Cerebral ischemia-reperfusion (CI/R) injury remains a major medical problem due to the lack of effective therapies. Previous studies have shown that increasing the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and gene targets in cell culture and stroke animal models is highly neuroprotective. Oxymatrine is the major quinolizidine alkaloid extracted from the root of Sophora flavescens AIT, and has been proved to be protective after ischemia in recent studies. The present study was designed to investigate the potential effect of oxymatrine in ischemia-reperfusion injury in rat's brain and to explore the possible role of oxymatrine in Nrf2 pathway. The results indicated that the ischemic infarct and edema were significantly reduced in rats that received oxymatrine, with a corresponding improvement in neurological function after CI/R. In immunohistochemistry and Western blotting analyses, Nrf2 and hemeoxygenase-1 (HO-1) were up-regulated in ischemic cortex, beginning at 6 h, peaking at 48 h and declining at 72 h after CI/R. Intraperitoneal injection of oxymatrine inhibited the production of lipid peroxidation and increased the activities of Nrf2 and HO-1 in rats brain after CI/R. Taken together, these results suggest that oxymatrine administered systemically protected brain against focal ischemia-reperfusion damage at the early stage of stroke, and that activating Nrf2/HO-1 pathway may contribute to the neuroprotective action of oxymatrine in rat focal brain ischemia-reperfusion model. Thus, treatment of stroke with oxymatrine may prevent severe consequences after brain attack.
Previously we developed dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) for use in small interfering RNA (siRNA) therapy. In the present study, mammalian target of rapamycin (mTOR) expression in cancer cells was silenced with mTOR-siRNA (simTOR) formulated in TEPA-PCL modified with Ala-Pro-Arg-Pro-Gly (APRPG), a peptide having affinity for vascular endothelial growth factor receptor-1 (VEGFR-1). We investigated the effects of inhibition of mTOR, focusing on the differences between cells treated with simTOR and those with rapamycin in terms of Akt (ser473) phosphorylation and antiproliferative effects. Rapamycin treatment is known to induce Akt (ser473) phosphorylation which attenuates the antiproliferative effects of rapamycin. As a result, knockdown of mTOR did not alter or only slightly reduced Akt (ser473) phosphorylation in phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null (LNCaP and MDA-MB-468 cells) and PTEN-positive (DU 145 and MDA-MB-231) cells, although rapamycin induced Akt (ser473) phosphorylation of these cells. Rapamycin suppressed the growth of PTEN-null cells, in which the rapamycin-sensitive mTOR complex 1 (mTORC1) is excessively activated. On the other hand, rapamycin did not suppress the growth of PTEN-positive cells possibly through a negative feedback mechanism via the rapamycin-insensitive mTOR complex 2 (mTORC2) signaling pathway. In contrast, simTOR significantly suppressed the growth of cancer cells regardless of the presence of PTEN, possibly through inhibition of both mTORC1 and mTORC2. These results indicate that mTOR knockdown using APRPG-TEPA-PCL/simTOR is likely to be an effective strategy for cancer siRNA therapy.
It has been reported that treatment with β2 adrenergic receptor (β2AR) agonist bronchodilators may result in airway β2ARs internalization and cardiac muscle cells apoptosis. This could lead to the loss of pharmacological effect of β2AR agonists and increase adverse cardiovascular events in asthma patients receiving β2AR agonist therapy. Glycyrrhizin, the major bioactive component of licorice root extract, has been reported to exhibit protective effect on respiratory system. Here, we investigate the effects of glycyrrhizin against β2AR agonist salbutamol-induced receptor internalization and cell apoptosis. In our study, the live cell confocal imaging and fixed-cell enzyme-linked immunosorbent assay (ELISA) assay revealed that glycyrrhizin significantly inhibited salbutamol-induced surface β2AR internalization. The underlying mechanisms were then identified to be that glycyrrhizin could reduce the association of β2ARs with β-arrestins and clathrin heavy chain as well as the level of G protein-coupled receptor kinase (GRK) mediated phosphorylation of β2ARs. The inhibition of receptor internalization by glycyrrhizin further lead to stabilization of the β2AR mRNA and protein expression, thus amplified the transmembrane signaling via the β2ARs. We also proved that glycyrrhizin could profoundly attenuate salbutamol-induced early cellular apoptosis by regulating the expressions of B-cell lymphoma 2 (Bcl-2) family genes. Taken together, our results suggest that glycyrrhizin exhibits protective effects against β2AR agonist-induced receptor internalization and cell apoptosis. These findings might have practical implications for future strategies of combined application of glycyrrhizin with β2AR receptor agonists to improve the efficacy of bronchodilators in patients with asthma and chronic obstructive pulmonary disease (COPD).
Advanced glycation end products (AGEs) have been shown to induce the proliferation of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis and diabetes. In the present study, we investigated the effects of pioglitazone, a peroxisome proliferator activated receptor gamma (PPARγ) agonist, on AGE-induced rat VSMC growth and the underlying mechanism. In cultured rat VSMCs, AGE treatment induced VSMC proliferation in time- and dose-dependent manner, while down-regulated the expression of PPARγ. Pretreatment of pioglitazone not only prevented the down-regulation of PPARγ, but inhibited VSMC proliferation and prevented S-phase entry of cell via a G0–G1 block in the presence of AGEs. Western blotting analysis showed that AGE treatment potentiated to activate extracelluar signal-regulated kinases (ERK1/2) by the induction of intracellular reactive oxygen species (ROS) production, since ROS scavenger N-acetyl-L-cysteine pretreatment significantly inhibited AGE-induced ERK1/2 activation. Further, pretreatment with either N-acetyl-L-cysteine or the inhibitor of ERK1/2 activation suppressed AGE-induced proliferation of VSMCs, suggesting a role of ROS/ERK1/2 signaling. Notably, we demonstrated that pretreatment of pioglitazone significantly attenuated AGE-induced ROS and ERK1/2 activation. Collectively, these results suggest that pioglitazone inhibits AGE-induced VSMC proliferation via increasing PPARγ expression and inhibiting ROS/ERK1/2 signaling pathway.
Surface expression levels of high-affinity immunoglobulin E (IgE) receptors (FcεRI) on mast cells are regulated by constitutive internalization from the plasma membrane, which is thought to be an important determinant of FcεRI-mediated signaling potential. However, molecular mechanism of FcεRI trafficking has remained poorly understood. Rab proteins are small guanosine 5′-triphosphatases (GTPases) involved in the regulation of membrane traffic. In particular, Rab5 has been shown to regulate transport in the early endocytic pathway, whereas it is not known whether the FcεRI surface expression levels are regulated by Rab5. In this study, we investigated the role of individual Rab5 isoforms in mast cells by small interfering RNA knockdown method. Our results demonstrate that Rab5a knockdown enhanced FcεRI-dependent mast cell activation and upregulated FcεRI surface expression in its steady state. In contrast, Rab5c knockdown caused suppression of the activation. These findings revealed modulatory and individual roles of Rab5 isoforms in mast cell functions.
Cerebral ischemia causes the depletion of oxygen and nutrition from brain tissues, and when persistent, results in irreversible damage to the cell function and survival. The cellular response to ischemic conditions and its mechanisms have been investigated widely in in vivo and in vitro experimental models, yet no study has addressed the response of a whole neuronal network to energy deprivation with the single-cell resolution. Observations at the level of network are necessary, because the activity of individual neurons is nonlinearly integrated through a network and thereby gives rise to unexpectedly complex dynamics. Here we used functional multineuron calcium imaging (fMCI), an optical recording technique with high temporal and spatial resolution, to visualize the activity of neuron populations in hippocampus CA1 region under ischemia-like conditions ex vivo. We found that, although neurons responded to oxygen and glucose deprivation with an increase in the event frequency, they maintained an asynchronous network state. This is in contrast with other well known pathological states, in which the network hyperexcitability is usually accompanied by an increase in synchrony. We suggest that under ischemic conditions, at least to some time point, the neuronal network maintains the excitatory and inhibitory balance as a whole, whether actively or as a consequence of the cellular response to energy deprivation.
Genome plasticity is a hallmark of Candida albicans and is believed to be an adaptation strategy. But the extent of such genomic variability is not well investigated. In this study, genetic contents of clinical C. albicans isolates were investigated at whole-genome level with array-based comparative genomic hybridization (array CGH) technology. It was revealed that C. albicans possessed variations of genetic contents, as well as aneuploidy. The variable genes were scattered across the chromosomes, as well clustered in particular regions, including sub-telomeric regions, retrotransposon-insertion sites and a variable region on chromosome 6.
Vibrio vulnificus possesses multiple iron-uptake systems which are mediated by VuuA (vulnibactin receptor), IutA (aerobactin receptor) and HupA (heme receptor). In this study, we determined the effect of a mutation of luxS encoding autoinducer-2 (AI-2) synthase on the expressions of the three receptors. A mutation and an in trans complementation of luxS did not affect the growing ability of V. vulnificus in iron-deficient conditions. Nevertheless, the luxS mutation slightly decreased vuuA expression, but slightly increased iutA and hupA expressions in the transcriptional reporter assay or Western blot analysis. These changes were all recovered by the luxS complementation. These results suggest that AI-2-mediated quorum sensing system may be involved in the fine modulation of V. vulnificus iron-uptake systems, positively affecting vuuA expression but negatively affecting iutA and hupA expressions.
The oral cavity contains almost half of the commensal bacterial population present in the human body. An increase in the number of these microorganisms may result in systemic diseases such as infective endocarditis and aspiration pneumonia as well as oral infections. It is essential to control the total numbers of these microorganisms in order to suppress disease onset. Thus, we examined the antimicrobial activity of a newly developed gel-entrapped catechin (GEC) preparation against oral microorganisms. The minimum inhibitory concentration (MIC) of GEC was determined based on the relationship between a modified agar diffusion method and a broth microdilution method. GEC inhibited the growth of the Actinomyces, periodontopathic bacteria and Candida strains tested, but did not inhibit the growth of the oral streptococci that are important in the normal oral flora. Commercially available moisture gels containing antimicrobial components showed antimicrobial activity against all of the tested strains. After a series of washes and after a 24-h incubation, GEC retained the antimicrobial activity of the catechins. Catalase prevented GEC-induced growth inhibition of Actinomyces naeslundii and Streptococcus mutans suggesting that hydrogen peroxide may be involved in the antimicrobial activity of catechins. These results suggest that GEC may be useful for controlling oral microorganism populations and reducing the accumulation of dental plaque, thereby helping to prevent periodontal disease and oral candidiasis.
The diabetic “lipotoxicity” hypothesis presents that fat-induced visceral white adipose tissue insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Berberine, a hypolipidemic agent, has been reported to have antidiabetic activities. The molecular mechanisms for this property are, however, not well clarified. Therefore in this study type 2 diabetic hamsters were induced by high-fat diet with low-dose streptozotocin. Then, we investigated the gene expression alterations and explored the molecular mechanisms underlying the therapeutic effect of berberine on fat-induced visceral white adipose tissue insulin resistance in diabetic hamsters by microarray analysis followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmation. Type 2 diabetic hamsters exhibited hyperglycemia and relative hyperinsulinemia, glucose intolerance, insulin resistance, intra-adipocyte lipid accumulation, significant increase in body weight and visceral white adipose tissue weight, abnormal serum adipokines levels, and deleterious dyslipidemia. Furthermore, they had increased sterol regulatory element-binding proteins (SREBPs) expression and decreased liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) expression in visceral white adipose tissue. After 9-week berberine treatment, fat-induced insulin resistance and diabetic phenotype in type 2 diabetic hamsters were significantly improved. Compared with diabetic hamsters, expression of LXRs and PPARs significantly increased and SREBPs significantly decreased in visceral white adipose tissue from berberine-treated diabetic hamsters. These results suggest that altered visceral white adipose tissue LXRs, PPARs, and SREBPs transcriptional programs are involved in the therapeutic mechanisms of berberine on fat-induced visceral white adipose tissue insulin resistance in type 2 diabetic hamsters.
The anti-inflammatory agent etodolac is used worldwide and it has a good gastrointestinal safety profile. Etodolac consists of two enantiomers, S- and R-etodolac. Here, we investigated the beneficial activities of racemic etodolac and its enantiomers. First, we compared S- and R-etodolac in terms of their inhibition of cyclooxygenase (COX) activity in vitro and their suppression of paw swelling in adjuvant-induced arthritic rats. The COX-2 inhibitory and anti-inflammatory effects of etodolac were found to be due to the S-enantiomer. We previously reported that etodolac attenuates allodynia in a mouse model of neuropathic pain by a COX-2-independent mechanism [N. Inoue et al., J. Pharmacol. Sci., 109, 600—605 (2009)]. In the present study, we showed that the anti-allodynic effects of etodolac in mice were also due to the S-enantiomer. In addition, we investigated the ulcerogenic activity of racemic etodolac and its enantiomers. At high doses, racemic etodolac showed a lower gastric lesion index in rats than the equivalent dose of S-etodolac. In contrast, R-etodolac showed no ulcerogenic activity and even showed protection against HCl/ethanol-induced gastric damage in rats. In conclusion, S-etodolac exhibited anti-inflammatory effects mediated by COX-2 inhibition and anti-allodynic effects that were independent of COX-2 inhibition, while R-etodolac showed gastroprotective effects that may contribute to the low gastrointestinal toxicity of racemic etodolac. Our results show that each enantiomer plays a different role in the efficacy and gastrointestinal safety of etodolac.
Curcumin is a polyphenol that is commonly used for its perceived health benefits. However, the absorption efficacy of curcumin is too low to exhibit beneficial effects. We have successfully developed a highly absorptive curcumin dispersed with colloidal nano-particles, and named it THERACURMIN. The absorption efficacy of THERACURMIN was investigated and compared with that of curcumin powder. The area under the blood concentration–time curve (AUC) after the oral administration of THERACURMIN was found to be more than 40-fold higher than that of curcumin powder in rats. Then, healthy human volunteers were administered orally 30 mg of THERACURMIN or curcumin powder. The AUC of THERACURMIN was 27-fold higher than that of curcumin powder. In addition, THERACURMIN exhibited an inhibitory action against alcohol intoxication after drinking in humans, as evidenced by the reduced acetaldehyde concentration of the blood. These findings demonstrate that THERACURMIN shows a much higher bioavailability than currently available preparations. Thus, THERACURMIN may be useful to exert clinical benefits in humans at a lower dosage.
Theophylline-associated convulsions are frequently exacerbated by fever, but the mechanisms behind it are still not completely understood. We investigated whether N-methyl-D-aspartic acid (NMDA) and gamma aminobutyric acid (GABA) receptors are involved in aminophylline (theophylline-2-ethylenediamine)-induced convulsions that are augmented by heat exposure-induced hyperthermia in mice. Mice exposed to 33 °C temperatures for 2 h had significantly increased body temperature (0.94 °C). Heat exposure significantly decreased time required for the onset of convulsions induced by an intraperitoneal (i.p.) injection of aminophylline (300 mg/kg). The shortened time for onset of convulsions was blocked by the NMDA receptor antagonist dizocilpine (0.1, 0.3 mg/kg, i.p.). However, the GABAA receptor agonist muscimol (1, 2 mg/kg, i.p.) did not have any effect. The pro-convulsant action of NMDA (100—125 mg/kg, i.p.) was enhanced by the heat exposure of 33 °C. However, the pro-convulsant actions of picrotoxin (3—4 mg/kg, i.p.), a GABAA receptor antagonist, were not affected by increased temperatures. These results suggest that NMDA receptors in the brain play a role in aminophylline-induced convulsions, which are augmented by heat exposure-induced hyperthermia in mice.
A reduced incretin effect is one of the well-known characteristics of patients with type 2 diabetes, and impaired release of glucagon-like peptide-1 (GLP-1) has been reported to be at least partly involved. In this study, we investigated the effect of nateglinide on GLP-1 release in vivo and in vitro. The GLP-1 level in the portal blood at 20 min after oral administration of nateglinide to Wistar rats was about twice that in vehicle-treated rats. To clarify whether this effect of nateglinide was related to direct stimulation of intestinal cells, in vitro studies were performed using human intestinal L cells (NCI-H716). Nateglinide stimulated GLP-1 release in a concentration-dependent manner from 500 μM, along with transient elevation of the intracellular calcium level. However, diazoxide, nitrendipine, and dantrolene did not block this effect of nateglinide. In addition, the major metabolite of nateglinide, tolbutamide, and mitiglinide, all of which augment insulin secretion by the pancreatic islets, had no effect on GLP-1 release by this cell line. On the other hand, capsazepine significantly inhibited the promotion of GLP-1 release by nateglinide in a concentration-dependent manner. These findings indicate that nateglinide directly stimulates GLP-1 release by intestinal L cells in a KATP channel-independent manner. A novel target of nateglinide may be involved in increasing intracellular calcium to stimulate GLP-1 release, e.g., the transient receptor potential channels. Taken together, the present findings indicate that promotion of GLP-1 release from intestinal L cells may be another important mechanism by which nateglinide restores early-phase insulin secretion and regulates postprandial glucose metabolism.
Allylmercapto glutathione S-conjugate, S-allylmercapto-L-cysteine (SAMC), which is biotransformed from allyl sulfides and from naturally occurring water-soluble garlic derivatives, has been known to inhibit tumorigenesis. We found that SAMC was able to induce apoptosis in gastric cancer cells in vitro. We report that SAMC inhibited tumor growth rate by 31.36% and 37.78% at doses of 100 and 300 mg/kg, respectively. Apoptosis in the implanted tumor cells was manifested by apoptotic characteristics, including morphological changes of chromatin crescent, cell shrinkage and membrane blebbing. The apoptosis index of 100 mg/kg and 300 mg/kg of SAMC was 20.74±2.50% and 30.61±2.42%, respectively, by terminal deoxy-nucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining. The positive rate of B-cell lymphoma 2 (bcl-2) protein expression of control, 100 mg/kg SAMC and 300 mg/kg SAMC was 15.20±1.67%, 10.94±1.57%, and 8.24±1.07%, respectively, by immunohistochemical staining. The positive rate of bax protein expression of control, 100 mg/kg SAMC and 300 mg/kg SAMC was 15.30±1.90%, 23.18±1.81%, and 25.26±3.03%, respectively. We also observed decreases in bcl-2 mRNA and increases in bax mRNA by SAMC in a dose-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that SAMC may regulate bcl-2 and bax to induce apoptosis in transplanted tumor cells.
We investigated the effects of dexamethasone on epidermal growth factor (EGF)-induced DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Isolated hepatocytes were cultured at a density of 3.3×104 cells/cm2 in Williams' medium E containing 5% bovine calf serum and various concentrations of dexamethasone for 1, 2 and 3 h. After the 3-h attachment period, the medium was changed, and cells were cultured in serum-free and dexamethasone-free Williams' medium E with or without glucocorticoid receptor antagonists. The growth-stimulating effects of EGF (20 ng/ml) on the primary cultured hepatocytes were time- and concentration-dependently inhibited by dexamethasone added to the culture medium. The mineral corticoid aldosterone (10−7 M) did not produce the same growth-inhibitory effects as dexamethasone (10−8 M). The inhibitory effects of dexamethasone were reversed by treatment with the glucocorticoid receptor antagonist mifepristone (RU486, 10−6 M) or a monoclonal antibody against glucocorticoid receptor (100 ng/ml). In addition, the growth-inhibitory effects of dexamethasone did not affect EGF-induced p42 mitogen-activated protein (MAP) kinase phosphorylation. These results indicate that dexamethasone concentration-dependently delays and inhibits the EGF-induced DNA synthesis and proliferation through its own intracellular receptor in primary cultures of adult rat hepatocytes.
We investigated the disposition of ARTCEREB® irrigation and perfusion solution (Artcereb) during intrathecal perfusion in a lateral ventricle-cisternal perfusion model in conscious rats. In this perfusion model, the perfusion rate was set at 0.35 ml/kg/h, taking into consideration the clinical perfusion rate (500 ml/60 kg/d). The influence of Artcereb on electrolytes in cerebrospinal fluid (CSF) and blood were then investigated. After 24 h of ventriculocisternal perfusion with Artcereb using the push-pull method, output of K+, Na+ and Cl− to the cistern magna was very similar to input of these electrolytes in Artcereb infused intraventricularly. Recovery rates of K+, Na+ and Cl− after perfusion were 102%, 105% and 100% when calculated using the recovered perfusion solution. In addition, concentrations of K+, Na+ and Cl− in blood remained almost constant at near baseline levels throughout perfusion. Thus, intrathecally perfused Artcereb did not affect electrolyte balance in the CSF and blood. To confirm the dynamics of Artcereb distribution, a whole body autoradiography study was performed at 1 and 6 h after perfusion with 14C-inulin-added Artcereb. Radioactivity was detected in the entire CSF space of the brain, and the cribriform plate in the nasal cavity, and the cerebrospinal cavity. Radioactivity was observed in the bladder, thus suggesting that some 14C-inulin was transferred to the bloodstream via a physiological route, and was excreted renally.
It is well known that metabolic syndrome (MS) is a risk factor for proteinuria and chronic kidney disease. Losartan (angiotensin II receptor blocker, ARB) and pioglitazone (peroxisome proliferator-activated receptor-γ, PPARγ agonist) have been shown to confer renoprotection. However, to date, whether or not an ARB and a PPARγ agonist have synergistic renoprotective effects remains controversial. Thus, the present study was designed to evaluate a combined treatment with losartan and pioglitazone in Sprague-Dawley rats fed with a high-fat, high-salt (HFS) diet and 20% sucrose solution for 16 weeks, an animal model of MS accompanying with renal lesions. Losartan, pioglitazone, and their combination were orally administered in the MS rats from 8 weeks to the end of this study. At 16 weeks, the MS rats showed the elevation in systolic blood pressure (SBP), urinary albumin excretion (UAE), and glomerulosclerosis (GS) score, but creatinine clearance, urinary protein excretion, and score of tubulointerstitial damage were not affected. Renal vascular endothelial growth factor (VEGF) protein level, mRNA and protein expression, which were respectively measured by enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis, were obviously decreased in the MS rats. Treatment with the combination of losartan and pioglitazone provided synergistic effects in reducing the SBP, UAE, and GS score when compared with monotherapy. These effects were not associated with ameliorated the downregulation of renal VEGF expression. Our data suggest that combined treatment with losartan and pioglitazone may offer additional advantages in treating MS nephropathy.
Glucocorticoids have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing their proliferation. Using human osteoblastic SaM-1 cells, we examined whether the effects of hydrocortisone on cellular proliferation are mediated by hepatocyte growth factor (HGF). Human osteoblasts constitutively express both HGF and c-Met, its receptor. Hydrocortisone decreased the gene and protein expression of HGF as well as proliferation in SaM-1 cells. These hydrocortisone (0.01—1 μM)-induced decreases in HGF synthesis and cellular proliferation occurred in a concentration-dependent manner. However, no hydrocortisone (0.01—1 μM)-induced decrease in cellular proliferation was observed in human osteosarcoma-derived cells (HOS and SaOS-2), which are not able to produce HGF. In the cellular proliferation in SaM-1 cells, the decrease was blocked concentration-dependently by exogenously applied HGF (0.01—3 ng/ml). Furthermore, SU11274 (1 μM), a highly specific inhibitor of c-Met, suppressed the proliferation of SaM-1 cells, but not HOS cells. From these results, we concluded that hydrocortisone inhibits the proliferation of SaM-1 cells by interrupting the autocrine/paracrine loop via the downregulation of HGF synthesis.
Methanol extract of Koreana stewartia leaves (SKE) stimulated collagen production in ultraviolet-B (UVB)-irradiated human fibroblast cells. An active compound was isolated from SKE by successive partitioning and chromatography, and the chemical structure was determined to be 3-O-β-D-glucopyranosylspinasterol (spinasterol-Glc) by spectroscopic characterization. Spinasterol-Glc increased collagen production in the supernatant of UVB-irradiated dermal fibroblast cell cultures in a dose-dependent manner. The effects of spinasteol-Glc on expression of procollagen and matrix metalloproteinase-1 (MMP-1) were further evaluated. We found that the compound stimulated collagen production in UVB-treated fibroblasts than in vehicle-treated control cells by about 3-fold. In addition, we also demonstrate that the compound increased the mRNA and protein levels of procollagen in UVB-treated fibroblast cells, while it inhibited expression of MMP-1. These results indicate that spinasterol-Glc protects fibroblast cells from the adverse effects of UV radiation via stimulation of procollagen synthesis as well as inhibition of MMP-1 expression. Spinasterol-Glc may be useful in the future development of therapeutic and cosmetic applications.
To elucidate the altered function of the lower esophageal sphincter (LES) in gastroesophageal reflux disease (GERD), we evaluated the motility proximal to LES using force transducers, contraction and relaxation responses to neurotransmitters in LES strips, and gene expression of neurotransmitter receptors in GERD rats. Force transducers were applied to the proximal LES, and contraction of the LES was monitored during free moving. In addition, LES was isolated from sham-operated and GERD rats to investigate the LES function in an organ bath, and to determine gene expression. The in vivo motility proximal to LES (% motility index) in conscious rats was decreased by atropine treatment and increased by cisapride (5-HT4 receptor agonist) treatment. Acetylcholine- and serotonin (5-HT)-induced LES contraction and sodium nitroprusside-induced relaxation in LES strips of GERD rats markedly decreased compared to sham-operated rats. The mRNA expressions of 5-HT4 and muscarinic acetylcholine 3 receptors were significantly reduced in esophageal LES strips of GERD rats compared with sham-operated rats. Intraperitoneal administration of cisapride improves the erosive damage in the esophagus in GERD rats. It is suggested that the reduction of 5-HT-induced contraction in LES strips in GERD rats may be partly due to the decrease in 5-HT4-receptor activation. The reduction of LES function may be due to the decrease in neurotransmitters signal transduction, leading to the deterioration of histopathological damage in GERD.
It is known that gentamicin (GM) could be a possible treatment for Duchenne Muscular Dystrophy (DMD). However, GM therapy has been hindered by several problems such as severe side effects of GM. In order to resolve these problems, we developed the drug delivery system (DDS) of GM using hybrid liposomes (HL) composed of L-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23) lauryl ether (C12(EO)23). The hydrodynamic diameters of HL including GM (GM-HL) were 60—90 nm with a narrow range of the size distribution and the sizes were kept almost constant for over 4 weeks, suggesting that GM-HL could avoid the reticuloendothelial system in vivo. Furthermore, GM-HL accumulated more to the skeletal muscle cells of X chromosome-linked muscular distrophy (mdx) mice as compared to those of normal mice. Significantly, we succeeded in increasing dystrophin positive fibers in skeletal muscle cells of mdx mice using GM-HL along with the reduction of ototoxicity. It is suggested that GM should be carried more efficiently into the muscular cells of mdx mice by HL. These results indicate that HL could be an effective carrier in the DDS of GM therapy for DMD.
Ephedrae herba has been used for treating colds, relieving coughs and asthma from ancient times. We previously reported the distribution of Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and Ephedra sp. in Mongolia, and among them E. sinica and E. equisetina were potential new resources of Ephedrae herba of Japanese pharmacopoeia grade, based on our field survey and subsequent molecular and chemical assessments. However, the Ephedra population in southwestern areas showed a high possibility of having hybrid origins. Further field surveys in southwestern areas, and sequence analysis of the partial nuclear internal transcribed spacer 1 (ITS1) region, besides trnK and 18S ribosomal RNA (rRNA) gene regions, were conducted in order to obtain detailed evidence of hybridization status. As a result, the distribution of E. glauca in western area and E. lomatolepis in western-most area was confirmed. The ITS sequences from all 8 Ephedra species collected in Mongolia were roughly divided into 5 types (types I—V). Type II sequence, having several additive nucleotides, was found in Ephedra sp., E. glauca, E. regeliana and E. sinica, which provided useful information for tracing hybrid origins. Morphological, genetic and distribution evidence suggested that the hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii and E. intermedia were involved in these events. Integrated with our previous report, trnK-, 18S- and ITS-types from pure lines of each species are proposed. In addition, we propose a practicable method for detecting additive peaks on a direct sequencing electropherogram.
Signal transducer and activator of transcription 3 (STAT3) plays an important role in regulating interleukin 6 (IL-6) related growth control of the liver. Our previous study demonstrated that a mixture containing Scutellaria baicalensis and Bupleurum scorzonerifolfium (S/B remedy) modulated the growth of hepatocytes during liver regeneration after 2/3 partial hepatectomy. The aim of this study was to investigate whether S/B remedy induced mouse hepatic STAT3 activation directly in hepatocytes or indirectly via non-parenchymal cell–hepatocyte interaction. Direct S/B remedy effects were studied using primarily isolated hepatocytes; while C57BL/6J mice were used to study indirect effects of S/B remedy using gadolinium chloride to deplete Kupffer cells' function. The results showed that S/B remedy and its active constituents did not directly activate growth-related signaling in primarily isolated hepatocytes. However, S/B remedy induced STAT3 and subsequently suppressor of cytokine signaling (SOCS3) activation in mouse liver and increased serum IL-6 level in a dose-dependent manner, which could be partially blocked by pretreatment with gadolinium chloride. Oligonucloetide microarray analysis from S/B remedy-treated peripheral blood leukocytes demonstrated an up-regulation of IL-6 gene expression. We conclude that S/B remedy did not directly induce STAT3 activation in vitro, but induced hepatic IL-6 related STAT3 activation through non-parenchymal cell-hepatocyte interaction in vivo. The results provide important information on the molecular mechanisms of S/B remedy for treatment of human liver diseases.
Cyclooxygenase enzymes (COX-1 and COX-2) catalyse the production of prostaglandins from arachidonic acid. Prostaglandins are important mediators in the inflammatory process and their production can be reduced by COX-inhibitors. Endocannabinoids, endogenous analogues of the plant derived cannabinoids, occur normally in the human body. The Endocannabinoids are structurally similar to arachidonic acid and have been suggested to interfere with the inflammatory process. They have also been shown to inhibit cancer cell proliferation. Anti-inflammatory effects of cannabinoids and endocannabinoids have been observed, however the mode of action is not yet clarified. Anti-inflammatory activity (i.e., inhibition of COX-2) is proposed to play an important role in the development of colon cancer, which makes this subject interesting to study further. In the present work, the six cannabinoids tetrahydrocannabinol (Δ9-THC), tetrahydrocannabinolic acid (Δ9-THC-A), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabigerolic acid (CBGA), isolated from Cannabis sativa, were evaluated for their effects on prostaglandin production. For this purpose an in vitro enzyme based COX-1/COX-2 inhibition assay and a cell based prostaglandin production radioimmunoassay were used. Cannabinoids inhibited cyclooxygenase enzyme activity with IC50 values ranging from 1.7·10−3 to 2.0·10−4 M.
Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).
The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis–Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the Km and Vmax values for the formation of 6-HNA in humans and rats were 640.0±30.9 and 722.9±111.7 μM, and 1167.5±33.0 and 1312.7±73.8 pmol min−1 mg protein−1, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.
Recently, transnasal drug delivery has attracted a great deal of attention as an administration route to deliver drugs directly to the central nervous systems (CNS) and drug targeting of the CNS is expected to increase. In the present study, we investigated the possibility of using a transnasal delivery system for milnacipran, a serotonin–noradrenaline reuptake inhibitor (SNRI), by evaluating the transport to the systemic circulation and cerebrospinal fluid (CSF) and the pharmacological effect after intranasal (i.n.) administration. Moreover, the effect of chitosan as a bioadhesive material on the transport to the systemic circulation and CSF and the pharmacological effect after i.n. administration were evaluated. As a result, i.n. administration of milnacipran was found to produce a higher direct delivery to the CNS as well as to the systemic circulation, suggesting that this is a promising route of administration and an alternative to peroral (p.o.) administration. Furthermore, the i.n. co-administration with chitosan led to increased plasma and CSF concentrations and an enhanced pharmacological effect, evaluated by means of the forced swimming test. The results suggested that chitosan produced a long residence time of milnacipran in the nasal cavity due to its bioadhesive effect, leading to the enhanced transport of milnacipran from the systemic circulation to the CNS via the blood–brain barrier by an increase in systemic absorption as well as direct transport to the CNS, resulting in a higher antidepressant effect compared to that with p.o. administration.
Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E2 (PGE2) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE2 secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE2 secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.
The aim of this study was to identify factors affecting the pharmacokinetics of mycophenolic acid (MPA) and its 7-O-glucuronide (MPAG) in systemic lupus erythematosus (SLE) patients. Thirty-one SLE patients in remission maintenance phase treated with mycophenolate mofetil (median 1500 mg/d) and prednisolone and followed-up for up to 56 months (median 13 months) were enrolled. Creatinine clearance and metal medication were significant predictors accounting for interindividual variability in the dose-normalized predose plasma concentration (C0) of MPA (adjusted R2=0.305, p=0.01) in a multivariate analysis. Dose-normalized MPAG C0 was significantly correlated with only creatinine clearance (adjusted R2=0.135, p=0.03). The free fraction of MPA was significantly correlated with only serum albumin (adjusted R2=0.700, p<0.01). The free fraction of MPAG was significantly correlated with serum albumin, metal medication, and age (adjusted R2=0.598, p=0.02). In conclusion, renal function and co-administered metal influenced the pharmacokinetics of MPA and MPAG in SLE patients in remission maintenance phase.