Cytological characteristics of histological types of lung cancer by cytomorphometric and flow cytometric analyses using liquid-based cytology materials.

BACKGROUND: Distinguishing the histological types of lung cancer is essential for determining treatment strategies in clinical practice. In this study, cytomorphological characteristics and proliferative activities were compared among histological types of lung cancer by cytomorphometric and flow cytometric analyses using liquid-based cytology (LBC) samples. METHODS: Scraped LBC samples from 73 surgically resected specimens were collected between August 2018 and November 2019. Papanicolaou-stained and paired Ki-67-stained slides were used for cytomorphometric analyses. Another sample for each case was analyzed using a flow cytometric system (LC-1000). The cell proliferation index (CPIx) was calculated to evaluate proliferative activity. RESULTS: In total, 73 cases, including cases of adenocarcinoma (n = 53), squamous cell carcinoma (n = 14), small cell carcinoma (n = 1), large cell neuroendocrine carcinoma (NEC; n = 3), and pleomorphic carcinoma (n = 2) were evaluated. Small cell carcinoma and large cell NEC were categorized into a single group, NEC. The adenocarcinoma group tended to have a larger nuclear area and longer perimeter than other histological types. The NEC group had a considerably higher Ki-67 labeling index and significantly higher CPIx than other histological types (p = .030). A significant positive correlation was observed between the Ki-67 labeling index and CPIx for all cases (r = 0.362, p = .002). CONCLUSION: The Ki-67 labeling index and flow cytometric analyses focus on proliferative activity for the distinction of histological types of lung cancer, thereby guiding clinical decision-making.

Impact of Gut Microbiota on the Pharmacokinetics of Glycyrrhizic Acid in Yokukansan, a Kampo Medicine.

Individual differences in gut microbiota can affect the pharmacokinetics of drugs. Yokukansan is a traditional Japanese kampo medicine used to treat peripheral symptoms of dementia and delirium. A study examining the pharmacokinetics of the components of yokukansan reported large individual differences in the pharmacokinetics of glycyrrhizic acid (GL). It is known that GL is metabolized by intestinal bacteria to glycyrrhetinic acid (GA), which is absorbed in the gastrointestinal tract. Thus, the gut microbiota may affect GL pharmacokinetics. We aimed to clarify the relationship between the gut microbiota composition and pharmacokinetics of GL in yokukansan. Mice were orally administered yokukansan, following the administration of various antibiotics, and the plasma concentration of GA and composition of gut microbiota were measured. The GA plasma concentration was low in mice treated with amoxicillin and vancomycin. The composition of gut microbiota revealed a different pattern from that of the control group. Mice with low plasma levels of GA had lower levels of the phylum Bacteroides and Firmicutes. Additionally, bacteria, such as those belonging to the genera Parabaceroides, Bacteroides, Ruminococcus and an unknown genus in families Lachnospiraceae and Ruminococcaceae, exerted positive correlations between the gene copies and plasma GA levels. These bacteria may contribute to the absorption of GA in the gastrointestinal tract, and multiple bacteria may be involved in GL pharmacokinetics. The pharmacokinetics of GL may be predicted by evaluating the composition of gut bacteria, rather than by evaluating the amount of a single bacterium.

Maoto, a traditional Japanese medicine, controls acute systemic inflammation induced by polyI:C administration through noradrenergic function.

Maoto, a traditional Japanese medicine (Kampo), is widely used to treat upper respiratory tract infections, including influenza virus infection. Although maoto is known to inhibit pro-inflammatory responses in a rodent model of acute inflammation, its underlying mechanism remains to be determined. In this study, we investigated the involvement of immune responses and noradrenergic function in the inhibitory action of maoto. In a mouse model of polyI:C-induced acute inflammation, maoto was administered orally in conjunction with intraperitoneal injection of PolyI:C (6 mg/kg), and blood was collected after 2 h for measurement of plasma cytokines by ELISA. Maoto significantly decreased PolyI:C-induced TNF-α levels and increased IL-10 production. Neither pretreatment with IL-10 neutralizing antibodies nor T-cell deficiency using nude mice modified the inhibitory effect of maoto, indicating that the anti-inflammatory effects of maoto are independent of IL-10 and T cells. Furthermore, the inhibitory effects of maoto on PolyI:C-induced TNF-α production were not observed in ex vivo splenocytes, suggesting that maoto does not act directly on inflammatory cells. Lastly, pretreatment with a ß-adrenergic receptor antagonist partially cancelled the anti-inflammatory effects of maoto. Collectively, these results suggest that maoto mediates its anti-inflammatory effects via ß-adrenergic receptors in vivo.

Predicting Inchinkoto efficacy, in patients with obstructive jaundice associated with malignant tumors, through pharmacomicrobiomics.

Inchinkoto (ICKT) is a popular choleretic and hepatoprotective herbal medicine that is widely used in Japan. Geniposide, a major ingredient of ICKT, is metabolized to genipin by gut microbiota, which exerts a choleretic effect. This study investigates the relationship between stool genipin-producing activity and diversity of the clinical effect of ICKT in patients with malignant obstructive jaundice. Fifty-two patients with malignant obstructive jaundice who underwent external biliary drainage were included. ICKT was administered as three packets per day (7.5 g/day) for three days and 2.5 g on the morning of the fourth day. Stool samples were collected before ICKT administration and bile flow was monitored on a daily basis. The microbiome, genipin-producing activity, and organic acids in stools were analyzed. The Shannon-Wiener (SW) index was calculated to evaluate gut microbiome diversity. The stool genipin-producing activity showed a significant positive correlation with the SW index. Stool genipin-producing activity positively correlated with the order Clostridia (obligate anaerobes), but negatively correlated with the order Lactobacillales (facultative anaerobes). Moreover, stool genipin-producing activity was positively correlated to the concentration valeric acid, but negatively correlated to the concentration of lactic acid and succinic acid. The change of bile flow at 2 and 3 days after ICKT administration showed significant positive correlation with genipin-producing activity (correlation coefficient, 0.40 and 0.29, respectively, P < 0.05). An analysis of stool profile, including stool genipin-producing activity, may predict the efficacy of ICKT. Modification of the microbiome may be a target to enhance the therapeutic effect of ICKT.

In Vivo Pharmacokinetic Analysis Utilizing Non-Targeted and Targeted Mass Spectrometry and In Vitro Assay against Transient Receptor Potential Channels of Maobushisaishinto and Its Constituent Asiasari Radix.

The Japanese traditional medicine maobushisaishinto (MBST) has been prescribed for treating upper respiratory tract infections, such as a common cold. However, its mode of action is poorly understood, especially concerning the MBST constituent Asiasari Radix (AR). In this study, we focused on AR, with an objective of clarifying its bioavailable active ingredients and role within MBST by performing pharmacokinetic and pharmacological studies. Firstly, we performed qualitative non-targeted analysis utilizing high-resolution mass spectrometry to explore the bioavailable ingredients of AR as well as quantitative targeted analysis to reveal plasma concentrations following oral administration of MBST in rats. Secondly, we performed in vitro pharmacological study of bioavailable AR ingredients in addition to other ingredients of MBST to confirm any agonistic activities against transient receptor potential (TRP) channels. As a result, methyl kakuol and other compounds derived from AR were detected in the rat plasma and showed agonistic activity against TRPA1. This study suggests that methyl kakuol as well as other compounds have the potential to be an active ingredient in AR and thus presumably would contribute in part to the effects exerted by MBST.

Temperature-Dependent Formation of N-Nitrosodimethylamine during the Storage of Ranitidine Reagent Powders and Tablets.

The purpose of this study was to elucidate the effect of high-temperature storage on the stability of ranitidine, specifically with respect to the potential formation of N-nitrosodimethylamine (NDMA), which is classified as a probable human carcinogen. Commercially available ranitidine reagent powders and formulations were stored under various conditions, and subjected to LC-MS/MS analysis. When ranitidine tablets from two different brands (designated as tablet A and tablet B) were stored under accelerated condition (40 °C with 75% relative humidity), following the drug stability guidelines issued by the International Conference on Harmonisation (ICH-Q1A), for up to 8 weeks, the amount of NDMA in them substantially increased from 0.19 to 116 ppm and from 2.89 to 18 ppm, respectively. The formation of NDMA that exceeded the acceptable daily intake limit (0.32 ppm) at the temperature used under accelerated storage conditions clearly highlights the risk of NDMA formation in ranitidine formulations when extrapolated to storage under ambient conditions. A forced-degradation study under the stress condition (60 °C for 1 week) strongly suggested that environmental factors such as moisture and oxygen are involved in the formation of NDMA in ranitidine formulations. Storage of ranitidine tablets and reagent powders at the high temperatures also increased the amount of nitrite, which is considered one of the factors influencing NDMA formation. These data indicate the necessity of controlling/monitoring stability-related factors, in addition to controlling impurities during the manufacturing process, in order to mitigate nitrosamine-related health risks of certain pharmaceuticals.

Cold Flow Evaluation in Transdermal Drug Delivery Systems by Measuring the Width of the Oozed Adhesive.

The objective of this study was to develop a simpler and more practical quantitative evaluation method of cold flow (CF) in transdermal drug delivery systems (TDDSs). CF was forcibly induced by loading a weight on a punched-out sample (bisoprolol and tulobuterol tapes). When the extent of CF was analyzed using the area of oozed adhesive as following a previously reported method, the CF profiles were looked different between the samples 12 mm in diameter subjected to a 0.5-kg weight and samples 24 mm in diameter subjected to a 2.0-kg weight despite an equal load per unit area (4.42 g/mm2). The width of oozed adhesive around the original sample was suggested to be an index that properly describes the relationship between the load per unit area and the extent of CF. Further, it was clarified that the average CF width over the entire circumference of the sample was the same whether the samples were round or square as long as the sample area and load were the same. We also observed a linear relationship between the CF width and the aspect ratio of oval and rectangular samples. These results indicated that the CF properties of typical TDDS products lacking CF-proof processing at the edges could be determined by testing samples cut from the product rather than the whole TDDS patch. The proposed width measuring method was simple and useful for optimizing the composition of the adhesive and for testing the quality of the product.

Remitting seronegative symmetrical synovitis with pitting oedema after surgical remission of Cushing's syndrome.

A 64-year-old woman with refractory cellulitis in the lower legs was referred for inadequate glycaemic control. Physical examination revealed cushingoid features including central obesity. CT of the abdomen revealed a right adrenal mass that was positive on 131I-adosterol imaging. Findings on endocrine evaluation confirmed a diagnosis of Cushing's syndrome, which was cured with a right adrenalectomy. Two months after surgery, the patient complained of pain and marked swelling of the hands during hydrocortisone replacement therapy (20 mg per day) given for postoperative adrenal insufficiency. Laboratory examination was unremarkable. However, contrast-enhanced T2-weighted MRI of the hands revealed enhanced signals surrounding the flexor tendons, leading to a diagnosis of remitting seronegative symmetrical synovitis with pitting oedema. Prednisolone (15 mg per day) was then initiated, and the symptoms disappeared within a few days. This case illustrates the possibility that successful treatment of Cushing's syndrome may trigger emergence of a glucocorticoid-responsive disease.

[Comparison of Adhesive Properties of Transdermal Patches Distributing in Japan-Tack and Peel Strength].

Forty-four brands of transdermal patches for twelve kinds of active pharmaceutical ingredients (APIs) are available in Japan as of April 30, 2018. Although approximately one-third of the corresponding pharmaceutical interview forms lack information on how to evaluate the adhesive properties of the patches, the peel test, probe tack test, or inclined ball tack test have generally been adopted. This means that it might be difficult to simply compare the adhesive properties among the patches because the testing methods are not unified in some cases. In this study, measurements of the adhesive properties of 38 transdermal patches of ten different APIs were performed using several unified testing methods (180° peel test, 90° peel test, self-adhesion test, and probe tack test) under unified experimental conditions. The adhesive properties were found to be quite different among the patches, even for the same API, dose, and size. For example, the ratios of the maximum to minimum measured values of tack and 180° peel strength for tulobuterol patches were 5 and 29, respectively. In the case of generic products for which the bioequivalence to a brand-name product is assured, the variation in adhesive properties can extend the range of choices for patients, which is advantageous. Providing information to medical experts on adhesive properties through, for example, pharmaceutical interview forms and package inserts, is considered to be useful for helping patients to make better choices.

Yokukansan, a Traditional Japanese Medicine, Enhances the Glutamate Transporter GLT-1 Function in Cultured Rat Cortical Astrocytes.

Astrocytes carry two glutamate transporters-GLAST and GLT-1-the latter of which is responsible for >90% of glutamate uptake activity in the brain; however, under culture conditions, the GLT-1 expression in astrocytes is exceedingly low, as is the glutamate uptake activity mediated by GLT-1. This study aimed to elucidate the effects of yokukansan (YKS) in relation to the GLT-1-mediated regulation of extracellular glutamate concentrations. Thus, we treated cultured astrocytes with tumor necrosis factor-α (TNF-α) and dibutyryl-cAMP (dBcAMP) (hereinafter, referred to as "TA") to increase GLT-1 expression and then functionally examined how YKS would affect glutamate uptake ability derived from GLT-1. Contrary to expectations, although the TA treatments did not affect the uptake activity, YKS significantly augmented it. Conversely, GLAST-derived glutamate uptake was significantly reduced by TA treatments but was unaffected by YKS. Subsequently, we analyzed the GLT-1 protein and mRNA levels and found that TA treatments had significantly increased them, which were then further augmented by YKS. These findings suggest that YKS enhances GLT-1-derived glutamate transport functions in TA-treated cultured astrocytes and that this process entails increased GLT-1 protein and mRNA levels. This type of mechanism may contribute to the YKS-mediated regulation of extracellular glutamate concentrations.

Time-dependent change in relapse sites of renal cell carcinoma after curative surgery.

We investigated time-dependent changes in the relapse features of renal cell carcinoma (RCC) after curative surgery. Between 1985 and 2015, 1398 patients with RCC (1226 clear cell RCC, 89 papillary RCC, and 53 chromophobe RCC) underwent curative surgery at Yokohama City University Hospital and its affiliated hospitals. We retrospectively reviewed the clinicopathologic factors of patients with relapse after surgery. Median follow-up was 56.3 months. Recurrence occurred in 245 patients (217 clear cell RCC, 12 papillary RCC, and 3 chromophobe RCC). Papillary RCC and chromophobe RCC had no recurrence beyond 5 years after surgery, but 20 cases of clear cell carcinoma had recurrence beyond 10 years after surgery. The typical recurrence sites of clear cell RCC were lung (46.6%), bone (17.9%), liver (7.6%), and lymph nodes (6.5%). The proportion of recurrences at these typical sites was 83.9% for recurrences within 5 years, 76.3% between 5 and 10 years, and 40.0% beyond 10 years. In contrast, the proportion of retroperitoneal organ recurrence, including contralateral kidney, pancreas, and adrenal glands, increased with increasing time after surgery. Interestingly, the hazard ratio of typical site relapse decreased whereas that of retroperitoneal organ relapse increased in a time-dependent manner. In summary, clear cell RCC showed potential to relapse beyond 10 years after surgery. Recurrence at typical sites decreased whereas retroperitoneal organ recurrence increased in a time-dependent manner. Clinicians should check for recurrence at various sites beyond 10 years, especially in clear cell RCC.

Peptide YY Causes Apathy-Like Behavior via the Dopamine D2 Receptor in Repeated Water-Immersed Mice.

Apathy is observed across several neurological and psychiatric conditions; however, its pathogenesis remains unclear. We clarified the involvement of brain-gut signaling in the disruption of goal-directed behavior. Male C57BL/6J mice were exposed to water immersion (WI) stress for 3 days. Food intake and nesting behavior were measured as indexes of motivation. Repeated WI caused decrease in food intake and nesting behavior. Plasma levels of peptide YY (PYY), IL-6, and ratio of dopamine metabolites in the striatum were significantly elevated after WI. PYY and IL-6 administration significantly decreased nesting behavior. The reductions in feeding and nesting behavior were blocked by PYY receptor (Y2R) antagonist or dopamine agonist. The ameliorative effect of the Y2R antagonist was diminished by the dopamine D2 receptor (D2R) antagonist. The reduction in goal-directed behavior is associated with dysfunction of D2R signaling via increased peripheral PYY, suggesting that PYY antagonism is a novel candidate for decline of motivation in several depressive diseases.

Deconstructing the traditional Japanese medicine "Kampo": compounds, metabolites and pharmacological profile of maoto, a remedy for flu-like symptoms.

Pharmacological activities of the traditional Japanese herbal medicine (Kampo) are putatively mediated by complex interactions between multiple herbal compounds and host factors, which are difficult to characterize via the reductive approach of purifying major bioactive compounds and elucidating their mechanisms by conventional pharmacology. Here, we performed comprehensive compound, pharmacological and metabolomic analyses of maoto, a pharmaceutical-grade Kampo prescribed for flu-like symptoms, in normal and polyI:C-injected rats, the latter suffering from acute inflammation via Toll-like receptor 3 activation. In total, 352 chemical composition-determined compounds (CCDs) were detected in maoto extract by mass spectrometric analysis. After maoto treatment, 113 CCDs were newly detected in rat plasma. Of these CCDs, 19 were present in maoto extract, while 94 were presumed to be metabolites generated from maoto compounds or endogenous substances such as phospholipids. At the phenotypic level, maoto ameliorated the polyI:C-induced decrease in locomotor activity and body weight; however, body weight was not affected by individual maoto components in isolation. In accordance with symptom relief, maoto suppressed TNF-α and IL-1ß, increased IL-10, and altered endogenous metabolites related to sympathetic activation and energy expenditure. Furthermore, maoto decreased inflammatory prostaglandins and leukotrienes, and increased anti-inflammatory eicosapentaenoic acid and hydroxyl-eicosapentaenoic acids, suggesting that it has differential effects on eicosanoid metabolic pathways involving cyclooxygenases, lipoxygenases and cytochrome P450s. Collectively, these data indicate that extensive profiling of compounds, metabolites and pharmacological phenotypes is essential for elucidating the mechanisms of herbal medicines, whose vast array of constituents induce a wide range of changes in xenobiotic and endogenous metabolism.

Protective effects of glycycoumarin and procyanidin B1, active components of traditional Japanese medicine yokukansan, on amyloid ß oligomer-induced neuronal death.

ETHNOPHARMACOLOGICAL RELEVANCE: Yokukansan, a traditional Japanese (Kampo) medicine, is composed of seven medicinal herbs, and has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Yokukansan and its constituent herbs, Glycyrrhiza and Uncaria Hook, have recently been shown to have protective effects against amyloid ß (Aß) oligomer-induced apoptosis by suppressing the activation of caspase-3 in primary cultured neurons. The aim of the present study was to identify the effective components of Glycyrrhiza and Uncaria Hook against Aß oligomer-induced neurotoxicity. We also attempted to clarify the mechanisms by which yokukansan and these herbs, as well as their components, suppressed the activation of caspase-3 in Aß oligomer-treated neurons. MATERIALS AND METHODS: Rat primary cultured cortical neurons were treated with Aß oligomer (3 µM). The protective effects of 16 components derived from Glycyrrhiza or Uncaria Hook against Aß oligomer-induced neurotoxicity were determined using the MTT reduction assay 48 h after the treatment. The suppressive effects of the test substances, i.e., yokukansan, Glycyrrhiza, Uncaria Hook, and screened components, on the Aß oligomer-induced activation of caspase-3(/7) were evaluated using the caspase-Glo assay 48 h after the Aß oligomer treatment. The suppressive effects of the test substances on the activation of caspase-8 and -9, both of which are located upstream of caspase-3, were also examined 24h after the Aß oligomer treatment. RESULTS: Two of the 16 components tested, glycycoumarin derived from Glycyrrhiza and procyanidin B1 derived from Uncaria Hook, significantly inhibited Aß oligomer-induced neuronal death in a dose-dependent manner. Glycyrrhiza, Uncaria Hook, and yokukansan significantly suppressed the Aß oligomer-induced activation of caspase-3 as well as caspase-8 and -9. Glycycoumarin also suppressed the activation of caspase-3, but not caspase-8 and -9. Procyanidin B1 suppressed the activation of caspase-3, -8, and -9. CONCLUSIONS: Our results demonstrated that glycycoumarin and procyanidin B1 had ameliorative effects on Aß oligomer-induced neurotoxicity. The neuroprotective effects of glycycoumarin are thought to be due to the attenuated activation of caspase-3, but not caspase-8 or -9. Procyanidin B1, as well as yokukansan, Glycyrrhiza, and Uncaria Hook, may attenuate the activation of caspase-3 by inhibiting that of caspase-8 and -9.

Specific binding and characteristics of geissoschizine methyl ether, an indole alkaloid of Uncaria Hook, in the rat brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Geissoschizine methyl ether (GM) is an indole alkaloid that is a component of Uncaria Hook, and has been identified as the active component responsible for the anti-aggressive effects of the Uncaria Hook-containing traditional Japanese medicine, yokukansan. Recently, GM was shown to reach the brain by crossing the blood-brain barrier in rats following the oral administration of yokukansan. This finding suggested that there may be specific binding sites for GM in the brain. Here we show evidence that tritium-labeled GM ([(3)H]GM) binds specifically to several brain areas of rats. MATERIALS AND METHODS: Male rats were used. [(3)H]GM was synthesized from a demethylated derivative of GM. Specific binding sites of [(3)H]GM on brain sections were determined by quantitative autoradiography, and maximum binding densities (Bmax) and dissociation constants (Kd) were calculated. Several chemical compounds were used to clarify the molecules that recognize [(3)H]GM in the completion-binding assay. Emulsion microautoradiography was also performed to identify the cells that bind [(3)H]GM. RESULTS: Specific binding of [(3)H]GM was observed in the frontal cortex, including the prefrontal cortical region (e.g., prelimbic cortex (PrL)), hippocampus, caudate putamen, amygdala, central medial thalamic nucleus, dorsal raphe nucleus (DR), and cerebellum. Bmax ranged between 0.65 and 8.79pmol/mg tissue, and Kd was between 35.0 and 232.6nM. Specific binding with relatively high affinity (Kd less than 62nM) was dense in the frontal cortical region, moderate in the DR, and sparse in the cerebellum. The specific binding of [(3)H]GM in the PrL was significantly replaced by the serotonin 1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), 5-HT2A receptor antagonist ketanserin, 5-HT2B receptor agonist BW 723C86, 5-HT2C receptor agonist RO60-0175, adrenergic α2A receptor antagonist yohimbine, L-type Ca(2+) channel blocker verapamil, and µ-opioid receptor antagonist naloxone. Similar results were obtained in the frontal cortex and DR, but not in the cerebellum. Microautoradiography revealed that [(3)H]GM signals were distributed throughout the frontal cortex, which included neuron-like large cells. CONCLUSION: These results demonstrate that specific binding sites for GM exist in rat brain tissue, and suggest that the pharmacological actions of GM are mainly associated with 5-HT receptors in the frontal cortex and DR. These results provide an insight into the neuropharmacology of GM and GM-containing herbal medicines.

Specific binding and characteristics of 18ß-glycyrrhetinic acid in rat brain.

18ß-Glycyrrhetinic acid (GA) is the aglycone of glycyrrhizin that is a component of Glycyrrhiza, and has several pharmacological actions in the central nervous system. Recently, GA has been demonstrated to reach the brain by crossing the blood-brain barrier in rats after oral administration of a Glycyrrhiza-containing traditional Japanese medicine, yokukansan. These findings suggest that there are specific binding sites for GA in the brain. Here we show evidence that [3H]GA binds specifically to several brain areas by quantitative autoradiography; the density was higher in the hippocampus, moderate in the caudate putamen, nucleus accumbens, amygdala, olfactory bulb, cerebral cortex, thalamus, and mid brain, and lower in the brain stem and cerebellum. Several kinds of steroids, gap junction-blocking reagents, glutamate transporter-recognized compounds, and glutamate receptor agonists did not inhibit the [3H]GA binding. Microautoradiography showed that the [3H]GA signals in the hippocampus were distributed in small non-neuronal cells similar to astrocytes. Immunohistochemical analysis revealed that immunoreactivity of 11ß-hydroxysteroid dehydrogenase type-1 (11ß-HSD1), a defined molecule recognized by GA, was detected mainly in neurons, moderately in astrocytes, and very slightly in microglial cells, of the hippocampus. These results demonstrate that specific binding sites for GA exist in rat brain tissue, and suggest that the pharmacological actions of GA may be related to 11ß-HSD1 in astrocytes. This finding provides important information to understand the pharmacology of GA in the brain.

Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

Glycyrrhiza and Uncaria Hook contribute to protective effect of traditional Japanese medicine yokukansan against amyloid ß oligomer-induced neuronal death.

ETHNOPHARMACOLOGICAL RELEVANCE: Yokukansan, a traditional Japanese (Kampo) medicine, composed of seven medicinal herbs has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Recently, this medicine has been reported to improve the behavioral and psychological symptoms of dementia (BPSD) that often become problematic in patients with Alzheimer's disease (AD). AIM OF THE STUDY: Amyloid ß (Aß) oligomers, which are extremely toxic to neurons, are involved in neurodegeneration in AD. In animals, yokukansan has been proven to improve memory impairments and BPSD-like behavior in transgenic mice overexpressing amyloid precursor protein and mice intracerebroventricularly injected with Aß oligomers. These results suggest that yokukansan is potentially able to reduce the neurotoxicity of Aß oligomers. Therefore, the present study aimed to explore the improving effects brought by yokukansan that consists of seven herbs for Aß oligomer-induced neurotoxicity in vitro and to identify the candidate herbs in yokukansan's action. MATERIALS AND METHODS: Primary cultured rat cortical neurons were used. Neurotoxicity induced by Aß oligomers (3µM) and improving effects of yokukansan (300-1000 µg/mL) and its constituent herbs were evaluated in MTT assay, DNA fragmentation analysis, and electron microscopic analysis at 48h after treatment with Aß oligomers and drugs. Moreover, changes in expression of genes related to endoplasmic reticulum (ER) stress and in caspase-3 activity that is the enzyme closely related to apoptosis were analyzed to investigate the underlying mechanisms. RESULTS: Yokukansan ameliorated Aß oligomer-induced neuronal damage in a dose-dependent manner in the MTT assay. This drug also suppressed DNA fragmentation caused by Aß oligomers. Electron microscopic analysis suggested that yokukansan reduced karyopyknosis and the expansion of rough ER caused by Aß oligomers. However, neither Aß oligomers nor yokukansan affected the mRNA expression of any ER stress-related genes, including CHOP and GRP78. On the other hand, yokukansan dose-dependently suppressed Aß oligomer-induced activation of caspase-3. Among the seven constituents of yokukansan, Glycyrrhiza and Uncaria Hook (60-200 µg/mL) suppressed Aß oligomer-induced neuronal damage, DNA fragmentation, karyopyknosis, and caspase-3 activation to almost the same extent as yokukansan. CONCLUSIONS: The present results suggest that yokukansan possesses an ameliorative effect against Aß oligomer-induced neuronal apoptosis through the suppression of caspase-3 activation. Glycyrrhiza and Uncaria Hook may, at least in part, contribute to the neuroprotective effect of yokukansan. These mechanisms may underlie the improving effects of yokukansan on memory impairment and BPSD-like behaviors induced by Aß oligomers.

Effects of geissoschizine methyl ether, an indole alkaloid in Uncaria hook, a constituent of yokukansan, on human recombinant serotonin 7 receptor.

Effects of seven alkaloids, geissoschizine methyl ether (GM), hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine, in Uncaria hook, a constituent of the kampo medicine yokukansan, on serotonin(7) (5-HT(7)) receptor were investigated using Chinese hamster ovary (CHO) cell membranes and human embryonic kidney 293 (HEK293) cells stably expressing the human recombinant 5-HT(7) receptor. A competitive binding assay using CHO membranes showed that GM (IC(50) = 0.034 µM) more strongly inhibited the binding of the radioligand [(3)H] LSD to 5-HT(7) receptor than the other alkaloids, suggesting that GM is bound to 5-HT(7) receptor. Agonistic/antagonistic effects of GM (1-50 µM) on the receptor were evaluated by measuring intracellular cAMP levels in HEK239 cells. GM (IC(50) = 6.0 µM) inhibited 5-HT-induced cAMP production in a concentration-dependent manner, as well as the specific 5-HT(7) receptor antagonist SB-269970 (0.1-1 µM). However, GM did not induce intracellular cAMP production as 5-HT did. These results suggest that GM has an antagonistic effect on 5-HT(7) receptor.

Ameliorative effect of yokukansan on vacuous chewing movement in haloperidol-induced rat tardive dyskinesia model and involvement of glutamatergic system.

Effects of yokukansan (YKS) on vacuous chewing movement (VCM), which is an index for tardive dyskinesia, were investigated in haloperidol decanoate-treated rats. Haloperidol decanoate was injected to a thigh muscle once every four weeks for 18 weeks. The rats which exhibited VCM eight times or more in 3min were selected on the 12th week, and examined. A significant increase in VCM on the 12th week continued until the 18th week. Oral administration of YKS (0.1 and 0.5g/kg) once a day for three weeks (21 days) from the 12th week to 15th week ameliorated the haloperidol decanoate-induced increase in VCM in a dose-dependent manner. The significant ameliorative effect observed in 0.5g/kg YKS-treated rats was abolished by stopping administration for three weeks from the 15th week to the 18th week. The extracellular glutamate concentration and glutamate transporter mRNA expression in the striatum were evaluated by microdialysis and real-time reverse-transcription polymerase chain reaction assays at the 15th week. The striatal glutamate level increased in haloperidol-treated rats, and the increase was inhibited by treatment with YKS. The striatal GLT-1 mRNA level showed a tendency to decrease in the haloperidol-treated rats. The GLT-1 mRNA level after treatment with YKS (0.5g/kg) was greater than the control level. These results suggest the effect of YKS may be involved in the extracellular glutamate level and GLT-1 mRNA expression in the striatum.