mice were allowed to continuously ingest water or Sarunashi juice from 7 weeks of age. At 8 weeks of age, MPTP (30 mg/kg/day) was intraperitoneally administered for 5 consecutive days. In the MPTP + water group, significant motor dysfunction was observed in the pole test, catalepsy test, beam walk test, and rotor rod test compared with the negative control (NC) group. However, in the MPTP + Sarunashi group, no impaired motor function was observed as compared with the NC group. The protein expression level of tyrosine hydroxylase (TH) in the MPTP + water group was lower than that in the NC group. On the other hand, TH expression in the MPTP + Sarunashi group was higher than that in the MPTP + water group. In conclusion, prophylactic administration of Sarunashi juice suppressed MPTP induced motor dysfunction mediated via decreased TH expression.

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increased single channel activities of transient receptor potential ankyrin-1 (TRPA1) in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and which was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared with allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through desensitization of TRPA1-expressing nociceptors.

Juzen-taiho-to, a traditional Chinese herbal medicine, is used for patients withanorexia and fatigue in human medicine. In our previous study, a granulated formof Juzen-taiho-to improved vincristine-induced gastrointestinal adverse effectsthrough increasing gastric motility in dogs. However, the effect of Hozen-S, thesweet liquid form of Juzen-taiho-to, on dog gastric motility has not beeninvestigated. Therefore, we examined whether the administration of Hozen-S todogs affects their gastric motility. To further elucidate the mechanism of theeffect of Hozen-S on gastric contraction, we assessed the plasma ghrelin levelof the dog. Finally, we assessed the Hozen-S palatability compared to granulatedJuzen-taiho-to in dogs and the effect on body weight in dogs. Administration ofHozen-S significantly increased gastric motility, plasma ghrelin concentration,and body weight in the dogs. As a result of the palatability evaluation, thesedogs preferred Hozen-S to the granulated Juzen-taiho-to. In conclusion, Hozen-Sadministration to dogs promoted gastric motility by raising the plasma ghrelinlevel. Considering these functional and palatability data, Hozen-S might replacethe granulated type Juzen-taiho-to and become a prominent traditional Chineseveterinary medicament.

Fibroblast growth factor 21 (FGF21), which structurally belongs to the FGF superfamily, acts as an endocrine factor and plays important roles in the regulation of energy homeostasis, glucose and lipid metabolism. The therapeutic administration of FGF21 analogs improves nonalcoholic steatohepatitis, the severe stage of non-alcoholic fatty liver disease, which is associated with obesity, metabolic syndrome, dyslipidemia and type 2 diabetes. In the present study, we screened crude extracts from 88 herbal drugs frequently used in Kampo prescriptions as potential anti-obesity agents by monitoring the production of FGF21 in C2C12 myotubes. Among these extracts screened, we found that 3-O-acetyloleanolic acid, an active constituent isolated from the fruits of Forsythia suspensa, stimulated FGF21 production in C2C12 myotubes. Additionally, significant increases in CRE-dependent luciferase activity were observed in cells overexpressing  bile acid receptor TGR5 in response to 3-O-acetyloleanolic acid treatment, which indicated that the responses caused by 3-O-acetyloleanolic acid was dependent on TGR5 activation. We observed that the phosphorylation of p38 was increased by 3-O-acetyloleanolic acid rapidly in C2C12 myotubes. Pretreatment with the selective p38 inhibitor SB203580 also significantly repressed the stimulatory effect of 3-O-acetyloleanolic acid on FGF21 secretion. These findings collectively indicated that TGR5 receptor signaling up-regulates FGF21 expression via p38 activation.

Purpose: Dihydromyricetin (DHM) is a flavonoid isolated from Hovenia dulcis. DHM have been reported to have hepatoprotective and anti-tumor effects. The purpose of this study was to elucidate the anti-tumor effect and the mechanism of DHM in human hepatocellular carcinoma cell line, Huh-7.

Methods: Huh-7 cells were cultivated with DHM (0 - 100 μM) for 72 hours. We evaluated that the cell proliferation in Huh-7 cells treated with DHM using a MTS assay. The mRNA and protein levels of Ki-67, cyclinD1, and caspase 3 were analyzed using a real time-RT-PCR or a western blot analysis. The protein levels and phosphorylation of ERK, c-fos, c-jun as MAPK signaling were confirmed.

Results and discussion: Although DHM (1 - 10 µM) did not changed cell proliferation, 30 µM and 100 µM of DHM significantly decreased in Huh-7 cells.

Ki-67 and cyclin D1 mRNA levels were decreased in DHM-treated Huh-7 cells. However, caspase 3 mRNA levels was not increased. Cyclin D1 protein levels were also significantly decreased and caspase 3 protein levels were not increased in DHM-treated with Huh-7 cells; these suggest that DHM inhibited cell proliferation and did not effect the apoptotic protein, caspase 3.

The total ERK and pERK were significantly decreased in Huh-7 treated with DHM. However, the level of p-c-fos, and p-c-jun were tended to decrease. DHM may related to the decreases of HCC cell proliferation through the ERK.

Toxicity of the compound on the nervous system has been reported in development of pharmaceuticals such as toxicity of the compound and medicinal efficacy evaluation. In nonclinical study for the purpose of pharmaceuticals development, in in vivo using model animals are being conducted to examine the efficacy of the drug. However, the labor required for the experiment is excessive and it has a challenges low reproducibility and accuracy. Therefore, there is a need to develop a high accuracy and objective method of pain evaluated, but there method are not established. In this study, we aimed to development of pain evaluation system to evaluate of effectiveness and side effect of drugs. Using complementary metal-oxide semiconductor microelectrode array (CMOS-MEA) with nerve cells electrical activity can measure an ultra-high temporal resolution, the response of single sensory neurons to pain was obtained. Furthermore, based on the electrical activity obtained from 236,880 electrodes, we constructed a method to identify single cell, calculation of conduction velocity of axons and profiling of firing pattern each single cells.

In vitro pharmacological assay based on human iPSC-derived neurons is a valuable alternative to the standard animal models. One of the main methods for this purpose is imaging drug-induced Ca²⁺ responses in neuronal networks cultured on multi-well plates. Preparing iPSC-derived neurons, however, requires costly optimization and lengthy culturing, and one possible solution to this problem would be to fabricate neuron plates that can be transported from the manufacturer to the user.

We have developed transportable neuron plates for high-throughput screening by optimizing the plate coating and culture conditions. Ca²⁺ imaging showed that human iPSC-derived neurons cultured in these plates exhibit excellent drug responses.

The transportable plates provide ready-to-use human neuron assay systems and would contribute to accelerate drug development.

In vitro microelectrode array (MEA) assessment using human induced pluripotent stem cell (iPSC)-derived neurons holds promise as a method of seizure and toxicity evaluation. However it is difficult to detect the response of drugs with different mechanisms of action with a single parameter, and the analysis method has become an issue. Therefore, in this study, we developed an artificial intelligence (AI) model that learned raster plot images of electrical activity acquired by multiple electrodes and an SVM model that learned parameters calculated from time-series data of electrical activity. We compared the accuracy of predicting convulsive toxicity for each of the developed models. To train the model, extracellular potential data of co-cultured samples of human iPSC-derived cortical neurons and stellate cells obtained using MED64 Presto were used. In the SVM model using the spike time-series information and burst-related parameters, the risk assessment of the positive and negative compounds of the trained data was achieved, but the untrained data of acetaminophen was judged to be positive. In contrast, AI accurately predicted seizure risk, even with unlearned well data. These results indicated that using raster plot features method is useful for predicting the seizure liability using hiPSC-derived neurons.

In vitro human iPSC-derived neuronal systems are an alternative platform for neurotoxicity testing to animal models and primary cultures. Microelectrode array (MEA), measurement system of the electrophysiological activity, are suitable to evaluate the neurotoxicity of compounds. We have previously reported the electrophysiological responses to known neurotoxicity compounds using MEA in human iPSC-derived neurons. However, the identification of analytical parameters to detecting toxicity of unknown compounds remains an important issue. We identify the analytical parameters enabling the separation of responses between neurotoxicity and negative control, and the separation the mechanism of action by using principal component analysis. By comparing the optimized analytical parameters of each testing compound with the standard deviation (SD) of negative control, we can predict general neurotoxicity in relatively quantitatively scale, for example, low risk for lower than SD range, medium risk for 2xSD, and high risk for over 2xSD. This predictability can help select appropriate concentration levels to avoid toxicity/adverse effects. In this study, we evaluated the toxicity risk of pesticides and industrial chemicals. We demonstrated that the toxicity risk can be detected by MEA measurement of human iPSC-derived neurons.

Benchmark Dose (BMD) approach is the way to calculate the risk at the lower dosage of chemical exposure, applying a mathematical model to the dose-response relationship. Although a number of the guidelines and software have been developed worldwide, the harmonization of BMD application is still undergoing. Before applying in actual risk assessment, it is essential to evaluate whether the BMD and NOAEL approaches give the same range of POD values and how different BMD values the major BMD software would give.

Here, we calculated the lower limit of BMD confidence interval (BMDL) from 201 tumorigenicity data publicized in the pesticide risk assessment reports by the Food Safety Commission of Japan (FSCJ). We applied three well-known BMD software, PROAST, BMDS, and BBMD, to compare their BMDLs to NOAELs and LOAELs and between the recently implemented methodologies such as model averaging (MA) or Bayesian inference.

Our result indicates that the BMD approach gives Point of Departure (POD) similar to the NOAEL approach if the data applied show a clear dose-response relationship. However, most of the datasets that resulted in failed calculation or extremely low BMDLs showed unclear dose-response relationships, such as non-monotonous and sporadic responses. We also noted that the Bayesian inference software gave failed calculation or extreme BMDLs less than the frequentist approaches.

Cigarette smoking is one of the risk factors in cardiovascular and respiratory diseases including atherosclerosis and chronic obstructive pulmonary disease (COPD). Although cigarette smoke mainstream consists of more than 4,500 chemical compounds, the compounds responsible for these diseases are still unknown. Cigarette smoke is divided in two phases: tar (particle) phase containing nicotine and gas phase. Airway epithelium cells are exposed both tar and gas phases. In this study, we have examined the effect of tar and gas phase of cigarette smoke on airway epithelium cells. The gas phase extract of cigarette smoke was prepared as previously described (Higashi et al., 2014, PLOS ONE 9: e107856). The tar phase extract of cigarette smoke was prepared by extracting tar phase on Cambridge filter with DMSO. Both tar and gas phases induced cell death in airway epithelial cells. The cell death induced by the gas phase was PKC-dependent, whereas the tar phase induced DNA double strand break and PKC-independent cell death. The pharmacological experiments revealed that the airway epithelium cell death by gas phase were ferroptosis. According to Yoshida et al., ferroptosis is involved in COPD pathogenesis (Yoshida et al., 2019, Nat Commun 10: 3145). Taken together, cigarette smoke gas phase might be a critical factor for cigarette smoking-induced COPD onset and development.

LAT1 (SLC7A5) is a type of amino acid transporter that transports many essential amino acids, including Leucine, into cells. High expression of LAT1 is associated with poor prognosis in a variety of carcinomas, and a selective inhibitor of LAT1 (JPH203) has been reported to inhibit cancer cell growth. Recently, we reported that LAT1 expression is upregulated in the cell line C4-2, a model of castration-resistant prostate cancer (CRPC). We hypothesized that JPH203 could be a novel therapeutic agent for CRPC. We examined the effects of JPH203 in C4-2 and PC-3 cell lines as a model of CRPC and LNCaP cell line as hormone-sensitive prostate cancer (HSPC). We performed [14C] Leucine uptake assay for functional analysis and WST-8 assay for cytotoxicity test. In addition, Migration/Invasion assay was performed using Corning™ Falcon™ Cell Culture Inserts to evaluate cell migration and invasion ability. To elucidate the molecular mechanism of the inhibitory effect on proliferation, the presence or absence of mTOR pathway inhibition was verified using Western blotting. We also investigated the tumor suppressive effect of JPH203 in mice injected subcutaneously with C4-2 cells. As a result, Leucine uptake was predominantly decreased by JPH203 in C4-2 and PC-3 cells, and the inhibitory effect on cell proliferation was also confirmed. In C4-2 cells, JPH203 inhibited the mTOR pathway. JPH203 suppressed tumor growth in vivo. These results suggest that JPH203 may have an antitumor effect on CRPC and may be a novel therapeutic agent.

Data for gene expression profile in the rat liver (after exposure to 150 chemical compounds) are available from Open TG-GATES database generated from the Toxicogenomics Project of Japan. With the 150 chemical compounds, several carcinogenic agents induced the expression of p75-NTR associated cell death executor (NADE) in the liver. NADE is known as a pro-apoptotic protein and induces apoptosis by associating with a partner protein, including p75-NTR, hamartin, or Smac, under certain physiological conditions. However, none of these genes were induced in these cases. As NADE expression starts to increase as early as three days of exposure to the carcinogenic agents, NADE is a potential early marker for chemical carcinogenesis. To evaluate whether NADE can be a carcinogenic marker, we examined the role of NADE in the process of chemical carcinogenesis. In our study, Sprague-Dawley rats were treated with a genotoxic carcinogen, diethylnitrosamine, for up to 8 weeks followed by another 4 weeks of no treatment. The liver was excised to obtain RNA and protein samples and paraffin embedded tissue sections. The expression of NADE and some cancer markers was quantified by real time PCR. By immunohistochemistry and TUNEL assay, the expression pattern of NADE, some cancer markers was visualised and apoptosis was detected. The NADE expression correlated with development and growth of cancerous cells, but not with apoptosis. Here, we discuss the role of NADE expression in cancerous growth and apoptosis.

We reported the enhanced liver-specific function and structure of HepG2 cells by oxygenation culture via a collagen vitrigel membrane (Oshikata-Miyazaki and Takezawa, 2016). The cells were conditioned in our laboratory for a long period, so their characteristics may change from the original HepG2 cells registered in RIKEN cell bank (RCB) with the number of 1648 (HepG2-RCB1648 cells). We named the conditioned HepG2-RCB1648 cells in our laboratory as HepG2-NIAS cells. The aim of the present study is to clarify the features of HepG2-NIAS cells by comparing to HepG2 cells with two different cell culture histories. HepG2-NIAS cells subjected to oxygenation culture grew as a monolayer and showed a high CYP3A4 activity which was equivalent to almost half level to that of differentiated HepaRG cells and potential to form bile canaliculus-like structure. On the other hand, HepG2-RCB1648 cells and HepG2-RCB1886 cells subjected to oxygenation culture formed large multicellular aggregates and almost no detectable CYP3A4 activity and potential to form bile canaliculus-like structure. Protein expression levels of sinusoidal drug uptake and canalicular transporters were notably higher in HepG2-NIAS cells than HepG2-RCB1648 cells and HepG2-RCB1886 cells subjected to oxygenation culture. In contrast, protein expression levels of sinusoidal drug efflux transporters were highest in HepG2-RCB1648 cells. In conclusion, it is expected that HepG2-NIAS cells subjected to oxygenation culture are useful for predicting drug metabolism and excretion in the liver.

Glycation stress is a concept of biological stress caused by excess sugars, aldehyde and its subsequent reactions in the body. Especially, it is thought that accumulation of Advanced Glycation End products (AGEs) cause skin yellowing, deep wrinkles and a decrease in skin elasticity due to crosslinking to collagen or elastin in the dermis.

We have reported that phlorotannins, polyphenols unique to brown algae, could inhibit the formation of N-ε-carboxymethyllysine resulting from glycation of collagen.

The aim of this study was to investigate the anti-glycation and skin protection effects against UV exposure by long-term intake of the brown algae extract powder containing phlorotannins as a supplement.

This study have performed a randomized, double-blind, placebo-controlled, parallel group study.

Forty-two healthy Japanese adult female were randomly divided into two groups: one group consuming supplements containing brown algae extract powder (Phlorotannins intake group) and the other group consuming supplements not containing brown algae extract powder (placebo group). In both groups, each supplement was consumed daily, and blood AGEs concentration and Minimal Erythema Dose were measured every 4 weeks. In addition, the condition of skin was analyzed by using the skin image analyzer.

In this presentation, we will introduce the skin protection effect against UV exposure and the anti-glycation effects by consuming the brown algae extract powder for eight weeks.

Introduction: Burning mouth syndrome and atypical odontalgia (BMS/AO) are chronic orofacial pain conditions in the absence of any identifiable odontogenic pathology. The pain is treatment-resistant and frequently causes depressive symptoms. Duloxetine (DLX), a serotonin-noradrenaline reuptake inhibitor, is not only used as therapy for depression, but also for chronic orofacial pain. Since their mechanisms in detail remains unknown, in this study, we focused on serotonin transporter (SERT), one of DLX action site, and investigated association between expression of SERT and effect of DLX on pain in BMS/AO.

Methods: The patients with BMS/AO, were assessed for severity of pain using the visual analog scale (VAS) and for signs of depression using the Hamilton Depression Rating Scale (HDRS). In their platelets before (baseline) and 12 weeks after DLX-treatment, the expression of total and ubiquitinated SERT proteins was confirmed by Western blot. This study was approved by the Ethics Review Committees of Nagoya, Aichi Gakuin, and Meijo Universities.

Results: The expression of total and ubiquitinated SERT protein at baseline in all patients were higher and lower, respectively, compared to those in controls. After the DLX-treatment, there was no difference in the total SERT protein levels between the patients and controls. The mean of VAS and HDRS scores or the expression of total SERT protein were significantly decreased after the treatment, compared to those at baseline. 

Conclusions: These results indicate that DLX relieves chronic orofacial pain in patients with BMS/AO, and such effect may be mediated via SERT downregulation.

In treatment of hepatitis B virus (HBV), it is usually difficult for us to control with emergence of drug resistance. As HBV often reactive after treatment was stopped, patients must keep it for long term. Recently, we have developed E-CFCP, as a candidate drug of HBV for patients with drug-resistant HBV. As it has high antiviral activity and the half-life also is longer, patients can take it in a once-weekly dosing. We expect that E-CFCP can greatly improve the quality of life of patients. However, effects of E-CFCP are unclear in renal. The aim of this study is to clarify the effects of E-CFCP in the kidney, especially organic acid transporter（Organic anion transporters：OATs, Organic cation transporter：OCT）. We conducted cell viability studies using mouse-derived renal cortical cells (S2, CCD, cTAL) and uptake studies using radioisotopes to determine the effects of E-CFCP on the kidneys. In cell viability studies, E-CFCP has no cytotoxicity in all cell lines. We also examined the effect of drugs at high concentration using S2 cells. E-CFCP has no cytotoxicity even at high concentrations. In the substrate uptake assay, there was no inhibition of substrate uptake by E-CFCP, the transporter is not involved in the intracellular transport of E-CFCP and is unlikely to cause cytotoxicity. In conclusion, E-CFCP, a novel HBV antiviral drug, is unlikely to cause renal damage. It may be a novel great candidate drug of HBV for patients with drug-resistant HBV.

It has been suggested that transient receptor potential melastatin 4 (TRPM4) cation channels are abundantly expressed in the prostate gland. However, the precise role of TRPM4 channels in prostatic smooth muscle contractility is still unknown. Here, we examined if TRPM4 channels are involved in the adrenergic contraction in mouse prostatic smooth muscles. Adrenergic contractile responses evoked by electrical field stimulation of intrinsic sympathetic nerve or exogenously applied noradrenaline (NA) were isometrically recorded and effects of 9-phenanthrol, a specific and potent TRPM4 channel inhibitor, on those contractile responses were investigated in mouse ventral prostate preparations. 9-phenanthrol (10 and 30 μM) concentration-dependently inhibited both sympathetic nerve-evoked contractions and NA-induced contractions. The percentage inhibition by 9-phenanthrol was much greater at lower stimulus frequencies and lower NA concentrations. However, the agent did not inhibit NA-induced contractile response due to release of stored Ca²⁺. These results suggest that TRPM4 channels are involved in the adrenergic contraction possibly through membrane depolarization by their opening and seems to be a potential candidate for the treatment of benign prostatic hyperplasia.

Hyaluronic acid (HA) is produced at extremely high levels in some breast cancer cells, which can then escape from apoptosis even if they become detached from the extracellular matrix. Activation of TRPV1, a non-specific cation channel protein, has been shown to cause apoptosis in these cells. Therefore, using MCF-7 cells, we investigated the expression of the HA receptor CD44 and TRPV1 when the cells were placed on a low-adhesive scaffold. Then, we examined the effects of the HA-synthesis inhibitor 4-methylumbelliferone (4-MU) on TRPV1 expression as well as the effect of TRPV1 modulators on 4-MU–induced apoptosis. We also tested the direct action of HA on TRPV1 activity by using a Ca imaging system. In cancer spheroids formed on the poly (2-hydroxyethylmethacrylate)-coated dishes, both HA production and CD44 mRNA expression were increased, whereas TRPV1 mRNA expression was decreased. 4-MU inhibited both HA production and CD44 expression but increased TRPV1 mRNA expression and protein production. The TRPV1 agonist capsaicin increased the amount of Annexin V/PI-positive cells, the action of which was inhibited by the TRPV1 antagonist AMG9810. Furthermore, 4-MU–induced apoptosis was strongly suppressed by AMG9810. HA itself inhibited the capsaicin-induced increase in Ca²⁺ in TRPV1-transfected HEK293 cells. These results suggest that during cell detachment, the HA-CD44 pathway is likely to be involved in the underlying mechanism of apoptosis avoidance by inhibiting the expression and function of TRPV1.

Overexpression of endothelin (ET)-1 and endothelin receptors (ETRs) are associated with human cancer malignancy. In cancer cells, ET-1 activates various signaling pathways, including mitogen-activated protein kinase pathway, phosphatidylinositol 3-kinase pathway, and protein kinase C pathway, through ETRs, although the mechanisms by which ET-1 activates these signaling pathways remain unclear. We previously reported that ETRs interacted with annexin A2, which is overexpressed in various cancers, and annexin A2 silencing suppressed ERK activation upon ET-1 stimulation in human umbilical vein cell line, EA.hy926 cells. Here we examined roles of annexin A2 in ET-1 signaling pathway of melanoma cells. In melanoma cells, ET-1 stimulation activated ATK, and phosphorylated serin residues of annexin A2. Annexin A2 silencing suppressed activation of AKT upon ET-1 stimulation. In addition, we found that serine residues mutant of annexin A2 suppressed activation of AKT. Our results suggested that phosphorylation of annexin A2 plays important roles in AKT activation of melanoma cells upon ET-1 stimulation.

[Purpose] GABAB receptors are responsible for inhibitory transmission by conjugating with Gi proteins. In addition, this receptor functions by forming a dimer of R1 and R2 subunits (R2). Phosphorylation of serine residue at position 892 (S892) of R2 suppresses receptor internalization and desensitization. We have investigated the possibility that protein phosphatase 5 (PP5) dephosphorylates S892. In this study, we investigated the effect of overexpression of PP5 on the phosphorylation level of S892.

[Methods] pCDH-CMV-MCS-EF1copGFP-PP5 was prepared, packaged in lentivirus, and infected with HEK293 cells. The PP5 and R2 genes were simultaneously introduced into HEK293 cells, and the phosphorylation level of S892 was confirmed by Western blotting.

[Results] Virus packaging of the R2 gene was confirmed, but sufficient infection was not obtained. On the other hand, when PP5 and R2 were transiently co-expressed in HEK293 cells, a decrease in the phosphorylation level of S892 was confirmed in the PP5 overexpression group.

[Conclusion] If the detailed function of internalization or inactivation mediated by GABAB receptor dephosphorylation due to overexpression of PP5 is clarified, multiple sclerosis and amyotrophic lateral sclerosis associated with GABAB receptors will be clarified. It may lead to detailed elucidation of pathological conditions such as sclerosis and development of new therapeutic agents.

G_q-protein-coupled histamine H₁ receptors play a key role for allergic and inflammatory reactions. We constructed two differential C-terminal mutants of human H₁ receptors, S487TR and S487A, in which the Ser487 residue of C-terminal was truncated or mutated to alanine, respectively. We have found that S487TR and S487A selectively couples to G_q-protein and β-arrestin, respectively. In this study, we investigated histamine-induced and G_q-protein/β-arrestin-mediated ERK phosphorylation in Chinese hamster ovary cells expressing S487TR and S487A. In cells expressing S487TR, histamine transiently induced ERK phosphorylation, which were suppressed by intracellular Ca²⁺ chelator and inhibitors of protein kinase C (PKC) but not inhibitors of G-protein-coupled receptor kinase (GRK), clathrin, Raf and MEK. In contrast, histamine sustainably induced ERK phosphorylation in cells expressing S487A, which were suppressed by inhibitors of GRK, clathrin, Raf and MEK but not intracellular Ca²⁺ chelator and PKC inhibitors. These results suggest that progressive processes of the H₁-receptor-mediated ERK phosphorylation might be differentially regulated by the G_q-protein/Ca²⁺/PKC- and GRK/β-arrestin/clathrin/Raf/MEK-dependent pathways.

Citalopram has been reported to have cardiac adverse effects. Although citalopram is known to be a racemic compound comprised of S-citalopram (escitalopram) and R-citalopram, it is still unclear which enantiomer is responsible for cardiac adverse effects induced by citalopram. In the present study, we investigated whether citalopram, escitalopram and R-citalopram had an electrophysiological effect on Nav1.5 voltage-gated sodium channel (VGSC) current and how their electrophysiological properties affected Nav1.5 VGSC. When whole-cell patch clamp was performed for analysis, the IC₅₀ of citalopram, escitalopram and R-citalopram were 89.2, 58.6 and 174.0 µM, respectively. In addition, treatment with 100 μM citalopram and escitalopram changed the voltage-dependence of activation and induced a negative shift of the voltage of half-maximal activation compared to 100 μM R-citalopram. In contrast, treatment with 100 μM citalopram and escitalopram changed the voltage-dependence of inactivation, and the voltage at half-maximal inactivation slightly shifted toward negative potential. These results suggest that the adverse cardiac effect produced be citalopram might result from modification of the electrophysiological properties of Nav1.5 VGSCs, and escitalopram might contribute more to this adverse effect than R-citalopram.

TRPA1 is a non-selective cation channel and has been shown to be activated by a wide variety of noxious compounds and physiological stressors. Interestingly, TRPA1 is reported to be stimulated by menthol, an agonist of TRPM8, with different dose dependencies between mouse and human. It has been suggested that this different reactivity may be attributable to three amino acid residues in the TM5 region of TRPA1, namely, S–T–V in human and S–T–G in mouse. In this study, to further investigate the importance of the TM5 region through comparison with other mammalian species, canine TRPA1 cDNA was cloned and its reactivity to several agonists was compared in dog and mouse by using recombinant proteins. The TM5 region of cloned canine TRPA1 showed high similarity to that of human TRPA1 and the S–T–V residues were conserved. HEK293T cells were transfected with mouse or canine TRPA1 and subjected to calcium influx imaging. Both mouse and canine TRPA1 were activated by the TRPA1 agonist allyl isothiocyanate and were inactivated by the TRPA1 antagonist HC-030031. In contrast, reactivity to menthol was observed to differ between these two species. Mouse TRPA1 was activated by 100 µM of menthol and showed transient Ca²⁺ influx when menthol was washed out ("off response"), whereas canine TRPA1 activation required a high (300 µM) concentration of menthol, but no off response was observed. These results showed that the reactivity of canine TRPA1 against menthol is similar to that of human TRPA1 but not to mouse TRPA1, which might reflect the similarity of their respective TM5 regions.

The carotid body is a peripheral chemoreceptor located in the branches of the internal and external carotid arteries, and has long been known to be involved in oxygen sensing. However, the precise molecular mechanism of oxygen sensing remains unclear. Our group has previously identified that TRPA1, a type of non selective cation channel, is activated in hypoxic conditions. To understand whether TRPA1 contributes to oxygen sensing in the carotid body, we have performed immunohistochemistry experiments, which revealed that TRPA1 is expressed in the carotid body. Furthermore, Ca²⁺ imaging experiments suggested that TRPA1 in the carotid body did not contribute to the response to severe hypoxia, but the respond to moderate hypoxia. Taken together, our results suggest that TRPA1 may play a role in the oxygen sensing mechanism in the carotid body.

Voltage (ΔΨ) across the inner mitochondrial membrane (IMM) controls a variety of mitochondrial function including ATP synthesis, thermogenesis, and cell death. Thus, maintenance of ΔΨ and stability of its magnitude are of a paramount significance for the physiology of the cell and the entire organism. Currently, the electron transport chain remains the only well-established mechanism for the ΔΨ maintenance. Here, we identify mitochondrial Cl⁻ channels as a crucial mechanism for the ΔΨ maintenance. We use the whole-IMM patch-clamp analysis, and demonstrate that mitochondria possess two distinct types of voltage-gated Cl⁻ channels (Cl_V), inactivating Cl_V activated at hyperpolarized voltage (hCl_V) and non-inactivating Cl_V activated at depolarized voltage (dCl_V). hCl_V is characterized by low activation threshold just below the physiological ΔΨ values and has fast inactivation. hCl_V is a novel mitochondrial Cl⁻ channel that has never been reported previously. In contrast, dCl_V activated only by profound membrane depolarization to ~ 0 mV and is completely lacking inactivation. dCl_V likely corresponds to the inner membrane anion channel or the 108-pS anion channel, but its detailed electrophysiological analysis was missing. Using optical methods and mitochondrial respiration assays, we demonstrate that mitochondrial Cl⁻ channels largely ameliorate ΔΨ depolarization induced by mitochondrial uncouplers (H⁺ leak) and Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter. Importantly, mitochondrial Cl⁻ channels profoundly delay the activation of mitochondrial permeability transition pore. Thus, hCl_V and dCl_V represent a previously unknown mechanism for ΔΨ maintenance, which could play a fundamental role in preserving mitochondrial integrity and function.

The prostanoid EP4 receptor belongs to the family of G protein-coupled receptors and it has been reported that the signaling pathway mediated by the EP4 receptor contributes to the cancer development. The cytoplasmic region of the EP4 receptor has several characteristic sites that are significantly different from other prostanoid receptors. In this study, we focused on one amino acid and examined the effects of point mutation on EP4 receptor signaling pathway. Thus, the point mutation-introduced EP4 receptor-stably expressing HEK cell line was prepared, and comparative experiment were conducted with wild-type EP4 receptor-expressing cell line. As a result, constitutive cAMP production and ligand-independent cell proliferation retardation were observed in the mutant EP4 receptor-expressing cell line when compared with wild-type EP4 receptor expressing cell line. These results suggested that the introducing only one mutation of the specific amino acid of the EP4 receptor would have a possibility to suppress the EP4 receptor-initiated cancer development by delaying the growth of cells.

The Gs protein coupled D type prostanoid (DP) receptors have been known to exhibit anti-inflammatory effects.

The DP receptors are well recognized as cognate receptors for prostaglandin (PG)D₂, which is known to be involved in inflammatory responses and allergies.

There are five endogeneous metabolites of PGD₂; PGJ₂, Δ¹²-PGJ₂, 13,14-dihydro-15-keto-PGD₂, 15-deoxy-Δ^12,14-PGD₂, and 15-deoxy-Δ^12,14-PGJ₂.

Previously, we showed that 15-keto-PGE₂, a metabolite of PGE₂, acts on EP2 and EP4 receptors as biased agonist, and plays a role on switching cellular signalings mediated from EP4 receptors to EP2 receptors.

Therefore, we here examined if PGD₂ and these five metabolites act on DP receptors as biased agonists, and found out they showed different profiles in terms of DP receptor activities.

Thus, PGD₂ and PGJ₂ acted as full agonists with similar potencies, whereas, Δ¹²-PGJ₂ acted as a partial agonist to the cAMP system and T cell factor/β-catenin transcriptional activity.

These results suggest that the metabolites of PGD₂ are not simply inactivated metabolites, but may have some physiological significance in the inflammatory response mediated by DP receptors.

G-protein-gated inwardly rectifying K⁺ (GIRK) channels control various physiological functions. For example, GIRK1/2 heterotetramers in the brain regulate neuronal excitability; GIRK1/4 heterotetramers in the heart regulate heart rate. GIRK channels are potential therapeutic targets for the treatment of several diseases, such as atrial fibrillation and addiction. In the present study, we aim to identify novel agonists/antagonists of GIRK channel and its disease-causing mutants. By electrophysiological recordings using Xenopus oocytes expressing different GIRK subunits without G-protein-coupled receptors, we screened the effect of a chemical library containing hundreds of natural products on GIRK currents. We observed that some plant alkaloids inhibit the current of wild-type GIRK1/2 and GIRK1/4 channels and some other plant alkaloids strongly inhibit a neurologic disorder-causing mutant of GIRK2 channel, G156S, which locates at the selectivity filter and induces the loss of K⁺ selectivity. Our data provided us with a clue toward the elucidation of the potential of natural products as sources of novel therapeutic agents on GIRK-targeted disease.

CC motif chemokine receptor 8 (CCR8), a G protein-coupled receptor (GPCR), is highly expressed in regulatory T cells, T helper 2 cells, and cancer cells. It plays important role in allergic inflammation and cancer development. Therefore, specific monoclonal antibodies (mAbs) for CCR8 would be useful for diagnostic and therapeutic purposes of the diseases. However, the production of mAbs for GPCRs has remained very difficult. We have developed a novel method for the development of mAbs, named the Cell-Based Immunization and Screening (CBIS) method. In the present study, an SD rat was immunized with mouse CCR8-overexpressed CHO-K1 cells (CHO/mCCR8). The hybridomas expressing anti-mCCR8 mAbs were screened by using CHO/mCCR8 and CHO-K1 cells. We obtained 73 strongly anti-mCCR8 mAb-expressing hybridomas from 1,916 hybridomas, and we finally established C₈Mab-2 (IgG_2b, kappa). C₈Mab-2 selectively reacted to CHO/mCCR8 cells in a dose-dependent manner, but not to CHO-K1 cells, in flow cytometry. C₈Mab-2 also recognized endogenous mCCR8 in a mouse lymphocyte-like cell line (P388) and a mouse macrophage-like cell line (J774-1). Furthermore, C₈Mab-2 visualized mCCR8 in CHO/mCCR8, P388, and J774-1 cells in immunocytochemistry. In conclusion, we developed the anti-mCCR8 mAb, C₈Mab-2, which is available for detecting endogenous and exogenous mCCR8 in flow cytometry and immunocytochemistry. C₈Mab-2 would be usable for diagnosis and medical treatment for allergic inflammation and cancer in mouse models.

The Mas-related receptor A3 (MRGPRA3), an orphan GPCR, is expressed specifically in the dorsal root ganglion (DRG) sensory neurons, and it has recently been attracting much attention as an itch inducer. While MRGPRA3 responds to chloroquine which is an anti-malaria drug and causes strong itch, chloroquine requires high concentrations to activate MRGPRA3 and also produces MRGPRA3-independent responses. Therefore, in order to unveil the role of MRGPRA3, a new tool that enables selective manipulation of MRGPRA3 function is needed. In this study, we screened a series of small-molecule chemical libraries (1,084 compounds) to explore agonists for MRGPRA3 by high-throughput Ca²⁺ imaging. We identified that papaverine, an opium alkaloid, specifically evoked Ca²⁺ responses in cells expressing MRGPRA3 without affecting responses of other MRGPRs subtypes. Papaverine evoked Ca²⁺ responses in a subpopulation of DRG neurons that responded to chloroquine. In addition, we found that intradermal injection of papaverine to the cheek produced scratching behavior in a histamine-independent manner and did not produce nociceptive wiping behavior. Furthermore, the papaverine-induced scratching behavior was suppressed by a selective ablation of MRGPRA3-expressing primary afferent neurons or a genetic knockout of gastrin-releasing peptide receptors (GRPR: a crucial receptor for spinal itch transmission). Taken together, these results uncover a new pharmacological action of papaverine that is a potent and selective agonistic effect for MRGPRA3, which could be a powerful tool for research investigating the biological role of MRGPRA3 and the physiology and pathology of itch.

Activation of glial cells contributes neural disorders like depression and neuropathic pain through the release of nitric oxide and cytokines. The release depends on intracellular Ca2+ levels. One of the Ca2+ entry pathways is Ca2+ release-activated Ca2+ (CRAC) channel. Anti-depressants are used for treatment of trigeminal neuralgia, but the effects of anti-depressants on CRAC channel have remained unsolved. We therefore examined the inhibitory effects of anti-depressants on the CRAC channel using patch-clamp technique. We recorded the CRAC channel current in rat basophil leukemia (RBL) cells instead of glial cells. The reasons are 1) RBL cells are model cells to record the CRAC channel current, and 2) glial cells have many kinds of ion channels. The CRAC channel was activated by adding 10-mM IP3 in the pipette solution. Membrane potential was held at 0 mV and was changed every 3 s from -120 mV to 80 mV with the 50-ms ramp wave. The membrane current showed the inward rectification, and its polarity reversed at >50 mV. These properties are hallmarks of the CRAC channel. Duloxetine, one of the anti-depressants, inhibited the CRAC channel current at -100 mV in a time-dependent and a concentration-dependent manners. The rank order of the inhibition was duloxetine = paroxetine > nortriptyline > imipramine. Blockade of the CRAC channel might be one of the pain relief mechanisms of duloxetine.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social communication and repetitive/restricted behaviors. Folate is essential for normal fetal development and growth. Our previous study has described that prenatal folate deficiency (FD) mice have exhibited ASD-like decreased sociability. Also, they have showed increase in convulsant-induced seizure susceptibility and decrease in anxiety-like behavior. In this study, we examined the changes in the relative expression of N-methyl-D-aspartate (NMDA)-type glutamate receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor, postsynaptic density protein 95 (PSD-95), and GABA-synthesizing enzymes (GAD65/67) in the prefrontal cortex (PFC). We found that the expression of NMDA receptor subunit 2b, AMPA receptor 1, PSD-95, and GAD65/67 were significantly decreased in FD mice compared with control mice. These results may suggest that decrease in both of excitatory and inhibitory synapse-related proteins in the PFC could have effects on behavioral impairment of FD mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by asynchronous development in several areas such as social behaviors, cognitive capabilities, and sensory responsiveness. Among the sensory abnormalities recognized as important features of ASD is either heightened or reduced sensitivity to pain. Individuals with ASD may experience pain in unusual ways, but mechanisms underlying altered pain sensitivity and processing in ASD remain unknown. Here we investigated the pain sensitivity in a prenatal valproic acid (VPA)-induced model of ASD and subsequently analyzed the pain signaling. Pregnant ICR mice were intraperitoneally injected with either VPA or saline on embryonic day 12.5. Male offspring of VPA-treated mothers showed mechanical allodynia. In the dorsal horn of the spinal cord in prenatal VPA-treated mice, the numbers and staining intensities of Iba1-positive cells were increased and the cell bodies became enlarged, indicating the microglial activation. Administration of PLX3397, a colony-stimulating factor 1 receptor inhibitor, resulted in a decreased number of spinal microglia and attenuated mechanical allodynia in prenatal VPA-treated mice. These findings suggest that prenatal VPA treatment causes allodynia and microglial activation might in part contribute to increased nociceptive responses.

Early-life social experience is critical for the development of social cognition, and early social deprivation induces alteration of sociality in animals and human. The medial orbitofrontal cortex (mOFC) to basolateral amygdala (BLA) pathway is one of the critical circuit for social behavior. Hence, the mOFC-BLA pathway may be functionally sensitive for early social environment, and its dysfunction may cause changes in social behaviors. Here, we examined effects of social isolation (SI) and resocialization (RS) on mOFC-BLA synaptic functions in mice. First, we isolated the mice during early (3-5 weeks of age) or late (6-8 weeks of age) adolescence and assessed sociality and mOFC-BLA synaptic properties using optogenetic and patch-clamp methods. SI in early-adolescent, but not late adolescent, decreased sociality and AMPA/NMDA current ratio in the mOFC-BLA synapse. Then, we examined the effects of RI during late adolescence or adulthood (9-11 weeks of age), on SI-induced synaptic and social deficit. RS in late adolescence, but not adulthood, recovered SI-induced social deficit and mOFC-BLA synaptic change. These results suggested that SI disrupted mOFC-BLA synaptic function in early adolescence and caused social deficit, and restorative effect of RS on SI-induced behavioral and synaptic change limited until late adolescence.

Unapproved use of some antiepileptic drugs has been reported to ameliorate core social deficits of autism spectrum disorder (ASD); however, the neural mechanisms remain unclear. Here, using brain-wide neuronal activation mapping in Arc-dVenus reporter mice, we showed that ASD model mice exhibited not only aberrant hyperactivation in multiple brain areas during social interaction but also disruption of prefrontal nodes in the brain network associated with social impairments. Three unapproved-use drugs for core ASD symptoms reversed the social deficits and dysfunction of the prefrontal nodes in the brain network. In addition, we identified a new unapproved use drug that reversed the social deficits and the dysfunction of prefrontal nodes during social interactions in ASD mice. These results suggest that prefrontal nodes in the brain network can be a diagnostic feature and a therapeutic target for the core symptoms of ASD and that its monitoring is useful to predict the therapeutic effect of ASD drug candidates.

It is known that N-methyl-D-aspartate (NMDA) receptor is essential for early brain development. NMDA receptor blockade during neonatal period causes various abnormal behaviors in later life. In the developing brain, NR2A- and NR2B-containing NMDA receptors show different expression patterns. However, the functions of these NMDA receptors in brain development are unknown. Therefore, we investigated the effects of pharmacological inactivation of NR2A- and/or NR2B-containing NMDA receptors on various behaviors in adulthood. We postnatally treated rats with an NR2A-preferring (PEAQX), an NR2B-selective (ifenprodil), or a nonselective NMDA receptor blocker (MK-801). Interestingly, neonatal treatment with PEAQX or MK-801 caused significant decrease in spontaneous alternation in the Y-maze test. In addition, PEAQX or MK-801 treatment increased startle response to acoustic stimuli. Neonatal PEAQX treatment also induced hypersensitivity to locomotor-stimulating effect of MK-801. On the other hand, neonatal ifenprodil treatment did not cause these behavioral alterations. Furthermore, PEAQX-treated but not ifenprodil-treated rats exhibited deformity of the hippocampal CA1 area, while neonatal NMDA receptor blockade did not alter the cell density in the hippocampus. In conclusion, our results suggest that the NR2A-containing NMDA receptor plays important roles in early brain development in rats, and hypofunction of this subunit during developmental period induces schizophrenia-related behaviors in adulthood.

The aim of the present study is to clarify emotional property focusing on the depressive-like behaviors, and its neuronal mechanism in the raphe nuclei which are involved in the mediation of emotional functions, in rats repeatedly administered ACTH during the early postnatal period.

Tetracosactide, the N-terminal 24 amino acids of the naturally occurring ACTH, were administered once a day at dose of 100 µg to male rat pups for 5 days on the day 21 after birth (3wACTH). Saline-injected rats were subjected as a littermate control.

Adult 3wACTH (10-12 weeks old) showed the decrease of sucrose consumption in the sucrose preference test, and reduction of time spent grooming after spraying a sucrose solution to the dorsal coat in the splash test. These abnormal behaviors in 3wACTH indicated the depressive-like/anhedonic-like behaviors, which were ameliorated by repeated administration of fluvoxamine, a selective serotonin reuptake inhibitor. Moreover, immunohistochemical studies revealed that the expression of parvalbumin, a marker of GABAergic neurons, significantly reduced in the raphe nuclei of adult 3wACTH.

These findings suggest that pharmacological stress during early postnatal period might produce depressive-like behaviors, which possibly implicated in dysfunction of the GABAergic neuronal systems in the raphe nuclei.