Circadian clocks are intrinsic, time-tracking systems that endow organisms with a survival advantage. Studies of animal models and human tumor samples have revealed that the disruption of circadian rhythms is an important endogenous factor that can contribute to mammalian cancer development. The core of the circadian clock mechanism is a cell-autonomous and self-sustained oscillator system mediated by a transcription/translation-based negative feedback loop that relies on positive and negative elements. Recent studies have implicated these core circadian components in the regulation of both the cell cycle and DNA damage responses (DDR). Indeed, the circadian feedback loop controls the timing of cell proliferation by regulating the expression of key cell cycle genes. Conversely, several intracellular signaling cascades and post-translational modifications that play important roles in the cell cycle and DDR are also essential for circadian clock regulation. Importantly, alteration of a cell's reduction–oxidation (redox) state triggers the transduction of photic signals that regulate circadian clock gene transcription, suggesting that cellular responses to photo-oxidative stress may have been the evolutionary origin of the circadian clock. This review describes selected regulatory aspects of circadian machinery that are evidence of a molecular link between the circadian clock and DDR, focusing particularly on the signaling cascades involved in the light entrainment of the zebrafish circadian clock.
Inflammation or hypoxia in gingival tissue can induce endoplasmic reticulum (ER) stress, which is related with autophagy. The autophagy is a catabolic process involving the degradation of a cell's own components. Although autophagy resulting in the total destruction of the cell is one of cell death types, no conclusive evidence exists for such a process. In order to examine the association of ER stress and autophagy in gingival system, ER stress agents brefeldin A, thapsigargin, and tunicamycin were exposed to human gingival cells. The ER stress agents induced cell death and the expression of ER stress proteins, glucose-regulated protein of 78 kDa (GRP78) and CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP). ER stress also increased the formation of autophagic vesicles with the expression of beclin and LC-3 (microtubule-associated protein1 light chain 3) II, two autophagic markers. ER stress induced the phosphorylation of p38MAPK (mitogen-activated protein kinase), and the p38MPAK inhibitor, SB203580, inhibited the resulting cell death and autophagy. In summary, ER stress induces cell death and autophagy through p38MAPK in human gingival cells.
The activity of acetylcholinesterase (AChE) is measured to obtain pathological information about the cholinergic system in various disease states and to assess the effect of AChE inhibitors. Using Ellman's method that is commonly used in such examinations, butyrylcholinesterase inhibitors must be added to measure AChE-specific activity because of low selectivity of AChE toward traditional substrates; however, such inhibitors also inhibit AChE. Therefore, it is desirable to obtain an AChE selective substrate that can be used with the Ellman's method. Here, we synthesized novel AChE substrates, 1-methyl-4-acetylthiomethylpiperidine and 1,1-dimethyl-4-acetylthiomethylpiperidine, and evaluated the hydrolysis rate and AChE selectivity by comparison with the results obtained when traditional substrates were used. The hydrolysis rate of the novel compounds by human AChE was one order of magnitude lower than that of the traditional substrates, acetylthiocholine and acetyl-β-methylthiocholine, whereas the hydrolysis rate using human butyrylcholinesterase was two orders of magnitude lower than that of the traditional substrates. This indicated that AChE showed selectivity towards the novel substrates which was one order of magnitude higher than that of the traditional substrates. The hydrolysis of the novel compounds in a rat cerebral cortical homogenate and a monkey whole blood was completely inhibited by 1 μM of the specific AChE inhibitor, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one, indicating the high specificity of AChE towards the novel substrates in a crude tissue sample. From these results, we conclude that the novel compounds developed would be suitable AChE-selective substrates for Ellman's method.
5-Hydroxyindole (5HI), a metabolite of tryptophan, is involved in learning and memory, central neuron system regulation, and anti-oxidant activity. However, its protective action in mitochondrial function is not clear. Here, we tested whether 5HI protects against tert-butylhydroperoxide (t-BHP)-induced oxidative damage and mitochondrial dysfunction in human fibroblast cells. 5HI significantly suppressed t-BHP-induced cytotoxicity as determined by intracellular reactive species generation, lipid peroxidation, glutathione depletion, and peroxynitrite (ONOO−) generation. In addition, 5HI reduced t-BHP-induced DNA condensation. Pretreatment with 5HI significantly restored mitochondrial membrane potential (Δψm), suggesting that it protected cells against t-BHP-induced apoptosis. Western blot analysis also revealed that 5HI markedly inhibited cytochrome c release and caspase-3 activation, but not caspase-9 activation. Our data suggest that 5HI protects cells by attenuating oxidative stress and consequently protects against mitochondrial dysfunction.
Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, is found at high levels in the blood of diabetic patients. MG induces oxidative stress and apoptosis. There is evidence that MG causes glutathione (GSH) depletion. However, it remains unknown whether GSH plays a protective role against the cytotoxic effect of MG. We examined the effect of DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, on the viability of bovine aortic endothelial cells (BAECs) exposed to MG. BAECs pretreated with BSO showed reduced ability to survive MG exposure. Flow cytometric analyses with annexin V and propidium iodide double staining revealed that BAECs exposed to MG after BSO pretreatment displayed features characteristic of apoptosis. Caspase-3 activation induced by MG was increased by BSO. Moreover, measurement of protein carbonyl levels showed that BSO promoted MG-induced oxidative stress. Taken together, these findings suggest that the depletion of GSH via BSO pretreatment promoted MG-induced apoptotic cell death and oxidative stress in BAECs.
Rat liver microsomal glutathione transferase (MGST1) is activated by limited proteolysis. Recently we purified a protease, hepsin, from rat liver microsomes that activates MGST1. In the present study the mechanism of MGST1 activation by hepsin was investigated. When MGST1 and hepsin were incubated at room temperature, MGST1 activity was markedly increased and the increase was decreased to the control level by further incubation with disulfide bond reducing agent dithiothreitol. MGST1 dimer was detected by electrophoresis after treatment of MGST1 with hepsin, instead of proteolytic product. MGST1 dimer formation accompanied by an increase in MGST1 activity was observed even in the presence of the protease inhibitor benzamidine. Furthermore, prolonged incubation of both enzymes caused the formation of MGST1 dimer and its proteolytic product. These results clearly show that the protease hepsin stimulates disulfide-linked MGST1 dimer formation resulting in activation of MGST1 and preferential degradation of MGST1 dimer. Since hepsin contains disulfide bonds in the scavenger receptor cysteine-rich (SRCR) domain, it was suggested that the SRCR domain interacts with MGST1 leading to thiol/disulfide exchange between the two enzymes followed by disulfide-linked MGST1 dimer formation.
A peptide antibiotic, edeine B1, exerts a lethal action in Bacillus subtilis causing filamentous morphology. This antibiotic assumes to inhibit cell division by interacting with FtsZ and inhibiting FtsZ polymerization. The temperature-sensitive mutant ftsZ ts1 was shown to be hypersensitive to the antibiotic as compared to the parent 168 with respect to its lethal action and the sensitivity to the antibiotic of the revertant of ftsZ ts1 was shown to be intermediate between those of the parent 168 and the ftsZ ts1. Alteration of FtsZ sequence may be responsible for sensitivity to edeine B1. The residues at 240, 278, 345 and 346 in the FtsZ sequence of the parent 168 were A240, A278, D345 and A346. Those of ftsZ ts1 were V240, V278, E345 and P346. Those of the revertant of ftsZ ts1 were A240, A278, E345 and P346. The difference in sensitivity to edeine B1 among these strains is presumably due to the difference in the residues at 240, 278, 345 and 346 in the FtsZ sequence. The sequential events of the inhibition of FtsZ assembly and the inhibition of protein biosynthesis by edeine B1 may progress synergistically, resulting in cell death.
Status epilepticus (SE) rendered selective neuronal loss and cognitive impairments. Previous studies proved that granulocyte colony-stimulating factor (G-CSF) acted as a neuroprotectant in some nervous diseases. However, no investigations were focused on whether G-CSF could protect the hippocampus from SE. In this study, we administered recombinant human G-CSF into Sprague-Dawley rats with lithium-pilocarpine-induced SE subcutaneously for three times. The Morris water maze was employed to determine spatial learning ability from the 15th to 20th days after the treatment. The quantitative terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining and levels of apoptosis-related molecules including cleaved caspase-3, Bcl-xL and Bax on hippocampal CA1 region were examined by immunohistochemical staining at the 3rd and the 5th day after the treatment. Moreover, the phosphorylation of AKT was evaluated with Western blot at the 6th, 24th and 48th hours after the treatment to explore apoptosis and detect the protective effects of G-CSF. We found G-CSF treatment prevented SE-induced cognitive impairments with the decreased escape latency time on the 17th (29.86±9.09 vs. 38.33±6.94, p<0.05) and 18th days (23.83±6.17 vs. 33.52±8.48, p<0.05). The reduced TUNEL staining demonstrated reduced neuronal apoptosis occurrences. The anti-apoptotic effects were associated with decreased cleaved caspase-3 and Bax expression and increased phosphorylation of AKT and Bcl-xL expression. Taken together, our results suggested that systemic G-CSF treatment conducted neuroprotective function following SE through an anti-apoptotic pathway and prevented cognitive impairments, which may provide novel insights into pathogenesis and treatment following SE injury.
Invariant natural killer T (iNKT) cells regulate multi-immune response through Th1/Th2 cytokine release triggered by the recognition of CD1d-restricted glycosphingolipid antigens. Here we report that acidic glycosphingolipids (AGLs) of mushroom (Hypsizigus marmoreus and Pleurotus eryngii) presented by murine CD1d-transfected rat basophilic leukocytes induced interleukin-2 (IL-2) release from iNKT hybridoma cells. AGL-1, one of the AGLs, containing mannose at the non-reducing ends, induced CD1d-dependent IL-2 release. Al-though α-galactosylceramide (α-GalCer) presented by CD11c-positive cells induced both interferon-γ (IFN-γ) and IL-4 release, all of AGLs presented by CD11c-positive cells and AGL-1 presented by B cells induced IL-4 release from iNKT hybridoma cells. A single intravenous injection of AGLs into B6 mice induced only a little elevation of IL-4 in serum but repeated intravenous injection of AGLs induced prolonged retention of IL-4 in serum; therefore, these results suggested that edible mushroom AGLs might contribute to the retention of immunohomeostasis through the minimum induction of iNKT cell activation in vivo.
The present study has been designed to investigate the modulatory role of dichlorobenzamil a Na+/Ca2+ exchanger inhibitor in ischemic post-conditioning-induced neuroprotection in mice. Bilateral carotid artery occlusion followed by reperfusion, produced a significant rise in cerebral infarct size along with impairment of memory and motor coordination. Ischemic post-conditioning involving three episodes of 10 s carotid artery occlusion with intermittent reperfusion of 10 s proceeding prolonged ischemic insult of 12 min, produced a significant decrease in cerebral infarct size along with reversal of ischemia-reperfusion-induced impairment of memory and motor coordination. Ischemic post-conditioning-induced neuroprotective effects were significantly abolished by pretreatment with dichlorobenzamil (DBZ 0.5, 1 mg/kg, intraperitoneal) a Na+/Ca2+ exchange inhibitor. It may be concluded that Na+/Ca2+ exchanger possibly plays an important role in ischemic post-conditioning-induced neuroprotection.
Phloridzin, a phloretin 2′-β-D-glucoside, belongs to dihydrochalcones and mainly exists in the fruits of Malus pumila Mill., Lithocarpus polystachyus REHD and the root skins, stems, tender leaves and fruits of Malus hupehensis. It has many pharmacological activities, such as regulating blood sugar level and blood pressure, protecting heart, scavenging of oxygen free radicals and antioxidant injuries. Thus, market demand of products containing phloridzin is increasing year by year. Our research results demonstrated that phloridzin is provided with a double directional adjusting function of estrogenic and antiestrogenic activities. It showed significant effects on the proliferation of estrogen sensitive estrogen receptor (ER) (+)MCF-7 cells in the absence of estrogen. When added with 17β-estradiol, phloridzin showed antagonism on estradiol-induced MCF-7 cell proliferation, but it did not significantly affect proliferation of estrogen insensitive ER (−)MDA-MB-231 cells. Phloridzin induced β-galactosidase activity in a yeast two-hybrid assay. Light increase of the uterine weight and serum estradiol content of mouse was observed when the glucoside was administered orally for 7 d. After oral administration, phloridzin was found mainly in the blood and a small part was metabolized to phloretin. Our investigation proved that phloridzin was distributed at the target organ and played the role of phytoestrogen.
It was previously reported that piperine (PIP) significantly blocks convulsions induced by intracerebroventricular injection of threshold doses of kainate, but had no or only slight effects on convulsions induced by L-glutamate, N-methyl-D-aspartate and guanidinosuccinate. In traditional Chinese medicine, black pepper has been used for epileptic treatment; however, the exact mechanism is still unclear. We reported here in that appropriate concentration of PIP effectively inhibites the synchronized oscillation of intracellular calcium in rat hippocampal neuronal networks and represses spontaneous synaptic activities in terms of spontaneous synaptic currents (SSC) and spontaneous excitatory postsynaptic currents (sEPSC). Moreover, pretreatment with PIP expects protective effect on glutamate-induced decrease of cell viability and apoptosis of hippocampal neurons. These data suggest that the neuroprotective effects of PIP might be associated with suppression of synchronization of neuronal networks, presynaptic glutamic acid release, and Ca2+ overloading.
Ginseng, the root of Panax ginseng C. A. MEYER, has been used as a food product and medicinal ingredient. In this study, we assessed the anti-arthritic effects of red ginseng saponin extract (RGSE), including ginsenosides Rg3, Rk1 and Rg5 as major components, on a murine type II collagen (CII)-induced arthritis (CIA), which is a valid animal model of human arthritis. Oral administration of RGSE at 10 mg/kg reduced the clinical arthritis score and paw swelling in the CIA mice, and inhibited joint space narrowing and histological arthritis, illustrating the severity of synovial hyperplasia, inflammatory cell infiltration, pannus formation, and erosion of cartilage. RGSE inhibited the expression of matrix metalloproteinase-3 and nitrotyrosine formation, and recovered the expression of superoxide dismutase in the joints of the CIA mice. Orally administered RGSE also reduced the levels of serum tumor necrosis factor-α and interleukin-1β in the CIA mice. CII- or lipopolysaccharide-stimulated cytokine production, in addition to CII-specific proliferation, was reduced in the spleen cells of the RGSE-treated CIA mice, as compared with those from vehicle-treated CIA mice. Furthermore, RGSE administration protected against CIA-induced oxidative tissue damage by restoring the increased malondialdehyde levels and the decreased glutathione levels and catalase activities almost to control levels. Therefore, RGSE may be a beneficial supplement which can improve human arthritis.
In the present study, we investigated the effects of a Kampo medicine Orento (TJ-120) on the production of prostaglandin E2 (PGE2), interleukin (IL)-6 and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide from Porphyromonas gingivalis (PgLPS). HGFs proliferation was dose-dependently decreased with Orento at days 3 and 7. However, treatment with PgLPS (10 ng/ml), Orento (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Orento suppressed PgLPS-induced PGE2 production in a dose-dependent manner but did not alter basal PGE2 level. In contrast, Orento did not alter PgLPS-induced IL-6 and IL-8 productions. These alterations by Orento were similar to those by a mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059. A Orento showed no effect on cyclooxygenase (COX)-1 and COX-2 activities, and increased cytoplasmic phospholipase A2 (cPLA2) expression and increased PgLPS-induced COX-2 expression. Orento suppressed PgLPS-induced mobility retardation of cPLA2 band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, that is cPLA2 phosphorylation and its activation, while Orento alone did not alter cPLA2 phosphorylation. Orento suppressed PgLPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA2 phosphorylation. These results suggest that Orento decreased PGE2 production by inhibition of cPLA2 phosphorylation and its activation via inhibition of ERK phosphorylation, and also that Orento may be useful to improve gingival inflammation in periodontal disease.
An imbalance between noradrenergic and dopaminergic systems is implicated in hyperactivity disorders, such as attention deficit/hyperactivity disorder (ADHD). We previously showed that the explosive jumping behavior elicited by intermittent rapid eye movement sleep deprivation (REMSD) may serve as a useful model of ADHD (see [Biogenic Amines, 20, 99—111]). Here, we investigated whether intermittent REMSD might cause changes in monoamine turnover in the mouse forebrain. Our main findings were as follows. Intermittent REMSD led to a significant decrease in dopamine turnover and a significant increase in noradrenaline turnover in the frontal cortex. The latter effect, but not the former, was attenuated by atomoxetine, which is used clinically to treat ADHD symptoms. These results suggest (a) that intermittent REMSD induces hypernoradrenergic and hypodopaminergic states within the frontal cortex, and (b) that the therapeutic effects of atomoxetine may include an inhibition of this hypernoradrenergic state.
α-Chaconine, a naturally occurring steroidal glycoalkaloid in potato sprouts, was found to possess anti-carcinogenic properties, such as inhibiting proliferation, migration, invasion, and inducing apoptosis of tumor cells. However, the effect of α-chaconine on tumor angiogenesis remains unclear. In the present study, we examined the effect of α-chaconine on angiogenesis in vitro. Data demonstrated that α-chaconine inhibited proliferation of bovine aortic endothelial cells (BAECs) in a dose-dependent manner. When treated with non-toxic doses of α-chaconine, cell migration, invasion and tube formation were markedly suppressed. Furthermore, α-chaconine reduced the expression and activity of matrix metalloproteinase-2 (MMP-2), which is involved in angiogenesis. Our biochemical assays indicated that α-chaconine potently suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), phosphatidylinositide-3 kinase (PI3K) and Akt, while it did not affect phosphorylation of extracellular signal regulating kinase (ERK) and p38. In addition, α-chaconine significantly increased the cytoplasmic level of inhibitors of κBα (IκBα) and decreased the nuclear level of nuclear factor kappa B (NF-κB), suggesting that α-chaconine could inhibit NF-κB activity. Furthermore, the treatment of inhibitors specific for JNK (SP600125), PI3K (LY294002) or NF-κB (pyrrolidine dithiocarbamate) to BAECs reduced tube formation. Taken together, the results suggested that α-chaconine inhibited migration, invasion and tube formation of BAECs by reducing MMP-2 activities, as well as JNK and PI3K/Akt signaling pathways and inhibition of NF-κB activity. These findings reveal a new therapeutic potential for α-chaconine on anti-angiogenic therapy.
Ginsenoside Rg1 (Rg1), one of the active components of Panax ginseng, has been reported to promote endogenous nitric oxide (NO) production in some tissues, and to inhibit left ventricular (LV) hypertrophy in rats. This study aimed to investigate whether Rg1-induced inhibition of rat LV hypertrophy is mediated by NO-production. Rat LV hypertrophy was induced by abdominal aorta coarctation. Rg1 15 mg/kg/d, L-arginine 200 mg/kg/d, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) 100 mg/kg/d used with the same dose of L-arginine or Rg1 were given starting from 1 d after surgery for 21 consecutive days. LV hypertrophy was evidenced by determining LV weight and mRNA expression of atrial natriuretic peptide, a marker of cardiac hypertrophic response, as well as by histopathology. Rg1 and L-arginine administration significantly reduced the elevated LV hypertrophic parameters independent of LV systolic pressure changing, and ameliorated the histopathology of LV myocardium and LV diastolic function. All the beneficial effects of Rg1 and L-arginine were abolished or blunted by L-NAME. Further to examine the role of NO in Rg1 inhibition on LV hypertrophy, expression of endothelial NOS was determined at the transcript levels. In our experimental conditions endothelial NOS mRNA expression in LV tissue was lowered by abdominal aorta coarctation, and upregulated by Rg1 administration. These results demonstrate that Rg1-induced protection against LV hypertrophy elicited by abdominal aorta coarctation in rats is mediated, at least in part, via endogenous NO production and release.
The aim of this study was to examine gene expression changes in the frontal cortex and hippocampus of animals with different susceptibility to stressful stimuli. Using a learned helplessness (LH) paradigm, mice received moderate current stimulation to induce different extents of LH behavior. Changes in mRNA expression were investigated by microarray and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Comparisons of expression profiles between LH and control or non-learned helplessness (non-LH) animals revealed that the signal transducers and activators of transcription 3 (Stat3)-interacting protein (StIP1) gene is downregulated in LH animals and the suppressor of cytokine signaling 3 (Socs3) gene is up-regulated in non-LH animals. Since both StIP1 and Socs3 regulate the activity of Stat3 gene, these results suggest that Stat3 systems may be involved in the pathogenesis of depressive disorders.
Astragaloside IV, the major active component extracted from Astragalus membranaceus, exerts multipotent activities under pathophysiological conditions. Hyperhomocysteinemia, an independent risk factor for cardiovascular disease, induces oxidative stress leading to endothelial dysfunction. We investigated the effect of astragaloside IV on acute phase endothelial dysfunction induced by homocysteine. In a concentration-dependent manner, endothelial dysfunction was induced by homocysteine. In organ bath experiment using rat aortic rings, treatment with astragaloside IV resulted in an improvement of the impaired endothelium-dependent vasorelaxation by homocysteine as reflected by the higher maximal vasorelaxation to acetylcholine. However, the presence of Nω-nitro-L-arginine methyl ester hydrochloride could abolish the protective effect of astragaloside IV on homocysteine-induced vasomotor dysfunction. In human umbilical vein endothelial cells culture experiment, exposure to astragaloside IV significantly ameliorated the homocysteine-induced inactivation of nitric oxide–nitric oxide synthase signal pathway via reducing oxygen species and increasing the activity of superoxide dismutase. Additionally, pretreatment with superoxide dismutase showed a similar effect to astragaloside IV on attenuation of the homocysteine-induced endothelial dysfunction. These data support the view that astragaloside IV might be advantageous in the treatment of endothelial dysfunction induced by disturbed nitric oxide–nitric oxide synthase pathway due to oxidative stress in hyperhomocysteinemia.
The present study was undertaken to clarify the effect of group I metabotropic glutamate receptor (mGluR) antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) on pentetrazol-induced kindled seizures. The mechanism of the anticonvulsant effect of AIDA was also studied. Mice were anesthetized with pentobarbital; the electrodes and guide cannula were chronically implanted into the cortex and lateral ventricle. In order to induce kindling, pentetrazol at a dose of 40 mg/kg was injected intraperitoneally once every 48 h. Behavioral and electroencephalographic (EEG) seizures were observed for 20 min following pentetrazol administration. Intracerebroventricular (i.c.v.) injection of AIDA (1000 nmol/site) resulted in a significant inhibitory effect on pentetrazol-induced kindled seizures, and this effect was antagonized by a group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine ((RS)-3,5-DHPG). The effect of AIDA (200 nmol/site) on pentetrazol-induced kindled seizures was augmented by the simultaneous use of γ-aminobutyric acid (GABA) mimetic drugs, such as NNC-711 and diazepam. Moreover, the effect of AIDA (1000 nmol/site) on pentetrazol-induced kindled seizures was antagonized by a GABAA receptor antagonist, bicuculline and a GABAC receptor antagonist, (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA). It can be concluded that AIDA had an anticonvulsant effect on pentetrazol-induced kindled seizures, which was partially mediated by the GABAergic mechanism through GABAA and GABAC receptors.
The present study was undertaken to examine whether distigmine, a therapeutic agent used to treat detrusor underactivity, binds directly to muscarinic and nicotinic receptors. We used radioreceptor binding assays and compared the effects of distigmine with those of neostigmine and donepedil. The inhibitory effect of distigmine on the blood acetylcholinesterase (AChE) activity was significantly weaker than that of neostigmine. Distigmine, neostigmine, and donepezil competed for specific binding sites of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS ) and [3H]oxotremorine-M in the bladder, submaxillary gland and cerebral cortex of rats in a concentration-dependent manner, indicating significant binding activity of muscarinic receptors. Distigmine displayed significantly higher affinity for binding sites of [3H]oxotremorine-M compared with those of [3H]NMS as revealed by large ratios of its Ki value for [3H]NMS to that for [3H]oxotremorine-M, suggesting that it has preferential affinity for agonist sites of muscarinic receptors. Distigmine seemed to bind to the agonist sites of muscarinic receptors in a competitive manner. Repeated oral administration of distigmine caused a significant decrease in the maximal number of binding sites (Bmax) for [3H]NMS in the bladder and submaxillary gland but not cerebral cortex. Distigmine also bound to nicotinic receptors in the rat cerebral cortex. In conclusion, distigmine shows direct binding to muscarinic receptors in the rat bladder, and repeated oral administration of distigmine causes downregulation of muscarinic receptors in the rat bladder. The observed direct interaction of distigmine with the bladder muscarinic receptors may partly contribute to the therapeutic and/or side effects seen in the treatment of detrusor underactivity.
CCl4 (0.5 ml/kg as CCl4) was orally administered to rats. Twelve hours after administration of CCl4, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, indicators of liver necrosis, were significantly higher than those in the control group showing that active liver necrosis took place. At the same time the level of liver vitamin C was decreased significantly compared to that in the control group. Oral administration of 100 mg/kg each of celecoxib 3 and 8 h after CCl4 treatment did not change plasma ALT and AST and liver vitamin C levels 12 h after CCl4 treatment, but 24 h after CCl4 treatment, significantly decreased plasma ALT and AST levels and elevated liver vitamin C level. These finding suggested that celecoxib effectively ameliorated the necrotic action and the oxidative stress induced by CCl4 in the second phase. Although the plasma levels of all ceramide species were significantly increased 24 h after CCl4 intoxication, treatment with celecoxib significantly reduced the total ceramide concentration in plasma. These results indicated that celecoxib significantly ameliorated the toxicity of CCl4 in the second phase.
RhoA plays an important role in Ca2+ sensitization of bronchial smooth muscle in antigen-induced airway hyperresponsiveness (AHR). Glucocorticoids are now the most effective anti-inflammatory treatment for asthma, and inhaled corticosteroids are the most effective long-term control therapy for persistent asthma. To determine the mechanism of the inhibitory action of glucocorticoids on AHR in allergic bronchial asthma, that of prednisolone on RhoA upregulation was investigated using cultured human bronchial smooth muscle cells (hBSMCs). The upregulation of RhoA induced by interleukin (IL)-13 and tumor necrosis factor (TNF)-α, major mediators for development of AHR, was observed in hBSMCs. Prednisolone partly inhibited the IL-13-induced RhoA upregulation and RhoA promoter activity, although prednisolone had no effects on the activations of signal transducers and activators of transcription (STAT)6 and nuclear factor (NF)-κB. Increased expression and promoter activity of RhoA induced by TNF-α was completely inhibited by prednisolone, although the activation of NF-κB failed to be inhibited by prednisolone in hBSMCs. These findings suggest that prednisolone might inhibit NF-κB-induced transcription via interaction between glucocorticoid receptor (GR), resulting in an inhibition of RhoA upregulation induced by IL-13 and TNF-α.
The enhancement of blood fluidity may lead to improvements in skin problems resulting from unsmooth circulation or blood stagnation. Since a 50% ethanolic extract (CH-ext) obtained from unripe Citrus hassaku fruits may be a useful ingredient in skin-whitening cosmetics, the present study was designed to examine the effect of CH-ext on blood fluidity. CH-ext concentration-dependently inhibited in vitro collagen-induced rabbit platelet aggregation and in vitro polybrene-induced rat erythrocyte aggregation. The CH-ext showed in vitro fibrinolysis activity in fibrin plate assay. Activity-guided fractionation of the CH-ext using antiplatelet activity, inhibitory activity of erythrocyte aggregation, and fibrinolysis activity revealed that these activities of CH-ext were attributable to naringenin-7-glycoside (prunin). Successive oral administration of CH-ext to rats inhibited the lipopolysaccharide (LPS)-induced decrease of blood platelets and fibrinogen, and LPS-induced increase of fibrin degradation products (FDP) in LPS-induced disseminated intravascular coagulation (DIC) model rats. Effects of CH-ext on blood fluidity were analyzed by a micro channel array flow analyzer (MC-FAN). Preventive oral administration of CH-ext to rats showed dose-dependent reduction of the passage time of whole blood flow of the DIC model rats in comparison with that of the vehicle control rats. These results imply that CH-ext may have effects which improve effects on blood fluidity.
A method was developed for rapid identification of Dendrobium species by a dot blot hybridization assay. The dot blot system is based on species-specific amplified fragments derived from the internal transcribed spacer (ITS) region of different Dendrobium species as target DNA, which were blotted as dots on the nylon membrane. A small aliquot of the ITS amplified product from a selected species or mixed ITS DNA from several species was labeled by peroxidase and used as a probe for hybridization to the dot blot membrane. Simply visualizing the positive hybridization reaction between the probe DNA and the target DNA could identify the selected species. The D. officinal-specific and D. nobile-specific ITS-based probes could separately identify D. officinal and D. nobile. Eight Dendrobium species, including D. salaccense, D. brymerianum, D. ellipsophyllum, D. chrysanthum, D. officinale, D. thyrsiflorum, D. nobile and D. chrysotoxum could be simultaneously detected using their complex ITS amplified products as a probe, and the ITS DNA probe amount for detection by the dot blot membrane is very small, just 2.5 ng. The assay can also be used to identify the resource species of Fengdou Shihu and Huangcao Shihu from the commercial market. The identification based on the ITS sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the polymerase chain reaction (PCR)-dot blot hybridization assay can be considered a reliable method for general identification of commercial Shihu.
The alkaloid, rhynchophylline (RHY), from the stems and hooks of Uncaria rhynchophylla was revealed in recent years to have protective effect on neuronal damage. The present research was carried out to investigate the in vivo metabolism of this bioactive alkaloid. After administering RHY to rats, LC-MS detected RHY in plasma, bile, brain, urine and feces, the glucuronides, 11-hydroxyrhynchophylline 11-O-β-D-glucuronide (M1) and 10-hydroxyrhynchophylline 10-O-β-D-glucuronide (M2) in bile, and 11-hydroxyrhynchophylline (M3) and 10-hydroxyrhynchophylline (M4) in urine and feces. Within 24 h, 78.0% of RHY was excreted into the feces and 12.6% into the urine of rats after oral administration of 37.5 mg/kg. Monitoring by LC-MS showed that 9.4% of RHY was metabolized to M3 and M4 in a ratio of about 1 : 1. RHY was also detected in the brain (0.650 ng/g) at 3 h after oral administration of the same dose. Cytochrome P450 (CYP) in rat liver microsomes played a key role in RHY hydroxylation. Specific inhibition of CYP isozymes indicated that CYP2D, CYP1A1/2 and CYP2C participated in RHY hydroxylation, but not CYP3A.
When the biological activites of hydrophobic drugs or xenobiotics are studied, it is important to clarify their effects on expression and function of multidrug resistance (MDR) protein. We therefore evaluated the effects of coptisine on MDR in comparison with the structurally related isoquinoline alkaloids berberine and palmatine. To achieve this, we investigated the effects of the three alkaloids on the expression and function of P-glycoprotein/MDR1, MDR1 gene products, in vascular smooth muscle cells (VSMCs). In A10 cells (a rat VSMC line), coptisine upregulated the mRNAs of Mdr1a and Mdr1b, rodent homologues of human MDR1, and these effects were completely abrogated by actinomycin D. Coptisine also induced Mdr1a/1b protein expression and enhanced the efflux of rhodamine 123 from A10 cells. In contrast, berberine and palmatine slightly upregulated the mRNAs of Mdr1a and Mdr1b, but failed to induce Mdr1a/1b protein expression or stimulate rhodamine 123 efflux. To clarify whether these effects occurred in other cells, the effects of the three alkaloids on Mdr1a/1b function were examined in 3Y1, dRLh-84 and B16 cells. Coptisine and berberine enhanced rhodamine 123 efflux in all three cell types, while palmatine inhibited it, based on the finding that palmatine efficiently activated the Mdr1a ATPase activity as a good substrate for Mdr1a. Therefore, the three isoquinoline alkaloids regulated MDR differently in cell type-specific manners. In particular, only coptisine induced Mdr1a/1b in A10 cells and stimulated rhodamine 123 efflux. Taken together, coptisine appears to exert VSMC-selective effects on Mdr1a/1b induction in contrast to berberine and palmatine.
The allergy-preventive activity of a 35% EtOH extract (IT) of flowers of Impatiens textori MIQ. was demonstrated in a continuing search for allergy-preventive substances from natural sources. The evaluation of its activity used an in vivo assay method for monitoring the blood flow decrease in the tail vein microcirculation of mice subjected to sensitization with hen-egg white lysozyme. Among the principal compounds in IT, apigenin (1), luteolin (3), and luteolin 7-glucoside (4) showed significant allergy-preventive effects.
Erythropoietin (EPO) was successfully loaded on self-dissolving micropile array (SDMA) chip using chondroitin sulfate as the base polymer. “Drug glue” was prepared by adding EPO solution to chondroitin sulfate solution and SDMA was formed by micromolding fabrication technology. One SDMA chip, 1.0×1.0 cm, contained 100 micropile arrays. Two types of SDMA, partially-loaded SDMA (p-SDMA) and fully-loaded SDMA (f-SDMA), were prepared. The mean lengths of the SDMAs were 474.8±8.1 μm for p-SDMA and 473.4±5.2 μm for f-SDMA. The diameters of the array basements were 288.4±4.5 μm (p-SDMA) and 294.6±3.2 μm (f-SDMA). EPO content was 25.0±3.8 IU (p-SDMA) and 125.9±26.7 IU (f-SDMA). After percutaneous administration of each SDMA chip to rats, maximum serum EPO concentrations (Cmax) were 30.5±4.2 mIU/ml for p-SDMA and 32.4±5.0 mIU/ml for f-SDMA. The mean areas under the serum EPO concentration vs. time curves (AUC) were 534.0±102.4 mIU·h/ml (p-SDMA) and 523.1±50.4 mIU·h/ml (f-SDMA). Bioavailability (BA) values of EPO delivered from SDMAs were calculated to be 39.4% for p-SDMA and 7.7% for f-SDMA. Dose-dependency of the serum EPO concentration vs. time curve was studied using p-SDMA chip containing less EPO, 11.6±1.06 IU. Good dose-dependency was observed for Cmax and AUC. The p-SDMA chip was also evaluated in dogs. One or two p-SDMA chips, where 1 chip contained 22.4 IU of EPO, were percutaneously administered to dogs. BA of EPO delivered from p-SDMA was 65.9—69.0%. These results suggest the usefulness of p-SDMA as a percutaneous drug delivery system (DDS) for EPO.
Phosphatidylcholine (PC) production is accelerated by glucocorticoid, such as dexamethasone (DEX), which enhances fetal lung maturation, promotes differentiation of alveolar type II (ATII) cells, and increases production of both lipid and protein components of lung surfactant. We previously demonstrated that inhibition of choline uptake by ATII cells leads to a decrease of PC synthesis. Since choline uptake may play a critical role in PC production and lung surfactant homeostasis for normal breathing, it is of interest to characterize transporters controlling the disposition of choline in ATII cells. Therefore, we studied the gene regulation and activity of choline transporters in A549 cells, a human ATII cell line. A549 cells were exposed to DEX for 24 h, and mRNA expression levels of choline transporters-like protein 1, (CTL1) and CTL2, were measured using real-time reverse transcription polymerase chain reaction. CTL1 and CTL2 mRNAs were strongly induced by DEX treatment of A549 cells, and the DEX-treated cells showed a significant increase in initial uptake rate of [3H]choline, which was assessed under ATP-depleted conditions to block the influence of consumption of choline by choline kinase. Transfection of A549 cells with either CTL1- or CTL2-small interfering RNAs significantly decreased [3H]choline uptake. In conclusion, choline transport in A549 cells is increased by treatment with DEX, and the increase is mediated by induction of functional choline transporters CTL1 and CTL2.
It was reported that coadministration of amiodarone with carvedilol increased the serum concentration to dose (C/D) ratio of S-carvedilol in patients with heart failure, but not of R-carvedilol. The aim of the present study was to investigate the effect of amiodarone and its metabolite on the metabolism of R- and S-carvedilol in human liver microsomes (HLM). Oxidation of carvedilol in HLM was evaluated in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH), whereas glucuronidation was evaluated in the presence of uridine 5′-diphosphate (UDP)-glucuronic acid. The oxidation and glucuronidation activities of HLM for S-carvedilol were approximately 2- and 4-fold greater, respectively, than those for R-carvedilol. In the presence of amiodarone (50 μM) and/or desethylamiodarone (25 μM), the oxidation activity for R- and S-carvedilol decreased significantly. In contrast, the glucuronidation activity for R-carvedilol was increased 1.6- and 1.4-fold by amiodarone and desethylamiodarone, respectively, whereas the glucuronidation of S-carvedilol was only slightly changed by amiodarone and desethylamiodarone. These results suggested that inhibition of S-carvedilol oxidation by amiodarone and/or desethylamiodarone is implicated in the increased C/D ratio of S-carvedilol associated with coadministration of amiodarone. On the other hand, the stimulative effect of amiodarone and/or desethylamiodarone on the glucuronidation of R-carvedilol may compensate for the inhibitory effect of those on R-carvedilol oxidation.
Zoledronic acid (ZDA) is commonly prescribed to treat and prevent skeletal complications in patients with multiple myeloma or bone metastases. Although hypocalcemia often develops by ZDA, there is little information about the risk factors for hypocalcemia mediated by ZDA. This study was conducted to assess the risk of ZDA-mediated hypocalcemia. We retrospectively reviewed the records of patients receiving ZDA in Mie University Hospital. The subjects were divided into two groups on the basis of whether hypocalcemia developed (19 patients) or not (30 patients). We compared patients' baseline characteristics between the two groups. The patients with hypocalcemia had lower albumin-adjusted serum calcium concentrations (median 9.2 mg/dl) before ZDA administration than the patients without hypocalcemia (median 9.8 mg/dl) (p< 0.01). Multivariate analysis revealed that an adjusted serum calcium concentration lower than 9.5 mg/dl before ZDA administration was an independent risk factor significantly contributing to the development of hypocalcemia (odds ratio 22.0, p< 0.01). Furthermore, the patients receiving corticosteroid had increased risk of ZDA mediated hypocalcemia (odds ratio 11.9, p<0.05). On the other hand, the patients with prostate cancer had a reduced risk for hypocalcemia after ZDA administration (odds ratio 0.06, p<0.05). In conclusion, a lower serum calcium concentration and co-administration of corticosteroid increased the risk of hypocalcemia after ZDA administration, while patients with prostate cancer might have a small risk of this incidence. These findings should provide useful information regarding the monitoring of serum calcium concentration in cancer patients receiving ZDA.
Naringin (Nar) is a flavonoid that has shown antigenotoxic effect against the chromosome damage induced by various compounds. The aims of the present investigation on Nar were threefold: a) to determine its DNA breaking potential in mouse hepatocytes and cardiocytes, b) to evaluate its capacity to inhibit the DNA damage induced by daunorubicin (Dau) in the same tissues, and c) to determine its capacity to trap free radicals in vitro using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method. For the two first purposes we applied the comet assay to three groups of animals administered with Nar by the oral route (50, 250, 500 mg/kg), and made the observations before the chemical administration and at 3, 12, and 21 h postadministration. Other three groups of mice were given equal doses of Nar, and 1 h later they were intraperitoneally injected with 1 mg/kg of Dau. The results showed that Nar did not induce DNA breakage in both types of studied cells, in contrast with the significant damage induced by Dau in hepatocytes and cardiocytes. Moreover, the administration of Nar protected the DNA damage produced by Dau, showing a maximum reduction of 71.3% and 51.1% in hepatocytes and cardiocytes, respectively. With respect to the antioxidant potential, 20 mM of Nar produced a free radical scavenging activity as high as 95%. Our study established a high DNA breaking potential of Dau, and a protective effect by Nar, probably related with its capacity to trap free radicals.
A new series of benzylidene-prop-2-ynyl-amines analogues have been synthesized and evaluated for their monoamine oxidase A and B inhibitory activity by determination of IC50 and selectivity index (SI). Among these inhibitors, benzhydrylidene-prop-2-ynyl-amine (2, IC50=32 nM) and (3, 4-dimethoxy-benzylidene)-prop-2-ynyl-amine (10, IC50=14 nM) provide the highest inhibitory potency toward monoamine oxidase (MAO) A and B respectively. (3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-prop-2-ynyl-amine (1, SI=58.96) and compound (2, SI=0.34) were proved to be the superior selective inhibitors toward MAO-A and MAO-B respectively. Docking studies show that the imine moiety is located in hydrophobic pocket, bringing the propargyl group close to FAD which indicates that the different inhibitory potency toward MAO-A may be ascribable to both the distance between alkynyl group and N5 of FAD, and hydrogen bonding interactions between inhibitors and enzymes.