Traditional Oriental medicines and health supplements have been empirically used to treat various ailments but most of them have not been evaluated objectively to prove their efficacies. We have been investigating the medical benefits of traditional Oriental medicines and health supplements as alternatives and their varied actions and mechanisms by pharmacological approaches. The study on airway inflammation has shown that even a Kampo preparation, Bakumondo-to, has anti-inflammatory, anti-allergic, immunomodulatory, secretory-modulating, and metabolic regulatory actions. All of its actions are based on the restoration of normal molecular and cellular functions through DNA transcriptional regulation. In other previous studies, we showed that a health supplement, royal jelly (RJ) has weak estrogenic activity. RJ competes with 17β-estradiol for binding to the human estrogen receptors α and β, although it is much weaker than diethylstilbestrol in binding affinity. Treatment of MCF-7 cells with RJ enhances proliferation, and concomitant treatment with tamoxifen blocked this effect. A reporter gene assay showed that RJ enhanced transcription of the luciferase gene through the estrogen-responsive element in MCF-7 cells. Furthermore, subcutaneous injection of RJ restored the expression of vascular endothelial growth factor gene in the uteri of ovariectomized rats. We suggest that the diverse pharmacological functions of RJ can be ascribed, in part, to its estrogenic effects. We hypothesize that traditional medicine, which has multiple actions, may be better than Western medicine of a single component to treat various diseases including “Mibyou” (presymptomatic disease). Our findings provide new ideas about the nature of disorders nd disease-state development that involve complicated mechanisms and will contribute to novel principles to prevent diseases and establish new treatments. Adoption of the means of translational research should provide an objective background for efficacy and stimulate broader application and usage of traditional medicines and health supplements.

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The cardiovascular diseases are closely related to circadian rhythm, which is under the control of the biological clock. Clock genes show circadian oscillation not only in the suprachiasmatic nucleus but also in peripheral tissues, suggesting the existence of the peripheral clock. We previously demonstrated that plasminogen activator inhibitor-1 (PAI-1) might be an output gene of the peripheral clock. To further elucidate the functional relevance of the peripheral clock in the cardiovascular system, we screened target genes of the peripheral clock by cDNA microarray analysis. A total of 29 genes including transcription factor, growth factors, and membrane receptors were upregulated by CLOCK/BMAL and showed circadian oscillation. These results suggest that cardiovascular systems have their own peripheral clocks, and at least in part, they may regulate the circadian oscillation of cardiovascular function directly. These results potentially provide a molecular basis for the circadian variation of cardiovascular function and novel strategies for the prevention and treatment of cardiovascular diseases.

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A circadian clock controls various physiological and behavioral rhythms. In mammals, a master circadian clock exists in the suprachiasmatic nucleus of the hypothalamus, and slave oscillators can be found in most tissues. These circadian oscillations are controlled by “clock genes”. The negative feedback loop is thought to function as a molecular mechanism of the circadian clock. It is plausible that clock genes may control lipid metabolism through so-called clock-controlled genes and that lipid metabolism-related clock-controlled genes may play important roles in the circadian change of lipid metabolism. Recently research has focused on the relationships between the clock system and lipid metabolism. In this review, we discuss the following items: 1) circadian clock system, 2) effect of the diet on clock gene expression, 3) effect of clock mutation on lipid metabolism, and 4) effect of streptozotocin-induced diabetes and ob mutation on clock gene expression and lipid metabolism. In this review we have summarized how the circadian clock affects lipid metabolism through the expression of lipid metabolism-related clock-controlled genes and at the same time discussed how abnormal metabolism of lipid affects the expression of clock genes. Further experiments are needed to elucidate the detailed mechanism of interaction between clock genes and lipid metabolisms.

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Bronchial asthma is a chronic inflammatory disorder of the airways, in which inflammation causes bronchial hyper-responsiveness and flow limitation in the presence of various stimuli. Pulmonary function in asthmatic patients frequently deteriorates between midnight and early morning, which has suggested a role for chronotherapy. Although relationships between bronchial asthma and the function of clock genes remain unclear, some medications given for asthma such as glucocorticoids or β₂-adrenoceptor agonists may influence clock genes in vivo. In our studies of clock gene mRNA expressions in human bronchial epithelial cells in vitro and peripheral blood cells in vivo, we demonstrated that glucocorticoid or β₂-adrenoceptor agonist treatment strongly induced human Per1 mRNA expression both in vitro and in vivo. Human peripheral blood cells provide a useful indication of peripheral clock gene mRNA expression in vivo.

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Genetic analyses of circadian rhythm sleep disorders (CRSD), such as familial advanced sleep phase syndrome (ASPS) and delayed sleep phase syndrome (DSPS), and morningness-eveningness revealed the relationship between variations in clock genes and diurnal change in human behaviors. Variations such as T3111C in the Clock gene are reportedly associated with morningness-eveningness. Two of the pedigrees of familial ASPS (FASPS) are caused by mutations in clock genes: the S662G mutation in the Per2 gene or the T44A mutation in the casein kinase 1 delta (CK1δ) gene, although these mutations are not found in other pedigrees of FASPS. As for DSPS, a missense variation in the Per3 gene is identified as a risk factor, while the one in the CK1ε gene is thought to be protective. These findings suggest that further, as yet unidentified, gene variations are involved in human circadian activity. Many of the CRSD-relevant variations reported to date seem to affect the phosphorylation status of the clock proteins. A recent study using mathematical models of circadian rhythm generation has provided a new insight into the role of phosphorylation in the molecular mechanisms of these disorders.

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The effectiveness and toxicity of antitumor drugs vary depending on dosing time associated with the 24-h rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Such chronopharmacological phenomena are influenced by not only the pharmacokinetics but also pharmacodynamics of medications. For example, the antitumor effect and/or toxicity of irinotecan hydrochloride, interferon, and antiangiogenic agents vary depending on the dosing time associated with the 24-h rhythm of their target enzyme, receptor, protein, and pharmacokinetics. Many of them are controlled by clock genes. Chronotherapy is especially relevant when the risk and/or intensity of the symptoms of disease vary predicably over time. In a randomized multicenter trial involving patients with previously untreated metastases from colorectal cancer, the chronomodulated infusion of oxaliplatin, fluorouracil (5-FU), and folinic acid is compared with a constant-rate infusion method. Side effects such as stomatitis, peripheral sensory neuropathy are lower and the objective response is higher in the chronotherapy as compared with the fixed-rate infusion. The merit of chronomodulated infusion is supported by the 24-h rhythm of DNA synthesis and the activity of dehydropyrimidine dehydrogenase, which brings about the intracellular catabolism of 5-FU. Although interferon (IFN) also alters the clock function, the disruptive effect of IFN on clock function can be overcome by devising a dosing regimen that minimizes adverse drug effects on clock function. Thus one approach to increasing the efficiency of pharmacotherapy is the administration of drugs at times at which they are most effective and/or best tolerated.

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The caspase activation cascade and mitochondrial changes are major biochemical reactions in the apoptotic cell death machinery. We attempted to clarify the temporal relationship between caspase activation, cytochrome c release, mitochondrial depolarization, and morphological changes that take place during tumor necrosis factor (TNF)-α-induced cell death in HeLa cells. These reactions were analyzed at the single-cell level with 0.5 – 1 min resolution by using green fluorescent protein (GFP)-variant-derived probes and chemical probes. Cytochrome c release, caspase activation, and cellular shrinkage were always observed in this order within 10 min in all dying cells. This sequence of events was thus considered a critical pathway of cell death. Mitochondrial depolarization was also observed in all dying cells observed, but frequently occurred after caspase activation and cellular shrinkage. Mitochondrial depolarization is therefore likely to be a reaction that does not induce caspase activation and subsequent cellular shrinkage. Mitochondrial changes are important for apoptotic cell death; moreover, cytochrome c release, and not depolarization, is a key reaction related to cell death. In addition, we also found that the apoptotic pathway proceeds only when cells are exposed to TNF-α. These findings suggest that the entire cell death process proceeds rapidly during TNF-α exposure.

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Differences of cell proliferation, cell cycle, and G₁/S transition regulatory proteins of gingival fibroblasts derived from nifedipine-reactive patient (NIFr) and nifedipine-non-reactive patient (NIFn) in the presence of basic fibroblast growth factor (bFGF) were investigated to elucidate the mechanism of gingival overgrowth associated with nifedipine, one of the Ca²⁺-channel blockers. The proliferation rate of NIFr cells in the presence of bFGF significantly increased than NIFn cells. The proportion of NIFr cells that had undergone progression to the S and G₂/M phases from the G₀/G₁ phase significantly increased compared to that in NIFn cells. Increases of pRB (Ser807/811), pCDK2 (Thr160), CDK2, and cyclin E protein levels in NIFr cells were greater than those in NIFn cells. The elevations of pRB (Ser780), RB, and cyclin A protein levels in NIFr cells did not differ from those of NIFn cells. The growth of NIFr cells was greater than that of NIFn cells as a result of the active G₁/S transition of NIFr cells, as assessed by the increments of cyclin E, pCDK2, and pRB (ser807/811) protein in NIFr cells.

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The most consistent behavioral changes caused by olfactory bulbectomy are hyperemotional responses such as hyperactivity in a novel environment. However, the changes in the emotional behavior of mice after undergoing olfactory bulbectomy have not yet been described in detail. The effects of methylphenidate on the hyperemotional behavior of olfactory bulbectomized (OBX) mice were examined by using the hole-board test. Mice (4-week-old) were subjected to olfactory bulbectomy, and the behavioral test was performed 2 weeks after surgery. OBX mice showed a significant increase in the number of head-dips as compared to the sham-operated mice. This increase was significantly decreased after treatment with methylphenidate (10 μg/kg, s.c.). The norepinephrine (NE) turnover ratio in the frontal cortex in OBX mice was significantly less than that in the sham-operated mice. However, the decreased NE ratio in OBX mice normalized after treatment with methylphenidate. Our results suggest that the increased head-dipping behavior in OBX mice might reflect an impulsive-like behavior. In addition, we proposed that the improvement in the noradrenergic abnormalities in the frontal cortex due to methylphenidate treatment may play a key role in the improvement of impulsive-like behaviors observed in OBX mice.

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The chronic atrioventricular block dog is a useful model for predicting the future onset of drug-induced long QT syndrome in clinical practice. To better understand the arrhythmogenic profile of this model, we recorded the action potentials of the isolated ventricular tissues in the presence and absence of the class III antiarrhythmic drug nifekalant. The action potential durations of the Purkinje fiber and free wall of the right ventricle were longer in the chronic atrioventricular block dogs than in the dogs with normal sinus rhythm. Nifekalant in concentrations of 1 and 10 μM prolonged the action potential durations of Purkinje fiber and the free wall in a concentration-dependent manner. The extent of prolongation was greater in the chronic atrioventricular block dogs than in the normal dogs. However, increase of temporal dispersion of ventricular repolarization including early afterdepolarization was not detected by nifekalant in either group of dogs, indicating lack of potential to trigger arrhythmias in vitro. These results suggest that the ventricular repolarization delay in the chronic atrioventricular block model by nifekalant may largely depend on the decreased myocardial repolarization reserve, whereas the trigger for lethal arrhythmia was not generated in the in vitro condition in contrast to the in vivo experiment.

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Cisplatin at 5 mg/kg, i.p. induced an acute (day 1) and delayed (days 2 and 3) emetic response in the ferret that was used to investigate the anti-emetic activity of the non-selective cyclooxygenase inhibitor indomethacin (3 – 30 mg/kg, i.p., three times per day) and two cyclooxygenase-2 inhibitors, DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone; 1 – 10 mg/kg, i.p. administered at 40 and 48 h] and L-745,337 [5-methanesulphonamido-6-(2,4-diflurothiophenyl)-1-indanone; 10 mg/kg, i.p., administered at 40 and 48 h]. Only indomethacin potentiated significantly cisplatin-induced retching + vomiting (P<0.05); DFU antagonized delayed emesis (P<0.05) but the action was not dose-related and L-745,337 was inactive (P>0.05). However, indomethacin alone (30 mg/kg) also induced emesis (P<0.05). The leukotriene biosynthesis inhibitor, MK-886 {3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid; 1 – 10 mg/kg, i.p., three times per day} had no action to modify cisplatin-induced emesis (P>0.05). The combination treatment of indomethacin (10 mg/kg, i.p., three times per day) with MK-886 (10 mg/kg, i.p., three times per day) did not antagonize cisplatin-induced acute delayed retching + vomiting and had a different profile compared to the action of dexamethasone (1 mg/kg, i.p., three times per day; P<0.05). Inhibition of the cyclooxygenase and lipoxygenase pathways does not account for the anti-emetic of dexamethasone.

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The present study was undertaken to investigate the effects of some H₁-antagonists on the sleep-wake cycle in sleep-disturbed rats in comparison with those of nitrazepam. Electrodes were chronically implanted into the frontal cortex and the dorsal neck muscle of rats for the electroencephalogram (EEG) and electromyogram (EMG), respectively. EEG and EMG were recorded with an electroencephalograph. SleepSign ver. 2.0 was used for EEG and EMG analysis. The total times of waking, non-rapid eye movement (non-REM), and rapid eye movement (REM) sleep were measured from 10:00 to 16:00. Nitrazepam showed a significant decrease in sleep latency, total waking time, and delta activity and an increase in the total non-REM sleep time. A significant decrease in the sleep latency was observed with diphenhydramine, chlorpheniramine, and cyproheptadine. Cyproheptadine also caused a significant decrease in the total waking time and increases in total non-REM sleep time, REM sleep time, slow wave sleep, and delta activity, although no remarkable effects were observed with diphenhydramine and chlorpheniramine. In conclusion, cyproheptadine can be useful as a hypnotic, having not only sleep inducing-effects, but also sleep quantity- and quality-increasing effects.

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Electropharmacological effects of chronically administered amiodarone and candesartan on atria that had been remodeled against congestive heart failure were assessed using dogs (about 10 kg in weight) with chronic atrioventricular block. Amiodarone was administered orally in a dose of 200 mg/body per day for the initial 7 days followed by 100 mg for the following 21 days (n = 7). Candesartan was administered in a dose of 12 mg/body per day for 28 days (n = 7). All animals survived the 4-week experimental period, indicating the lack of risks for inducing cardiohemodynamic collapse or torsade de pointes by these drugs. The plasma amiodarone concentration was 353 ng/ml at 4 weeks of treatment. Before candesartan treatment (control), intravenous administration of 30 ng/kg of angiotensin II increased the mean blood pressure by 18 mmHg, which was significantly decreased to 1 mmHg by 4 weeks of treatment. Amiodarone prolonged the atrial effective refractory period without affecting inter-atrial conduction time and decreased the duration of the burst pacing-induced atrial fibrillation, whereas candesartan hardly affected these variables. These results indicate that amiodarone should become a pragmatic pharmacological strategy against atrial fibrillation in patients with chronically compensated heart failure and suggest that a much higher dose of candesartan may be needed to exert its efficacy in this model.

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Tenryocha, rooibos, and guava teas are widely consumed as herbal beverages, especially as a therapy against pollen allergy. To investigate the possible herbal tea-drug interaction the effect of continuous ingestion of these teas on cytochrome P450 (CYP) 3A were studied. Rats (n = 6) were allowed free access to either tea (experimental groups) or water (control) for two weeks. Midazolam (MDZ) (20 mg/kg) was orally administered and the serum concentration was determined. The area under the serum concentration-time curve (AUC_0–∞) and the maximum serum concentrations (C_max) of MDZ were reduced by more than 60% after the treatment of tenryocha and rooibos tea (P<0.05). Intestinal MDZ 1'- and 4-hydroxylation activities mediated by CYP3A were increased in tenryocha and rooibos tea-treated group by 50% compared to the control group, although the results were not statistically significant. Furthermore, the Western blot analysis showed that CYP3A content was significantly increased in the intestine after the treatment of these teas (P<0.05). Hepatic MDZ hydroxylation and CYP3A content were slightly increased by these teas. The results suggested that two weeks ingestion of tenryocha and rooibos tea reduced serum concentration of MDZ by the induction of intestinal CYP3A. The possible interaction between tenryocha or rooibos tea and medicines mediated by CYP3A was suggested.

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The cardioprotective effect of caldaret, a novel intracellular Ca²⁺ handling modulator that acts through reverse-mode Na⁺/Ca²⁺ exchanger inhibition and potential sarcoplasmic reticulum (SR) Ca²⁺ uptake enhancement, against reperfusion injury was investigated. We employed a canine model of myocardial infarction induced by 90-min occlusion of left circumflex (LCX) coronary artery followed by 4 h of reperfusion. Intravenously infused caldaret (3 or 30 μg/kg per hour) for 30 min at LCX-reperfusion markedly reduced infarct size (by 51.3% or 71.9%, respectively). This cardioprotection was accompanied by an acceleration of left ventricular (LV) contraction and relaxation during reperfusion, but not by an increase in ischemic regional transmural myocardial blood flow (TMBF) or endocardial/epicardial blood flow ratio (Endo/Epi ratio) or a reduction in double-product throughout the protocol. Diltiazem (2000 μg/kg per hour) also reduced infarct size (by 36.1%), but unlike caldaret, was accompanied by the significant increase in Endo/Epi ratio in the ischemic region and decrease in double-product. There were significant inverse relationships between infarct size and ischemic regional TMBF in all groups. Caldaret, but not diltiazem shifted the regression line downward with a flatter slope. These results suggest that the amelioration of intracellular Ca²⁺ handling dysfunction achieved by caldaret leads to cardioprotective effects against reperfusion injury following prolonged ischemia.

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The aim of this study was to investigate the effect of A-3922, a dihydrobenzofuran derivative, on linoleic acid hydroperoxide (LHP)-induced corneal neovascularization (NV) in a rabbit model. Male New Zealand rabbits received intraperitoneal (i.p.) injections of 10 or 30 mg/kg per day A-3922 or its vehicle as control for 3 days. One day after i.p. injections, LHP was injected with a 30-gauge needle into the corneal stroma of the superior quadrant 4.5-mm below the limbus. Photographs of the vessels were taken for digital analysis with a surgical microscope. Vascular endothelial growth factor (VEGF) was measured using an immunoassay kit, and matrix metalloproteinase (MMP)-9 was measured by gelatin zymography in corneal samples. At 7 days post-LHP injection, the total vessel length was 26.7 ± 3.8 mm in the control animals (n = 8), 16.1 ± 0.8 mm in the A-3922 (10 mg/kg)-treated group (n = 5), and 11.4 ± 2.1 mm in the 30 mg/kg group (n = 8, P<0.01 vs control), respectively. After LHP injection, the content of VEGF and MMP-9 activity were increased in the superior cornea, but these were not influenced by A-3922 treatments. These results indicate that LHP-induced corneal NV is inhibited by treatment with A-3922 and therefore may represent a potential pharmacological intervention for ocular neovascularization disorders.

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Involvement of the Na⁺/Ca²⁺ exchanger in ouabain-induced inotropy and arrhythmogenesis was examined with a specific inhibitor, SEA0400. In right ventricular papillary muscle isolated from guinea-pig ventricle, 1 μM SEA0400, which specifically inhibits the Na⁺/Ca²⁺ exchanger by 80%, reduced the ouabain (1 μM)-induced positive inotropy by 40%, but had no effect on the inotropy induced by 100 μM isobutyl methylxantine. SEA0400 significantly inhibited the contracture induced by low Na⁺ solution. In HEK293 cells expressing the Na⁺/Ca²⁺ exchanger, 1 μM ouabain induced an increase in intracellular Ca²⁺, which was inhibited by SEA0400. The arrhythmic contractions induced by 3 μM ouabain were significantly reduced by SEA0400. These results provide pharmacological evidence that the Na⁺/Ca²⁺ exchanger is involved in ouabain-induced inotropy and arrhythmogenesis.

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In this study, we evaluated the antiproliferative and proapoptotic effects of the isothiocyanate iberin, a bioactive agent in Brassicaceae species, in human glioblastoma cells. The human glioblastoma cell cultures were treated with different concentrations of iberin and tested for growth inhibition, cytotoxicity, induction of apoptosis, and activation of caspases. Iberin inhibited growth of tumor cells in cell proliferation assays, enhanced cytotoxicity, and induced apoptosis by activation of caspase-3 and caspase-9. Findings from this study could provide a basis for potential usefulness of the diet-derived isothiocyanate iberin as a promising therapeutic micronutrient in the prevention/intervention of brain tumors.

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