Suppressive Activity of Boiogito, a Japanese Traditional Kampo Medicine, on Periostin Secretion in Human Fibroblast-Like Synoviocytes In Vitro.

Background Knee osteoarthritis (KOA) is a prevalent degenerative disease that affects the knee joints, particularly among individuals aged over 40 years. It leads to pain, stiffness, and reduced quality of life; affects approximately 300 million individuals worldwide; and is increasing, particularly in developed nations. Although treatments for KOA range from conservative measures to surgical interventions, such as total knee arthroplasty (TKA), the financial burden of TKA in many countries underscores the urgent need for effective conservative therapies. The pathophysiology of KOA involves articular cartilage degeneration, increased subchondral bone turnover, synovitis, and periarticular soft tissue contracture. Abnormal bone turnover, intensified by factors, such as weight gain and knee injury, precedes cartilage degeneration. Synovitis, characterized by inflammation in the synovial tissue, plays a crucial role in perpetuating the disease by triggering a cascade of catabolic and proinflammatory mediators, including cytokines, such as interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-13. Periostin, an extracellular matrix protein, is implicated in KOA progression, with its levels increasing with disease severity. Materials & methods In this study, the preventive effect of boiogito (BOT), a traditional herbal medicine, on periostin secretion in human fibroblast-like synoviocytes (hFLS) stimulated by IL-13 was investigated. Synoviocyte Growth Medium and recombinant human IL-13 were used for cell culture and stimulation. BOT was dissolved in phosphate-buffered saline and applied to cell cultures. Periostin secretion and mRNA expression were measured using enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction, respectively. Cell viability was assessed using an MTT assay, and signal transducer and activator of transcription factor 6 (STAT6) phosphorylation was examined using Western blotting. Results IL-13 stimulation of hFLS significantly increased periostin secretion, with levels rising above 20 ng/mL after 72 h of stimulation. Pretreatment with BOT dose-dependently suppressed periostin secretion, with doses of 1,000 µg/mL significantly reducing periostin levels. Furthermore, BOT inhibited periostin mRNA expression and STAT6 phosphorylation in IL-13-stimulated hFLS, suggesting its potential in modulating IL-13-mediated inflammatory pathways in KOA. Conclusion This study demonstrated the preventive effect of BOT on periostin secretion in IL-13-stimulated hFLS, highlighting its potential as a therapeutic agent for KOA. By inhibiting periostin production and downstream signaling pathways, BOT may offer a promising conservative treatment option for KOA, addressing the inflammatory cascade implicated in disease progression. Further research is warranted to elucidate the specific herbal components responsible for the therapeutic effects of BOT and to validate its efficacy in clinical settings.

Duration of the preemptive analgesic effects of low- and high-frequency transcutaneous electrical nerve stimulation in rats with acute inflammatory pain.

Transcutaneous electrical nerve stimulation (TENS) activates various pathways to induce antinociceptive effects, based on the frequencies used. This study evaluates the preemptive analgesic effects and their duration of low- (LT: 4 Hz) and high-frequency TENS (HT: 100 Hz) using a rat model of acute inflammatory pain. Acute inflammation was induced by injecting 1% formalin into the hind paws of rats. LT or HT was applied for 30 min before formalin injection. Pain-related behaviors, such as licking, flinching, and lifting, were recorded for 60 min postinjection. Immunohistochemistry was used to assess the number of phosphorylated extracellular signal-regulated kinase (pERK)- and c-fos-positive cells in the spinal cord. Naloxone, a µ-opioid receptors (MORs) antagonist, and naltrindole, a δ-opioid receptors (DORs) antagonist, were administered before TENS application. Pain behavior duration and pERK- and c-fos-positive cell expression were then measured. LT and HT pretreatment significantly reduced both pain behaviors and the number of pERK- and c-fos-positive cells postformalin injection. Naloxone and naltrindole partially reversed the effects of LT and HT, respectively. Notably, HT's analgesic effect lasted up to 120 min whereas that of LT persisted for 90 min. LT and HT effectively exerted their preemptive analgesic effects on acute inflammatory pain by inhibiting pERK and c-fos expression in the spinal cord. HT presented a longer-lasting effect compared to LT. MOR and DOR activation may contribute to LT and HT's analgesic mechanisms, respectively.

The Preventive Effects of Platelet-Rich Plasma Against Knee Osteoarthritis Progression in Rats.

BACKGROUND: In recent years, the intra-articular administration of platelet-rich plasma (PRP), a novel therapeutic strategy for knee osteoarthritis (KOA), has gained attention. However, the efficacy of PRP in inhibiting degenerative joint changes remains unclear. The current study aimed to evaluate the therapeutic effect of the intra-articular administration of PRP in rats with induced KOA. MATERIALS AND METHODS: PRP was prepared from the whole blood of nine-week-old male Wistar rats via centrifugation at 25°C, 200 × g, for seven minutes. KOA was induced in the right knees of the rats via destabilization of the medial meniscus (DMM) surgery. The animals were divided into the control, sham, DMM, and DMM + PRP groups (n = 5 each). The rats in the DMM + PRP group received 50 µL of intra-articular PRP in the right knee joint four weeks after surgery. The rotarod test was conducted to assess locomotive function. Eight weeks after DMM surgery, the degree of medial meniscus extrusion was measured via computed tomography (CT) images on the right knee. Then, a histological analysis of the harvested knees was conducted. KOA progression was assessed using the Osteoarthritis Research Society International (OARSI) score. The number of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the subchondral bone was counted via histological analysis. RESULTS: The degree of medial meniscus extrusion did not significantly differ between the DMM and DMM + PRP groups. Similarly, there were no significant differences in the walking time based on the rotarod test between the DMM and DMM + PRP groups. However, the DMM group had a significantly higher OARSI score than the DMM + PRP group. The number of TRAP-positive osteoclasts in the subchondral bone of the DMM group increased over time, peaking four weeks after surgery. The DMM + PRP group had a higher number of TRAP-positive osteoclasts in the subchondral bone than the control group. However, there was no significant difference between the number of TRAP-positive osteoclasts between the DMM group and the control and sham groups. CONCLUSION: The intra-articular administration of PRP may inhibit KOA progression in a rat model, especially in the articular cartilage degradation and osteophyte formation. The results can provide further evidence about the efficacy of PRP against KOA progression and can contribute to the current practice of healthcare professionals based on accurate knowledge.

Extracellular high molecular weight α-synuclein oligomers induce cell death by disrupting the plasma membrane.

α-Synuclein (αS), the causative protein of Parkinson's disease and other α-synucleinopathies, aggregates from a low molecular weight form (LMW-αS) to a high molecular weight αS oligomer (HMW-αSo). Aggregated αS accumulates intracellularly, induces intrinsic apoptosis, is released extracellularly, and appears to propagate disease through prion-like spreading. Whether extracellular αS aggregates are cytotoxic, damage cell wall, or induce cell death is unclear. We investigated cytotoxicity and cell death caused by HMW-αSo or LMW-αS. Extracellular HMW-αSo was more cytotoxic than LMW-αS and was a crucial factor for inducing plasma membrane damage and cell death. HMW-αSo induced reactive oxygen species production and phospholipid peroxidation in the membrane, thereby impairing calcium homeostasis and disrupting plasma membrane integrity. HMW-αSo also induced extrinsic apoptosis and cell death by activating acidic sphingomyelinase. Thus, as extracellular HMW-αSo causes neuronal injury and death via cellular transmission and direct plasma membrane damage, we propose an additional disease progression pathway for α-synucleinopathies.

Shoseiryuto Ameliorated TDI-Induced Allergic Rhinitis by Suppressing IL-33 Release from Nasal Epithelial Cells.

Toluene diisocyanate (TDI) is a major cause of occupational asthma and rhinitis. Shoseiryuto (SST) is one of the traditional herbal medicines (Kampo medicine) and has long been used as a natural medicine for allergic diseases such as allergic rhinitis (AR) and asthma. Recent studies have shown that the expression and release of IL-33, which regulates the TH2 cytokine response in epithelial cells, is an important step in developing the inflammatory response of the nasal mucosa. In this study, we investigated whether SST may ameliorate the TDI-induced AR-related symptoms in rats and inhibit IL-33 release from nasal epithelial cells. An AR rat model was generated by sensitization and induction with TDI. SST was administered during the sensitization period. AR-related symptoms in rats were evaluated, and IL-33 release was measured both in vivo and in vitro. SST suppressed symptoms appearing in TDI-induced AR model rats, such as elevated serum histamine and IL-33 levels in nasal lavage fluid (NLF)/serum, which were suppressed by SST administration. TDI-induced IL-33 release from the nasal epithelial cell nuclei was also observed and suppressed in SST-treated rats and cultured nasal epithelial cells. These results suggest that SST ameliorates the symptoms of TDI-induced AR at least partially by inhibiting IL-33 release from nasal epithelial cells.

Yokukansan Inhibits the Development of Morphine Tolerance by Regulating Presynaptic Proteins in DRG Neurons.

Opioids, such as morphine, are used in clinical settings for the management of acute and chronic pain. However, long-term use of morphine leads to antinociceptive tolerance and hypersensitivity. The cellular and molecular mechanisms of morphine tolerance seem to be quite complex, with suggestions including internalization of the µ-opioid receptor (MOR), neuroinflammation with activation of microglia and astrocytes, and changes in synaptic function in the central nervous system. Yokukansan (YKS), a traditional Kampo medicine consisting of seven herbs, has been used to treat emotional instability, neurosis, and insomnia. Interestingly, recent studies have begun to reveal the inhibitory effect of YKS on the development of morphine tolerance. In the present study, we determined the effect of YKS on morphine tolerance formation and its mechanisms in a rat model, focusing on the synapses between primary sensory neurons and spinal dorsal horn secondary neurons. We found that morphine tolerance formation was significantly inhibited by YKS (0.3 or 1.0 g/kg/day) preadministration for 7 days. Repeated administration of morphine (10 mg/kg/day) increased the expression of presynaptic proteins, including synaptotagmin I, in the spinal cord, which was suppressed by YKS. Furthermore, these changes in presynaptic protein expression were more pronounced at isolectin B4 (IB4)-positive excitatory synapses around the lamina II of the dorsal horn. These results suggest that YKS suppresses the development of morphine tolerance by inhibiting the enhancement of presynaptic function of dorsal root ganglia neurons projecting to spinal dorsal horn neurons caused by continuous morphine administration.

Kamikihito rescued depressive-like behaviors and hippocampus neurogenesis in chronic restraint stress rats.

Background and aim: Substantial evidence suggests the effectiveness of plant-based medicine in stress-related diseases. Kamikihito (KKT), a Japanese traditional herbal medicine (Kampo), has been used for anemia, insomnia, and anxiety. Recent studies revealed its ameliorating effect on cognitive and memory dysfunction in several animal models. We, therefore, determined whether daily supplementation of KKT has an antidepressant-like effect on the stress-induced behavioral and neurological changes in rats. Experimental procedure: The effect of KKT against the stress-induced changes in anxiety- and depressive-like behaviors and hippocampal neurogenesis were determined using a rat model of chronic restraint stress (CRS). KKT was orally administered daily at 300 or 1000 mg/kg during 21 consecutive days of CRS (6 h/day). The effect of CRS and KKT on physiological parameters, including body weight gain, food/water consumptions, plasma corticosterone (CORT) levels, and percentage of adrenal gland weight to body weight, were firstly measured. Anxiety- and depressive-like behaviors in rats were assessed in the open field test (OFT), sucrose preference test (SPT), and forced swimming test (FST). Hippocampal neurogenesis was determined by immunohistochemistry. Results and conclusion: CRS for 21 days caused a significant decrease in body weight gain and increase in plasma CORT levels and percentage of adrenal gland weight to body weight, which were rescued by KKT treatment. KKT also suppressed the CRS-induced anxiety- and depressive-like behaviors and impairment of hippocampal neurogenesis. These results suggest that daily treatment of KKT has a protective effect against physiological, neurological, and behavioral changes in a rat model of depression.

Brain-derived neurotrophic factor predominantly regulates the expression of synapse-related genes in the striatum: Insights from in vitro transcriptomics.

AIM: The striatum, a main component of the basal ganglia, is a critical part of the motor and reward systems of the brain. It consists of GABAergic and cholinergic neurons and receives projections of dopaminergic, glutamatergic, and serotonergic neurons from other brain regions. Brain-derived neurotrophic factor (BDNF) plays multiple roles in the central nervous system, and striatal BDNF has been suggested to be involved in psychiatric and neurodegenerative disorders. However, the transcriptomic impact of BDNF on the striatum remains largely unknown. In the present study, we performed transcriptomic profiling of striatal cells stimulated with BDNF to identify enriched gene sets (GSs) and their novel target genes in vitro. METHODS: We carried out RNA sequencing (RNA-Seq) of messenger RNA extracted from primary dissociated cultures of rat striatum stimulated with BDNF and conducted Generally Applicable Gene-set Enrichment (GAGE) analysis on 10599 genes. Significant differentially expressed genes (DEGs) were determined by differential expression analysis for sequence count data 2 (DESeq2). RESULTS: GAGE analysis identified significantly enriched GSs that included GSs related to regulation and dysregulation of synaptic functions, such as synaptic vesicle cycle and addiction to nicotine and morphine, respectively. It also detected GSs related to various types of synapses, including not only GABAergic and cholinergic synapses but also dopaminergic and glutamatergic synapses. DESeq2 revealed 72 significant DEGs, among which the highest significance was observed in the apolipoprotein L domain containing 1 (Apold1). CONCLUSIONS: The present study indicates that BDNF predominantly regulates the expression of synaptic-function-related genes and that BDNF promotes synaptogenesis in various subtypes of neurons in the developing striatum. Apold1 may represent a unique target gene of BDNF in the striatum.

Analgesic Effect of Combined Therapy with the Japanese Herbal Medicine "Yokukansan" and Electroacupuncture in Rats with Acute Inflammatory Pain.

Background: Japanese herbal medicine, called Kampo medicine, and acupuncture are mainly used in Japanese traditional medicine. In this experiment, the analgesic effect of Yokukansan (YKS) alone and a combination of YKS and electroacupuncture (EA) on inflammatory pain induced by formalin injection were examined. Methods: Animals were divided into four groups: a control group, formalin injection group (formalin), YKS-treated formalin group (YKS), and YKS- and EA-treated formalin group (YKS + EA). The duration of pain-related behaviors and extracellular signal-regulated protein kinase (ERK) activation in the spinal cord after formalin injection in the right hind paw were determined. Results: The duration of pain-related behaviors was dramatically prolonged in the late phase (10-60 min) in the formalin group. The YKS treatment tended to reduce (p = 0.08), whereas YKS + EA significantly suppressed the pain-related behaviors (p < 0.01). Immunohistochemical and Western blot analyses revealed that the number of phosphorylated ERK1/2 (pERK1/2)-positive cells and the pERK expression level, which were increased by formalin injection, were significantly inhibited by YKS (p < 0.05) and YKS + EA (p < 0.01). Conclusions: The YKS + EA combination therapy elicited an analgesic effect on formalin-induced acute inflammatory pain.

Analgesic effect of voluntary exercise in a rat model of persistent pain via suppression of microglial activation in the spinal cord.

In this study, we employed a rodent model for persistent allodynia and hyperalgesia to determine whether voluntary exercise could exert analgesic effects on these pain symptoms. Rats were subcutaneously injected with formalin into the plantar surface of the right hind paw to induce mechanical allodynia and hyperalgesia. We assessed the analgesic effects of a voluntary wheel running (VWR) using the von Frey test and investigated microglial proliferation in the dorsal horn of the spinal cord. We also determined the effect of formalin and VWR on the protein expression levels of brain-derived neurotrophic factor (BDNF), its receptor TrkB, and K+-Cl- cotransporter 2 (KCC2), which play a key role in inducing allodynia and hyperalgesia. Rats with access to the running wheels showed beneficial effects on persistent formalin-induced mechanical allodynia and hyperalgesia. The effects of VWR were elicited through the suppression of formalin-induced microglial proliferation, TrkB up-regulation, and KCC2 down-regulation in the spinal cord. BDNF, however, might not contribute to the beneficial effects of VWR. Our results show an analgesic effect of voluntary physical exercise in a rodent model with persistent pain, possibly through the regulation of microglial proliferation and TrkB and KCC2 expression in the spinal cord.

Small noncoding vault RNA modulates synapse formation by amplifying MAPK signaling.

The small noncoding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. Emerging evidence suggests that vtRNAs may be involved in the regulation of a variety of cellular functions when unassociated with the vault complex. Here, we demonstrate a novel role for vtRNA in synaptogenesis. Using an in vitro synapse formation model, we show that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. mvtRNA is transported to the distal region of neurites as part of the vault complex. Interestingly, mvtRNA is released from the vault complex in the neurite by a mitotic kinase Aurora-A-dependent phosphorylation of MVP, a major protein component of the vault complex. mvtRNA binds to and activates MEK1 and thereby enhances MEK1-mediated ERK activation in neurites. These results suggest the existence of a regulatory mechanism of the MAPK signaling pathway by vtRNAs as a new molecular basis for synapse formation.

Behavioral and neurological improvement by Cydonia oblonga fruit extract in chronic immobilization stress rats.

It is known that chronic stress is a contributing factor to several physical and mental diseases. In this study, we examined the effect of hydroethanolic extract of Cydonia oblonga fruit (HECO, 300 mg/kg) in chronically immobilized rats on physiological and behavioral parameters by the open field test (OFT), sucrose preference test (SPT), and forced swimming test (FST) and on neurological alterations by analysis of the hippocampal neurogenesis. A daily 6 hr exposure to chronic immobilization stress (CIS) for 21 consecutive days induced anxiety- and depressive-like behaviors in rats' concomitant with decreased weight gain and increased plasma corticosterone (CORT) levels, rats also showed atrophy in the CA3 subregion of the hippocampus and a decreased number of Ki67 and DCX positive cells in the dentate gyrus (DG). Treatment with HECO successfully suppressed the physiological and behavioral markers of the CIS and prevents the structural abnormality and the impaired cell proliferation in the hippocampus. Moreover, the daily administration of HECO improved the mood function in normal rats. Taking together, our findings demonstrate, for the first time, the anti-depressive effect of C. oblonga fruit by enhancing the hippocampal neurogenesis in the rat model of depression.

Analgesic Efficacy of a Combination of Fentanyl and a Japanese Herbal Medicine "Yokukansan" in Rats with Acute Inflammatory Pain.

Background: Fentanyl can induce acute opioid tolerance and postoperative hyperalgesia when administered at a single high dose; thus, this study examined the analgesic efficacy of a combination of fentanyl and Yokukansan (YKS). Methods: Rats were divided into control, formalin-injected (FOR), YKS-treated+FOR (YKS), fentanyl-treated+FOR (FEN), and YKS+FEN+FOR (YKS+FEN) groups. Acute pain was induced via subcutaneous injection of formalin into the paw. The time engaged in pain-related behavior was measured. Results: In the early (0-10 min) and intermediate (10-20 min) phases, pain-related behavior in the YKS+FEN group was significantly inhibited compared with the FOR group. In the late phase (20-60 min), pain-related behavior in the FEN group was the longest and significantly increased compared with the YKS group. We explored the influence on the extracellular signal-regulated kinase (ERK) pathway in the spinal cord, and YKS suppressed the phosphorylated ERK expression, which may be related to the analgesic effect of YKS in the late phase. Conclusions: These findings suggest that YKS could reduce the use of fentanyl and combined use of YKS and fentanyl is considered clinically useful.

Vexin is upregulated in cerebral cortical neurons by brain-derived neurotrophic factor.

AIM: Chromosome 8 open reading frame 46 (C8orf46), a human protein-coding gene, has recently been named Vexin. A recent study indicated that Vexin is involved in embryonic neurogenesis. Additionally, some transcriptomic studies detected changes in the mRNA levels of patients with psychiatric and neurological diseases. In our previous study, we sought for target genes of brain-derived neurotrophic factor (BDNF) in cultured rat cortical neurons, finding that BDNF potentially leads to the upregulation of Vexin mRNA. However, its underlying mechanisms are unknown. In the present study, we assessed the regulatory mechanisms of the BDNF-induced gene expression of Vexin in vitro. METHODS: We reanalyzed ChIP-seq data in various human organs provided by the ENCODE project, evaluating acetylation levels of the 27th lysine residue of the histone H3 (H3K27ac) at the Vexin locus. The transcriptomic effects of BDNF on rat Vexin (RGD1561849) were evaluated by real-time quantitative PCR (RT-qPCR) in primary cultures of cerebral cortical neurons, in the presence or absence of inhibitors for signaling molecules activated by BDNF. RESULTS: The Vexin locus and its promoter region in the brain angular gyrus show higher acetylation levels of the H3K27 than those in other organs. Stimulation of cultured rat cortical neurons, but not astrocyte, with BDNF, led to marked elevations in the mRNA levels of Vexin, which was inhibited in the presence of K252a and U0126. CONCLUSION: The upregulated H3K27ac in the brain may be associated with the enriched gene expression of Vexin in the brain. It is indicated that BDNF induces the gene expression of Vexin in the cortical neurons via the TrkB-MEK signaling pathway.

Adipose-Derived Stem Cells and Ceiling Culture-Derived Preadipocytes Cultured from Subcutaneous Fat Tissue Differ in Their Epigenetic Characteristics and Osteogenic Potential.

BACKGROUND: Adipose-derived stem cells and ceiling culture-derived preadipocytes can be harvested from subcutaneous adipose tissue. Little is known about the epigenetic differences, which may contribute to differences in osteogenic potential, between these cell types. The purpose of this study was to address the osteogenic potential and underlying epigenetic status of adipose-derived stem cells and ceiling culture-derived preadipocytes. METHODS: Adipose-derived stem cells and ceiling culture-derived preadipocytes were cultured from abdominal subcutaneous fat tissues of four metabolically healthy, lean female patients. After 7 weeks of culture, cellular responses to osteogenic differentiation media were examined. To evaluate the osteogenic potentials of undifferentiated adipose-derived stem cells and ceiling culture-derived preadipocytes, two types of epigenetic assessment were performed using next-generation sequencing: DNA methylation assays with the Human Methylation 450K BeadChip, and chromatin immunoprecipitation assays for trimethylation of histone H3 at lysine 4. RESULTS: Human ceiling culture-derived preadipocytes showed greater osteogenic differentiation ability than did adipose-derived stem cells. In an epigenetic survey of the promoters of four osteogenic regulator genes (RUNX2, SP7, ATF4, and BGLAP), the authors found a general trend toward decreased CpG methylation and increased trimethylation of histone H3 at lysine 4 levels in ceiling culture-derived preadipocytes as compared to adipose-derived stem cells, indicating that these genes were more likely to be highly expressed in ceiling culture-derived preadipocytes. CONCLUSIONS: The surveyed epigenetic differences between adipose-derived stem cells and ceiling culture-derived preadipocytes were consistent with the observed differences in osteogenic potential. These results enhance the authors' understanding of these cells and will facilitate their further application in regenerative medicine.

Actions of Brain-Derived Neurotrophin Factor in the Neurogenesis and Neuronal Function, and Its Involvement in the Pathophysiology of Brain Diseases.

It is well known that brain-derived neurotrophic factor, BDNF, has an important role in a variety of neuronal aspects, such as differentiation, maturation, and synaptic function in the central nervous system (CNS). BDNF stimulates mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), phosphoinositide-3kinase (PI3K), and phospholipase C (PLC)-gamma pathways via activation of tropomyosin receptor kinase B (TrkB), a high affinity receptor for BDNF. Evidence has shown significant contributions of these signaling pathways in neurogenesis and synaptic plasticity in in vivo and in vitro experiments. Importantly, it has been demonstrated that dysfunction of the BDNF/TrkB system is involved in the onset of brain diseases, including neurodegenerative and psychiatric disorders. In this review, we discuss actions of BDNF and related signaling molecules on CNS neurons, and their contributions to the pathophysiology of brain diseases.

Basic fibroblast growth factor increased glucocorticoid receptors in cortical neurons through MAP kinase pathway.

Prolonged and intense stress chronically increases blood concentration of glucocorticoids, which in turn causes downregulation of glucocorticoid receptor (GR) in the central nervous system (CNS). This process has been suggested to be involved in the pathogenesis of major depressive disorder (MDD). Here, we found that basic fibroblast growth factor (bFGF) increased the expression of GR in the rat cerebral cortex and cultured cortical neurons and restored the reduced GR expression caused by glucocorticoid exposure. Among intracellular signaling pathways stimulated by bFGF, extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway was responsible for the upregulation of GR. The bFGF-induced GR was functional as a transcription factor to enhance transcription of a target gene. Because high stress augments bFGF levels in the brain, it is likely that bFGF plays a compensating role for reduced GR expression after stress and thus should be studied as a therapeutic target for the treatment of MDD.

[Evaluation of Dissolution Profiles of Famotidine from OTC Drugs].

　In recent years, self-medication has started to receive more attention in Japan owing to increasing medical costs and health awareness among people. One of the main roles of pharmacists in self-medication is to provide appropriate information regarding OTC drugs. However, pharmacists promoting the proper use of OTC drugs have little information on their formulation properties. In this study, we performed dissolution tests on both OTC drugs and ethical drug (ED) containing famotidine, and evaluated the differences in their dissolution profiles. Marked differences in dissolution profiles of OTC drugs were observed in test solutions at pH 1.2, 4.0, and 6.8 and in water. To evaluate the differences quantitatively, we calculated the lag time and dissolution rate constant from the dissolution profiles. Significant differences in lag times and dissolution rate constants between some OTC drugs and ED were observed. We also used similarity factor (f2), to quantify the similarity between dissolution profiles of OTC drugs and ED. f2 values less than 42 were observed in some OTC drugs, suggesting that these differences might influence absorption in vivo resulting in differences in their onset time and efficacy. The findings of this study will provide useful information for the promotion of proper use of OTC drugs.

Corticotropin-releasing hormone-binding protein is up-regulated by brain-derived neurotrophic factor and is secreted in an activity-dependent manner in rat cerebral cortical neurons.

A recent study revealed that corticotropin-releasing hormone (CRH) in the cerebral cortex (CTX) plays a regulatory role in emotional behaviors in rodents. Given the functional interaction between brain-derived neurotrophic factor (BDNF) and the CRH-signaling pathway in the hypothalamic-pituitary-adrenal axis, we hypothesized that BDNF may regulate gene expression of CRH and its related molecules in the CTX. Findings of real-time quantitative PCR (RT-qPCR) indicated that stimulation of cultured rat cortical neurons with BDNF led to marked elevations in the mRNA levels of CRH and CRH-binding protein (CRH-BP). The BDNF-induced up-regulation of CRH-BP mRNA was attenuated by inhibitors of tropomyosin related kinase (Trk) and MEK, but not by an inhibitor for PI3K and Phospholipase C gamma (PLCγ). The up-regulation was partially blocked by an inhibitor of lysine-specific demethylase (KDM) 6B. Fluorescent imaging identified the vesicular pattern of pH-sensitive green fluorescent protein-fused CRH-BP (CRH-BP-pHluorin), which co-localized with mCherry-tagged BDNF in cortical neurons. In addition, live-cell imaging detected drastic increases of pHluorin fluorescence in neurites upon membrane depolarization. Finally, we confirmed that tetrodotoxin partially attenuated the BDNF-induced up-regulation of CRH-BP mRNA, but not that of the protein. These observations indicate the following: In cortical neurons, BDNF led to gene expression of CRH-BP and CRH. TrkB, MEK, presumably ERK, and KDM6B are involved in the BDNF-induced gene expression of CRH-BP, and BDNF is able to induce the up-regulation in a neuronal activity-independent manner. It is suggested that CRH-BP is stored into BDNF-containing secretory granules in cortical neurons, and is secreted in response to membrane depolarization.

The Sox2 promoter-driven CD63-GFP transgenic rat model allows tracking of neural stem cell-derived extracellular vesicles.

Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments.