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Abstract 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.

Adjuvant chemoradiotherapy, molecular targeted therapy, and immunotherapy are frequently employed to extend the survival of patients with advanced gastric cancer (GC). However, most of these treatments have toxic side effects, drug resistance, and limited improvements in survival and quality of life. Therefore, it is crucial to discover and develop new medications targeting GC that are highly effective and have minimal toxicity. In previous studies, the total terpene extract from the stem of Celastrus orbiculatus demonstrated anti-GC activity; however, the specific mechanism was unclear. Our research utilising co-immunoprecipitation-mass spectrometry (Co-IP-MS), polypyrimidine tract binding protein 1 (ptbp1) clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9)-knockout (KO) mouse model, tissue microarray, and functional experiments suggests that alpha actinin-4 (ACTN4) could be a significant biomarker of GC. PTBP1 influences actin cytoskeleton restructuring in GC cells by interacting with ACTN4. Celastrus orbiculatus stem extract (COE) may directly target ACTN4 and affect the interaction between PTBP1 and ACTN4, thereby exerting anti-GC effects.

Abstract Background Polypyrimidine tract binding protein 1 (PTBP1) has been found to play an important role in the occurrence and development of various tumors. At present, the role of PTBP1 in gastric cancer (GC) is still unknown and worthy of further investigation. Methods We used bioinformatics to analyze the expression of PTBP1 in patients with GC. Cell proliferation related experiments were used to detect cell proliferation after PTBP1 knockdown. Skeleton staining, scanning electron microscopy and transmission electron microscopy were used to observe the changes of actin skeleton. Proliferation and actin skeleton remodeling signaling pathways were detected by Western Blots. The relationship between PTBP1 and proliferation of gastric cancer cells was further detected by subcutaneous tumor transplantation. Finally, tissue microarray data from clinical samples were used to further explore the expression of PTBP1 in patients with gastric cancer and its correlation with prognosis. Results Through bioinformatics studies, we found that PTBP1 was highly expressed in GC patients and correlated with poor prognosis. Cell proliferation and cycle analysis showed that PTBP1 down-regulation could significantly inhibit cell proliferation. The results of cell proliferation detection related experiments showed that PTBP1 down-regulation could inhibit the division and proliferation of GC cells. Furthermore, changes in the morphology of the actin skeleton of cells showed that PTBP1 down-regulation inhibited actin skeletal remodeling in GC cells. Western Blots showed that PTBP1 could regulate proliferation and actin skeleton remodeling signaling pathways. In addition, we constructed PTBP1 Cas9-KO mouse model and performed xenograft assays to further confirm that down-regulation of PTBP1 could inhibit the proliferation of GC cells. Finally, tissue microarray was used to further verify the close correlation between PTBP1 and poor prognosis in patients with GC. Conclusions Our study demonstrates for the first time that PTBP1 may affect the proliferation of GC cells by regulating actin skeleton remodeling. In addition, PTBP1 is closely related to actin skeleton remodeling and proliferation signaling pathways. We suppose that PTBP1 might be a potential target for the treatment of GC. Graphical abstract

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.

Abstract 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.

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.

Complex regional pain syndrome (CRPS) is a chronic pain syndrome that occurs in tissue injuries as the result of surgery, trauma, or ischemia. The clinical features of this severely painful condition include redness and swelling of the affected skin. Intriguingly, it was recently suggested that transient receptor potential ankyrin 1 (TRPA1) is involved in chronic post-ischemia pain, a CRPS model. TRPA1 is a non-selective cation channel expressed in calcitonin gene-related peptide (CGRP)-positive primary nociceptors that becomes highly activated in ischemic conditions, leading to the generation of pain. In this review, we summarize the history of TRPA1 and its involvement in pain sensation, inflammation, and CRPS. Furthermore, bone atrophy is also thought to be a characteristic clinical sign of CRPS. The altered bone microstructure of CRPS patients is thought to be caused by aggravated bone resorption via enhanced osteoclast differentiation and activation. Although TRPA1 could be a target for pain treatment in CRPS patients, we also discuss the paradoxical situation in this review. Nociceptor activation decreases the risk of bone destruction via CGRP secretion from free nerve endings. Thus, TRPA1 inhibition could cause severe bone atrophy. However, the suitable therapeutic strategy is controversial because the pathologic mechanisms of bone atrophy in CRPS are unclear. Therefore, we propose focusing on the remission of abnormal bone turnover observed in CRPS using a recently developed concept: senso-immunology.

The neuromodulatory effects of brain stimulation therapies notably involving repetitive transcranial magnetic stimulation (rTMS) on nocturnal sleep, which is critically disturbed in major depression and other neuropsychiatric disorders, remain largely undetermined. We have previously reported in major depression patients that prefrontal rTMS sessions enhanced their slow wave activity (SWA) power, but not their sigma power which is related to sleep spindle activity, for electrodes located nearby the stimulation site. In the present study, we focused on measuring the spindle density to investigate cumulative effects of prefrontal rTMS sessions on the sleep spindle activity. Fourteen male inpatients diagnosed with medication-resistant unipolar or bipolar depression were recruited and subjected to 10 daily rTMS sessions targeting the left dorsolateral prefrontal cortex (DLPFC). All-night polysomnography (PSG) data was acquired at four time points: Adaptation, Baseline, Post-1 (follow-up after the fifth rTMS session), and Post-2 (follow-up after the tenth rTMS session). Clinical and cognitive evaluations were longitudinally performed at Baseline, Post-1, and Post-2 time points to explore associations with the spindle density changes. The PSG data from 12 of 14 patients was analyzed to identify sleep spindles across the sleep stages II–IV at four electrode sites: F3 (frontal spindle near the stimulation site), F4 (contralateral homologous frontal region), P3 (parietal spindle in the hemisphere ipsilateral to the stimulation site), and P4 (contralateral parietal region). Statistical analysis by two-way ANOVA revealed that spindle density at F3 increased at Post-1 but decreased at Post-2 time points. Moreover, the local and transient increase of spindle density at F3 was associated with the previously reported SWA power increase at F3, possibly reflecting a shared mechanism of thalamocortical synchronization locally enhanced by diurnal prefrontal rTMS sessions. Clinical and cognitive correlations were not observed in this dataset. These findings suggest that diurnal rTMS sessions transiently modulate nocturnal sleep spindle activity at the stimulation site, although clinical and cognitive effects of the local changes warrant further investigation.

Kampo medicine has been practiced as traditional medicine (TM) in Japan. Kampo medicine uses Kampo formulae that are composed of multiple crude drugs to make Kampo formulae. In Japan, Kampo formulae are commonly used instead of or combined with Western medicines. If drug therapy that follows the guidelines for neuropathic pain does not work or cannot be taken due to side effects, various Kampo formulae are considered as the next line of treatment. Since Kampo formulae are composed of two or more kinds of natural crude drugs, and their extracts contain many ingredients with pharmacological effects, one Kampo formula usually has multiple effects. Therefore, when selecting a formula, we consider symptoms other than pain. This review outlines the Kampo formulae that are frequently used for pain treatment and their crude drugs and the basic usage of each component. In recent years, Yokukansan (YKS) has become one of the most used Kampo formulae for pain treatment with an increasing body of baseline research available. We outline the known and possible mechanisms by which YKS exerts its pharmacologic benefits as an example of Kampo formulae’s potency and holistic healing properties.

Boiogito (BO), a Japanese traditional herbal medicine, has been reported to prevent knee osteoarthritis (KOA) development in in vivo studies. In the early stage of KOA, osteoclasts proliferate in the subchondral bone. This study aimed to investigate the preventive effect of BO on osteoclast proliferation, which remains unclear, in a KOA-induced rat model. KOA was induced in 12-week-old male Wistar rats using surgical destabilization of the medial meniscus (DMM). BO was mixed with powdered chow, applying 1%, 3%, and 5% of the total feed, and administered to KOA-induced rats. The rats were divided into 6 groups: control, sham, DMM, DMM + BO 1%, DMM + BO 3%, and DMM + BO 5%. Rotarod tests were performed each week to assess the locomotor function, and the right knees were harvested 28 days after surgery for histological analysis. Oral administration of BO significantly inhibited the decrease in the latency to fall off in the rotarod test, which was aggravated in the DMM group. Furthermore, KOA development was significantly prevented in the BO-administrated groups as assessed by the Osteoarthritis Research Society International score. The number of multinucleated activated osteoclasts in the subchondral bone was decreased in the BO-treated groups, which was increased in the DMM group. Therefore, oral administration of BO may reduce articular cartilage degeneration, osteoclast differentiation and proliferation in the KOA patients.

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.

Objective: To investigate the effect of ethyl acetate extract from Celastrus orbiculatus (COE) on gastric cancer cell apoptosis and reveal its underlying molecular mechanism. In addition, it was aimed to stablish a theoretical basis for the clinical application of Celastrus orbiculatus in the gastric cancer treatment.Material and Methods: Western blot and RT-qPCR were used to detect mRNA and protein expression of PHB in gastric cancer and adjacent tissues. MTT method was used to detect the COE effect on the proliferation of AGS cells and to determine the 50% inhibitory concentration COE on these cells. COE effect on AGS apoptosis was evaluated by flow cytometry. Changes in apoptosis-related proteins expression in AGS cells were detected by western blot and changes in mitochondrial membrane potential were detected by JC-1 fluorescence staining. PHB expression was knocked down in AGS cells by lentiviral-mediated RNA interference. The COE antitumor effect was assessed in vivo using a subcutaneous transplantation tumor model in nude mice and in vivo fluorescence tracing technique in small animals.Results: The clinical samples analysis results showed that the PHB expression in gastric cancer samples was significantly higher than in corresponding adjacent tissues. MTT results showed that the AGS cell proliferation was significantly inhibited. RT-qPCR and western blot results showed that COE can significantly inhibit the PHB mRNA and protein expression, respectively. Flow cytometry analysis showed that COE was able to significantly promote AGS cell apoptosis. Western blot results also indicated that apoptosis-related protein expression changed significantly; BCL-2 expression significantly reduced while the Caspase-3 and Bax expression significantly increased after COE treatment. JC-1 fluorescence staining results showed that COE changed the mitochondrial membrane potential and activated the mitochondrial apoptosis pathway. Furthermore, in vivo experiments results demonstrated that the growth of subcutaneous transplanted tumor was significantly inhibited by the PHB knockdown and by the COE intragastric administration.Conclusion: COE can significantly promote apoptosis of human gastric cancer cells, which can be achieved by inhibiting PHB expression, thus altering the structure and function of mitochondria and activating the mitochondria apoptosis pathway. The antitumor effect of COE has also been proved in vivo.

The transmembrane 16 (TMEM16) family contains 10 subtypes, and the function of each protein is different. TMEM16A is a calcium-activated chloride channel involved in physiological and pathological situations. Liquiritigenin is an aglycone derived from Glycyrrhiza glabra, and it is generated via the metabolism of enterobacterial flora. It has been known that liquiritigenin reduces pain sensation involving TMEM16A activation in primary sensory neurons. In addition, other pharmacological effects of liquiritigenin in physiological functions involving TMEM16A have been reported. However, the relationship between TMEM16A and liquiritigenin is still unknown. Therefore, we hypothesized that TMEM16A is inhibited by liquiritigenin. To confirm this hypothesis, we investigated the effect of liquiritigenin on TMEM16A currents evoked by intracellular free calcium in HEK293T cells transfected with TMEM16A. In this study, we found that liquiritigenin inhibited the mouse and human TMEM16A currents. To further confirm its selectivity, we also investigated its pharmacological effects on other ion channels, including transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), which are non-selective cation channels involved in pain sensation. However, liquiritigenin did not inhibit the currents of TRPV1 and TRPA1 induced by capsaicin and allyl isothiocyanate, respectively. Therefore, our findings indicate that selective TMEM16A inhibition could be one molecular mechanism that explains liquiritigenin-induced pain reduction. Additionally, we also investigated the inhibitory effects of estrogens on TMEM16A because liquiritigenin reportedly binds to the estrogen receptor. In this study, a pregnancy-dependent estrogen, estriol, significantly inhibited TMEM16A. However, the efficacy was weak. Although there is a possibility that TMEM16A activity could be suppressed during pregnancy, the physiological significance seems to be small. Thus, the inhibitory effect of estrogen might not be significant under physiological conditions. Furthermore, we investigated the effect of dihydrodaidzein, which is an analog of liquiritigenin that has a hydroxyphenyl at different carbon atom of pyranose. Dihydrodaidzein also inhibited mouse and human TMEM16A. However, the inhibitory effects were weaker than those of liquiritigenin. This suggests that the efficacy of TMEM16A antagonists depends on the hydroxyl group positions. Our finding of liquiritigenin-dependent TMEM16A inhibition could connect the current fragmented knowledge of the physiological and pathological mechanisms involving TMEM16A and liquiritigenin.

Background and Objectives: The Japanese herbal medicine Yokukansan (YKS) has analgesic properties and is used for various pain disorders. The purpose of the present study was to investigate the effects of YKS in Hunner-type interstitial cystitis (HIC) using an experimental rat model of HIC and to explore its antioxidant activity and role as the underlying mechanism of action. Materials and Methods: The antioxidant capacity of YKS was evaluated by determining its hydroxyl radical (·OH) scavenging capacity using electron spin resonance (ESR). Next, the effects of YKS administration were explored using a toll-like receptor-7 agonist-induced rat model of HIC. The von Frey test was performed to assess bladder pain. Three days after HIC induction, the bladder was removed, and the expression of oxidative stress parameters in the bladder wall was investigated (reactive oxygen metabolites (ROMs), ·OH, and 8-hydroxy-2’-deoxyguanosine (8-OhdG)). Results: YKS had a ·OH scavenging capacity according to the ESR study. In the von Frey test, a significant decrease in the withdrawal threshold was observed in the HIC group compared with the control group; however, the decrease was ameliorated by the administration of YKS. Oxidative stress parameters showed increasing tendencies (ROMs test and 8-OHdG) or a significant increase (·OH) in the HIC group compared with the control group; however, the increase was significantly suppressed by the administration of YKS. Conclusions: These findings suggest that YKS is effective against HIC and that its antioxidant activity is involved in the mechanism of action.

Background: Yokukansan (YKS), a traditional herbal (Kampo) medicine consisting of seven herbs, is effective in the treatment of pain disorders, such as headache, postherpetic neuralgia, fibromyalgia, and trigeminal neuralgia, and we have previously shown it to be effective against morphine analgesic tolerance in rats. It has been reported that orexin receptor antagonists prevent the development of morphine tolerance and that YKS inhibits the secretion of orexin A in the hypothalamus. This study examined whether the inhibition of the secretion of orexin A by YKS is one mechanism underlying its effect against morphine analgesic tolerance. Methods: Male Wistar rats were administered a subcutaneous injection of morphine hydrochloride (10 mg/kg/day) for 5 days. One group was preadministered YKS, starting 3 days before the morphine. The withdrawal latency following thermal stimulation was measured daily using a hot plate test. On day 5, the levels of orexin A in the plasma and the midbrain were measured, and the appearance of activated astrocytes in the midbrain was examined by immunofluorescence staining. Results: The preadministration of YKS prevented the development of morphine tolerance. The repeated administration of morphine significantly increased the plasma and midbrain levels of orexin A and the activation of astrocytes. These increases were significantly inhibited by the preadministration of YKS. Conclusion: These results suggest that the preadministration of YKS attenuated the development of antinociceptive morphine tolerance and that the inhibition of orexin A secretion may be one mechanism underlying this phenomenon. Keywords: Astrocyte, Kampo medicine, Morphine tolerance, Orexin A, Yokukansan

In recent years, various new drugs such as molecularly targeted drugs and immune checkpoint inhibitors have been developed. Liquid biopsy is becoming increasingly important as a guide for selecting these new drugs and determining their efficacy. In urological cancers, given the lack of serum markers for kidney cancer or urothelial cancers, the development of liquid biopsy is strongly desired. Liquid biopsy is less invasive than conventional tissue biopsy, enabling frequent biopsies, and is therefore considered effective for monitoring of the treatment course. Liquid biopsy is largely divided into three types: circulating tumor cells (CTCs), cell-free DNA, and exosomes, each of which has its own set of advantages and disadvantages with regard to the identification method and utility. In the present article, we focus on CTCs and discuss issues in their identification method as well as recent findings.

Boiogito (BO), a Japanese traditional herbal medicine, has been proven to be clinically effective against knee osteoarthritis (KOA)-associated pain. However, the therapeutic mechanism of BO remains unclear. Thus, we investigated the analgesic mechanism of BO using a rat KOA model. KOA was induced by destabilization of the medial meniscus (DMM). Rats were allocated into the following four groups: control, sham, DMM, and DMM + BO groups. Rotarod test was performed to evaluate the pain-related locomotive dysfunction. Expression of phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) in the spinal dorsal horn was examined using immunofluorescence staining and Western blotting on days 1 and 28 after DMM surgery. A mitogen-activated protein kinase inhibitor, U0126, was intrathecally injected and rotarod test and Western blotting were performed. The rotarod test revealed hampered locomotive function in the DMM group, which was significantly improved upon BO administration. The number of pERK1/2-positive cells was increased in the DMM group, whereas it was significantly decreased in the DMM + BO group. U0126 significantly inhibited ERK1/2 phosphorylation and increased walking time in the rotarod test, suggesting that the DMM-related pain was associated with ERK1/2 phosphorylation in the spinal dorsal horn. In conclusion, BO administration improved the pain-related locomotive dysfunction by suppressing ERK1/2 phosphorylation.

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.

Background: Angiogenesis is well known to be an important event in the tissue remodeling observed in allergic diseases. Although there is much evidence that quercetin, one of the most abundant dietary flavonoids, exerts anti-allergic effects in both human and experimental animal models of allergic diseases, the action of quercetin on angiogenesis has not been defined. Therefore, in this study, we first examined the action of quercetin on the secretion of angiogenic factors from murine mast cells in vitro. We also examined the action of quercetin on angiogenic factor secretion in the murine allergic rhinitis model in vivo. Methods: Mast cells (1 × 105 cells/mL) sensitized with ovalbumin (OVA)-specific murine IgE were stimulated with 10.0 ng/mL OVA in the presence or the absence of quercetin for 24 h. The concentrations of angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-α, IL-6 and IL-8 in the supernatants were examined by ELISA. BALB/c male mice immunized with OVA were challenged intranasally with OVA every other day, starting seven days after the final immunization. These mice were then orally administered quercetin once a day for five days, starting seven days after the final immunization. Clinical symptoms were assessed by counting the number of sneezes and nasal rubbing behaviors during the 10 min period just after OVA nasal provocation. The angiogenic factor concentrations in the nasal lavage fluids obtained 6 h after nasal antigenic provocation were examined by ELISA. Results: Quercetin significantly inhibited the production of angiogenetic factors induced by IgE-dependent mechanisms at 5.0 µM or more. Oral administration of 25.0 mg/kg quercetin into the mice also suppressed the appearance of angiogenetic factors in nasal lavage fluids, along with the attenuation of nasal symptoms. Conclusions: These results strongly suggest that the inhibitory action of quercetin on angiogenic factor secretion may be implicated in the therapeutic action of quercetin on allergic diseases, especially allergic rhinitis.

Background: Animal models have shown that glial cells (microglia and astrocytes) in the spinal cord undergo activation following peripheral injury associated with chronic pain, suggesting the involvement of these cells in pain diseases. We have previously reported that Yokukansan (YKS), a Japanese traditional herbal (Kampo) medicine, is effective against chronic pain through the suppression of spinal glial cell activation. Morphine is a widely-used opioid analgesic for relieving severe pain, but its repeated administration leads to the development of antinociceptive tolerance. The development of morphine tolerance is also reported to be caused by spinal glial cells activation. In the present study, we investigated the inhibitory effects of YKS on the development of morphine tolerance and the activation of the spinal microglia and astrocytes using a rat model. Methods: Male Wistar rats received a subcutaneous injection of morphine hydrochloride (10 mg/kg/d) for 7 days, and the withdrawal latency to thermal stimulation was measured daily using a hot plate test. Thereafter, the appearance of activated microglia and astrocyte in the spinal cord (L5) was examined by immunofluorescence staining. Ionized calcium binding adapter molecule-1 (Iba-1) staining was used to label microglia and glial fibrillary acidic protein (GFAP) staining was performed to label astrocytes. YKS was administered mixed with powdered rodent chow at a concentration of 3%. Results: The preadministration of YKS (started 3 d before the morphine injection) prevented the development of morphine tolerance. The repeated administration of morphine increased Iba-1 and GFAP immune reactivities in the spinal cord; however, these activations were inhibited by the preadministration of YKS. Conclusion: These results suggest that the preadministration of YKS attenuates the development of antinociceptive morphine tolerance, and the suppression of spinal glial cell activation may be one mechanism underlying this phenomenon.

Background: Considering the anti-inflammatory properties of the Japanese traditional Kampo medicine Boiogito (BO), we aimed to investigate the therapeutic effect of BO to prevent the development of knee osteoarthritis (KOA) in rats with surgically induced KOA. Methods: Destabilization of the medial meniscus (DMM) was performed to induce osteoarthritis in the right knees of 12-week-old Wistar rats under general anesthesia. The rats were orally administered 3% BO in standard powder chow for 4 weeks after surgery (controls: n = 6; sham group: n = 6; DMM group: n = 5; DMM + BO group: n = 5). During this period, the rotarod test was performed to monitor locomotive function. After 4 weeks, histological assessment was performed on the right knee. Results: Oral administration of BO improved locomotive function in the rotarod test. Walking time on postoperative days 1, 14, or later was significantly longer in the DMM + BO group than in the DMM group. Histologically, the DMM group showed significant progression of KOA, which, in the DMM + BO group, was strongly suppressed, as assessed by the Osteoarthritis Research Society International score. Conclusions: Our results showed that oral administration of BO had a clinically preventive effect on early stage posttraumatic KOA.

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.

Gastric cancer is the fifth most common tumor and has the third-highest mortality rate among various malignant tumors, and the survival rate of patients is low. Celastrus orbiculatus extract has been shown to inhibit the activity of a variety of tumors. This study explored the inhibitory effect of the oleanane-type triterpenoid acid 28-hydroxy-3-oxoolean-12-en-29-oic acid molecule from Celastrus orbiculatus extract on gastric cancer cell invasion and metastasis and determined its mechanism. 28-Hydroxy-3-oxoolean-12-en-29-oic acid was first diluted to various concentrations and then used to treat SGC-7901 and BGC-823 cells. Cell proliferation was assessed by an MTT (thiazole blue) assay. Transwell and wound healing assays were used to assess cell invasion and migration. High-content imaging technology was used to further observe the effects of the drug on cell invasion and migration. Western blotting was used to assess the effects on the expression of matrix metalloproteinases (MMPs) and the effects on epithelial−mesenchymal transition (EMT)-related proteins and phosphorylation-related proteins. We found that 28-Hydroxy-3-oxoolean-12-en-29-oic acid inhibited the migration and invasion of SGC-7901 and BGC-823 gastric cancer cells in a dose-dependent manner. Consequently, 28-hydroxy-3-oxoolean-12-en-29-oic acid decreased the expression of EMT-related proteins and MMPs in gastric cancer cells and reduced protein phosphorylation, inhibiting the migration and invasion of gastric cancer cells.

purpose: Liquid biopsy is becoming increasingly important as a guide for selecting new drugs and determining their efficacy. In urological cancer, serum markers for renal cell and urothelial cancers has made the development of liquid biopsy for these cancers strongly desirable. Liquid biopsy is less invasive than conventional tissue biopsy is, enabling frequent biopsies and, therefore, is considered effective for monitoring the treatment course. Circulating tumor cells (CTCs) are a representative liquid biopsy specimen. In the present study, we focused on developing our novel technology for capturing renal cell cancer (RCC)-CTCs using an anti-G250 antibody combined with new devices. Basic experiments of our technology showed that it was possible to detect RCC-CTC with a fairly high accuracy of about 95%. Also, RCC-CTC was identified in the peripheral blood of actual RCC patients. Additionally, during the treatment course of the RCC patient, change in the number of RCC-CTC was confirmed in one case. We believe that the technology we developed will be useful for determining the treatment efficacy and drug selection for the treatment of renal cell cancer (RCC). In order to solve issues such as thresholds setting of this technology, large-scale clinical trials are expected.

Stomatitis is occasionally multiple, recurrent, and refractory. Currently, mucositis induced by chemotherapy and radiation therapy in patients with cancer has become a significant clinical problem. Effective treatments have not been established and the treatment of numerous cases remains a challenge for physicians. Traditional Japanese herbal medicines termed Kampo formulae (i.e., Hangeshashinto, Orengedokuto, Inchinkoto, Orento, Byakkokaninjinto, Juzentaihoto, Hochuekkito, and Shosaikoto) are used for treating various types of stomatitis and mucositis. Its use has been based on the Kampo medical theories—empirical rules established over thousands of years. However, recently, clinical and basic research studies investigating these formulae have been conducted to obtain scientific evidence. Clinical studies investigating efficacies of Shosaikoto and Orento for the treatment of cryptogenic stomatitis and acute aphthous stomatitis and those investigating the effects of Hangeshashinto, Orengedokuto, and Juzentaihoto on chemotherapy- or radiotherapy-induced mucositis have been conducted. The Kampo formulae comprise several crude drugs, whose mechanisms of action are gradually being clarified. Most of these drugs that are used for the treatment of stomatitis possess anti-inflammatory, analgesic, and antioxidative properties. In this review, we introduce the clinical applications and summarize the available evidence on the Kampo formulae for the treatment of stomatitis and oral mucositis.

Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasive activity of celastrol on LPS-stimulated human RA-FLSs, and to elucidate the mechanism involved. We investigated the effect of celastrol on LPS-induced FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that celastrol suppressed LPS-stimulated FLS migration and invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppressing the binding activity of NF-κB in the MMP-9 promoter, and suppressed the TLR4/MyD88/NF-κB pathway. Administration of celastrol (0.5 mg/kg and 1 mg/kg, intraperitoneally) daily for 3 weeks in a collagen-induced arthritis rat model markedly alleviated the clinical signs, synovial hyperplasia and inflammatory cell infiltration of joints. In conclusion, celastrol might inhibit FLS migration and invasion induced by LPS by suppressing TLR4/NF-κB-mediated MMP-9 expression, providing a theoretical foundation for the clinical treatment of RA with celastrol.