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1.  Effect of chitosan chewing gum on reducing serum phosphorus in hemodialysis patients: a multi-center, randomized, double-blind, placebo-controlled trial 
BMC Nephrology  2014;15:98.
HS219 (40 mg chitosan-loaded chewing gum) is designed to bind salivary phosphorus as an add-on to available phosphorus binders. We performed a randomized, placebo-controlled, double-blind study to evaluate the efficacy and safety of HS219 in hemodialysis (HD) patients with hyperphosphatemia as an add-on to phosphorus binders.
Sixty-eight HD patients who were maintained on calcium carbonate (n = 33) or sevelamer hydrochloride (n = 35) were enrolled. The primary end point was a change in serum phosphorus levels. Secondary end points included changes in levels of salivary phosphorus, serum calcium, parathyroid hormone (PTH), and intact fibroblast growth factor (iFGF) 23.
Sixty-three patients chewed either HS219 (n = 35) or placebo (n = 28) for 30 min, three times a day, for 3 weeks. HS219 was well tolerated and safe. However, HS219 was not superior to placebo with additional reduction of serum phosphorus with respect to phosphorus binders at the end of the chewing period. There were no significant effects of HS219 on reduction of salivary phosphorus, serum calcium, iPTH, or iFGF23 levels.
The chitosan-loaded chewing gum HS219 does not affect serum and salivary phosphorus levels in Japanese HD patients with hyperphosphatemia. Our findings do not support previous findings that 20 mg of chitosan-loaded chewing gum reduces serum and salivary phosphorus levels.
Trail registration NCT01039428, 24 December, 2009.
PMCID: PMC4080692  PMID: 24968790
Chewing gum; Chitosan; Clinical trial; Hemodialysis; Hyperphosphatemia; Phosphorus binders
2.  Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light 
Scientific Reports  2014;4:5223.
Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm2). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light.
PMCID: PMC4048889  PMID: 24909301
3.  Protective effects of bilberry and lingonberry extracts against blue light-emitting diode light-induced retinal photoreceptor cell damage in vitro 
Blue light is a high-energy or short-wavelength visible light, which induces retinal diseases such as age-related macular degeneration and retinitis pigmentosa. Bilberry (Vaccinium myrtillus L.) and lingonberry (Vaccinium vitis-idaea) contain high amounts of polyphenols (anthocyanins, resveratrol, and proanthocyanidins) and thus confer health benefits. This study aimed to determine the protective effects and mechanism of action of bilberry extract (B-ext) and lingonberry extract (L-ext) and their active components against blue light-emitting diode (LED) light-induced retinal photoreceptor cell damage.
Cultured murine photoreceptor (661 W) cells were exposed to blue LED light following treatment with B-ext, L-ext, or their constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin B2). 661 W cell viability was assessed using a tetrazolium salt (WST-8) assay and Hoechst 33342 nuclear staining, and intracellular reactive oxygen species (ROS) production was determined using CM-H2DCFDA after blue LED light exposure. Activation of p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor-kappa B (NF-κB), and LC3, an ubiquitin-like protein that is necessary for the formation of autophagosomes, were analyzed using Western blotting. Caspase-3/7 activation caused by blue LED light exposure in 661 W cells was determined using a caspase-3/7 assay kit.
B-ext, L-ext, NAC, and their active components improved the viability of 661 W cells and inhibited the generation of intracellular ROS induced by blue LED light irradiation. Furthermore, B-ext and L-ext inhibited the activation of p38 MAPK and NF-κB induced by blue LED light exposure. Finally, B-ext, L-ext, and NAC inhibited caspase-3/7 activation and autophagy.
These findings suggest that B-ext and L-ext containing high amounts of polyphenols exert protective effects against blue LED light-induced retinal photoreceptor cell damage mainly through inhibition of ROS production and activation of pro-apoptotic proteins.
PMCID: PMC3992157  PMID: 24690313
Anthocyanin; Bilberry; Blue LED light; Lingonberry; Proanthocyanidin; Resveratrol; Retinal photoreceptor
4.  Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation 
PLoS ONE  2014;9(1):e84387.
Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm2 (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm2 (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm2 (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.
PMCID: PMC3879303  PMID: 24392131
5.  The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells 
Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.
PMCID: PMC3876711  PMID: 24416064
6.  Involvement of Mincle and Syk in the changes to innate immunity after ischemic stroke 
Scientific Reports  2013;3:3177.
Accumulating evidence shows that post-ischemic inflammation originated by Toll-like receptors (TLR) plays critical roles in ischemic stroke. However, the functions of other innate immune receptors are poorly understood in cerebral ischemia. Macrophage-inducible C-type lectin, Mincle, is one of the innate immune receptor C-type lectin-like receptor (CLR) to response against dying cells. In the present study, we showed that Mincle, its ligand SAP130, and its downstream phospho-Syk/Syk were upregulated after ischemia, and that Mincle is expressed in immune and non-immune cells in the ischemic brains of mice and human. We treated mice with piceatannol, a Syk inhibitor, and consequently the infarct volume and swelling were suppressed by piceatannol. The levels of phospho-Syk, MMP9 and ICAM-1 were downregulated, and the level of Claudin5 was uplegurated in piceatannol-treated groups. These data indicate that innate immune system, such as Mincle and Syk plays a pivotal role in the pathogenesis after the ischemia and reperfusion.
PMCID: PMC3822396  PMID: 24212132
7.  The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment 
To improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice.
In vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells.
We found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells.
PGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.
PMCID: PMC3765381  PMID: 23972823
Cerebral ischemia-reperfusion; Inflammation; Progranulin; Neuroprotection; Neutrophil recruitment
8.  Mild Endoplasmic Reticulum Stress Promotes Retinal Neovascularization via Induction of BiP/GRP78 
PLoS ONE  2013;8(3):e60517.
Endoplasmic reticulum (ER) stress occurs as a result of accumulation of unfolded or misfolded proteins in the ER and is involved in the mechanisms of various diseases, such as cancer and neurodegeneration. The goal of the present study was to clarify the relationship between ER stress and pathological neovascularization in the retina. Proliferation and migration of human retinal microvascular endothelial cells (HRMEC) were assessed in the presence of ER stress inducers, such as tunicamycin and thapsigargin. The expression of ER chaperone immunoglobulin heavy-chain binding protein (BiP), known as Grp78, was evaluated by real time RT-PCR, immunostaining, and Western blotting. Tunicamycin or thapsigargin was injected into the intravitreal body of oxygen-induced retinopathy (OIR) model mice at postnatal day 14 (P14) and retinal neovascularization was quantified at P17. The expression and localization of BiP in the retina was also evaluated in the OIR model. Exposure to tunicamycin and thapsigargin increased the proliferation and migration of HRMEC. Tunicamycin enhanced the expression of BiP in HRMEC at both the mRNA level and at the protein level on the cell surface, and increased the formation of a BiP/T-cadherin immunocomplex. In OIR model mice, retinal neovascularization was accelerated by treatments with ER stress inducers. BiP was particularly observed in the pathological vasculature and retinal microvascular endothelial cells, and the increase of BiP expression was correlated with retinal neovascularization. In conclusion, ER stress may contribute to the formation of abnormal vasculature in the retina via BiP complexation with T-cadherin, which then promotes endothelial cell proliferation and migration.
PMCID: PMC3609792  PMID: 23544152
9.  Kinetics of neurodegeneration based on a risk-related biomarker in animal model of glaucoma 
Neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases progress slowly and steadily over years or decades. They show significant between-subject variation in progress and clinical symptoms, which makes it difficult to predict the course of long-term disease progression with or without treatments. Recent technical advances in biomarkers have facilitated earlier, preclinical diagnoses of neurodegeneration by measuring or imaging molecules linked to pathogenesis. However, there is no established “biomarker model” by which one can quantitatively predict the progress of neurodegeneration. Here, we show predictability of a model with risk-based kinetics of neurodegeneration, whereby neurodegeneration proceeds as probabilistic events depending on the risk.
We used five experimental glaucomatous animals, known for causality between the increased intraocular pressure (IOP) and neurodegeneration of visual pathways, and repeatedly measured IOP as well as white matter integrity by diffusion tensor imaging (DTI) as a biomarker of axonal degeneration. The IOP in the glaucomatous eye was significantly increased than in normal and was varied across time and animals; thus we tested whether this measurement is useful to predict kinetics of the integrity. Among four kinds of models of neurodegeneration, constant-rate, constant-risk, variable-risk and heterogeneity models, goodness of fit of the model and F-test for model selection showed that the time course of optic nerve integrity was best explained by the variable-risk model, wherein neurodegeneration kinetics is expressed in an exponential function across cumulative risk based on measured IOP. The heterogeneity model with stretched exponential decay function also fit well to the data, but without statistical superiority to the variable-risk model. The variable-risk model also predicted the number of viable axons in the optic nerve, as assessed by immunohistochemistry, which was also confirmed to be correlated with the pre-mortem integrity of the optic nerve. In addition, the variable-risk model identified the disintegrity in the higher-order visual pathways, known to underlie the transsynaptic degeneration in this disease.
These findings indicate that the variable-risk model, using a risk-related biomarker, could predict the spatiotemporal progression of neurodegeneration. This model, virtually equivalent to survival analysis, may allow us to estimate possible effect of neuroprotection in delaying progress of neurodegeneration.
PMCID: PMC3599096  PMID: 23331478
Diffusion tensor; Neurodegenerative mechanisms; Biomarker; Kinetic model; Glaucoma
10.  Pharmacological inhibition of TLR4-NOX4 signal protects against neuronal death in transient focal ischemia 
Scientific Reports  2012;2:896.
Recent data have shown that TLR4 performs a key role in cerebral ischemia-reperfusion injury which serves as the origin of the immunological inflammatory reactions. However, the therapeutic effects of pharmacological inhibitions of TLR4 and its immediate down-stream pathway remain to be uncovered. In the present study, on mice, intracerebroventricular injection of resatorvid (TLR4 signal inhibitor; 0.01 μg) significantly reduced infarct volume and improved neurological score after middle cerebral artery occlusion and reperfusion. The levels of phospho-p38, nuclear factor-kappa B, and matrix metalloproteinase 9 expressions were significantly suppressed in the resatorvid-treated group. In addition, NOX4 associates with TLR4 after cerebral ischemia-reperfusion seen in mice and human. Genetic and pharmacological inhibitions of TLR4 each reduced NOX4 expression, leading to suppression of oxidative/nitrative stress and of neuronal apoptosis. These data suggest that resatorvid has potential as a therapeutic agent for stroke since it inhibits TLR4-NOX4 signaling which may be the predominant causal pathway.
PMCID: PMC3508453  PMID: 23193438
11.  Laxative effects and mechanism of action of Brazilian green propolis 
Brazilian green propolis is reported to have wide range of biological properties including antibacterial, anti-inflammatory, anti-influenza, and antioxidant activities. In the digestive system, a protective effect of propolis on gastric ulcer has been reported, but a laxative effect has not yet been reported. We investigated the effect and the mechanism of action of water and ethanol extracts of Brazilian green propolis.
We examined the laxative effect of propolis on stool frequency by administering orally an ethanol extract of propolis (EEP) or a water extract of propolis (WEP) at 10, 50, 100, or 500 mg/kg to normal mice. We then investigated the effects of propolis using constipation model mice induced by two types of drugs, loperamide (a μ opioid receptor agonist) and clonidine (an α-2 adrenergic receptor agonist). We also investigated the effects of WEP on gastrointestinal transit and contractional tension of the ileum to uncover the mechanism of action of WEP.
Treatment with WEP, but not with EEP, significantly increased the weight of stools (p<0.01 at 500 mg/kg). WEP treatment significantly restored stool frequency and stool weight in clonidine-induced constipation model mice, but not in loperamide-induced constipation model mice. WEP treatment did not affect gastro-intestinal transit, but significantly increased the contractional tension of the isolated ileum of guinea pigs. This increase was inhibited by an acetylcholine receptor antagonist (atropine), but not by a 5-HT receptor antagonist (GR113808).
These findings indicate that WEP has laxative effects both in normal mice and in clonidine-induced constipation model mice. The laxative effects of WEP might be mediated by increased contractional tension of the ileum exerted at least in part via activation of an acetylcholine receptor.
PMCID: PMC3487869  PMID: 23088672
Propolis; Laxative; Acetylcholine receptor; Water extract
12.  The potential of GPNMB as novel neuroprotective factor in amyotrophic lateral sclerosis 
Scientific Reports  2012;2:573.
Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by the loss of motor neurons. Despite substantial research, the causes of ALS remain unclear. Glycoprotein nonmetastatic melanoma protein B (GPNMB) was identified as an ALS-related factor using DNA microarray analysis with mutant superoxide dismutase (SOD1G93A) mice. GPNMB was greatly induced in the spinal cords of ALS patients and a mouse model as the disease progressed. It was especially expressed in motor neurons and astrocytes. In an NSC34 cell line, glycosylation of GPNMB was inhibited by interaction with SOD1G93A, increasing motor neuron vulnerability, whereas extracellular fragments of GPNMB secreted from activated astrocytes attenuated the neurotoxicity of SOD1G93A in neural cells. Furthermore, GPNMB expression was substantial in the sera of sporadic ALS patients than that of other diseased patients. This study suggests that GPNMB can be a target for therapeutic intervention for suppressing motor neuron degeneration in ALS.
PMCID: PMC3417778  PMID: 22891158
13.  Diacylglycerol Kinase β Knockout Mice Exhibit Attention-Deficit Behavior and an Abnormal Response on Methylphenidate-Induced Hyperactivity 
PLoS ONE  2012;7(5):e37058.
Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. DGKβ is one of the subtypes of the DGK family and regulates many intracellular signaling pathways in the central nervous system. Previously, we demonstrated that DGKβ knockout (KO) mice showed various dysfunctions of higher brain function, such as cognitive impairment (with lower spine density), hyperactivity, reduced anxiety, and careless behavior. In the present study, we conducted further tests on DGKβ KO mice in order to investigate the function of DGKβ in the central nervous system, especially in the pathophysiology of attention deficit hyperactivity disorder (ADHD).
Methodology/Principal Findings
DGKβ KO mice showed attention-deficit behavior in the object-based attention test and it was ameliorated by methylphenidate (MPH, 30 mg/kg, i.p.). In the open field test, DGKβ KO mice displayed a decreased response to the locomotor stimulating effects of MPH (30 mg/kg, i.p.), but showed a similar response to an N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801 (0.3 mg/kg, i.p.), when compared to WT mice. Examination of the phosphorylation of extracellular signal-regulated kinase (ERK), which is involved in regulation of locomotor activity, indicated that ERK1/2 activation induced by MPH treatment was defective in the striatum of DGKβ KO mice.
These findings suggest that DGKβ KO mice showed attention-deficit and hyperactive phenotype, similar to ADHD. Furthermore, the hyporesponsiveness of DGKβ KO mice to MPH was due to dysregulation of ERK phosphorylation, and that DGKβ has a pivotal involvement in ERK regulation in the striatum.
PMCID: PMC3349656  PMID: 22590645
14.  Morphological and Functional Changes in the Retina after Chronic Oxygen-Induced Retinopathy 
PLoS ONE  2012;7(2):e32167.
The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment.
PMCID: PMC3279421  PMID: 22348151
15.  An Alteration in the Lateral Geniculate Nucleus of Experimental Glaucoma Monkeys: In vivo Positron Emission Tomography Imaging of Glial Activation 
PLoS ONE  2012;7(1):e30526.
We examined lateral geniculate nucleus (LGN) degeneration as an indicator for possible diagnosis of glaucoma in experimental glaucoma monkeys using positron emission tomography (PET). Chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the left eyes of 5 cynomolgus monkeys. Glial cell activation was detected by PET imaging with [11C]PK11195, a PET ligand for peripheral-type benzodiazepine receptor (PBR), before and at 4 weeks after laser treatment (moderate glaucoma stage). At mild, moderate, and advanced experimental glaucoma stages (classified by histological changes based on the extent of axonal loss), brains were stained with cresyl violet, or antibodies against PBR, Iba-1 (a microglial marker), and GFAP (an activated astrocyte marker). In laser-treated eyes, IOP was persistently elevated throughout all observation periods. PET imaging showed increased [11C]PK11195 binding potential in the bilateral LGN at 4 weeks after laser treatment; the increase in the ipsilateral LGN was statistically significant (P<0.05, n = 4). Immunostaining showed bilateral activations of microglia and astrocytes in LGN layers receiving input from the laser-treated eye. PBR-positive cells were observed in LGN layers receiving input from laser-treated eye at all experimental glaucoma stages including the mild glaucoma stage and their localization coincided with Iba-1 positive microglia and GFAP-positive astrocytes. These data suggest that glial activation occurs in the LGN at a mild glaucoma stage, and that the LGN degeneration could be detected by a PET imaging with [11C]PK11195 during the moderate experimental glaucoma stage after unilateral ocular hypertension. Therefore, activated glial markers such as PBR in the LGN may be useful in noninvasive molecular imaging for diagnosis of glaucoma.
PMCID: PMC3267730  PMID: 22299044
16.  Unoprostone reduces oxidative stress- and light-induced retinal cell death, and phagocytotic dysfunction, by activating BK channels 
Molecular Vision  2011;17:3556-3565.
Unoprostone isopropyl (unoprostone) is a docosanoid currently used as an antiglaucoma agent. Unoprostone is known to have neuroprotective effects and to activate large conductance Ca2+-activated K+ (BK) channels. Recently, unoprostone has been tested in clinical studies as a therapeutic agent for retinitis pigmentosa (RP) and studies have demonstrated an improvement in retinal sensitivity and in the protection of central retinal sensitivity with its use. However, the mechanism of action underlying unoprostone’s protective effect in RP is not fully known. It is well known that the pathogenesis of RP can be accelerated by oxidative stress or light irradiation. Therefore, the current study investigated the effects and the underlying mechanism of action of unoprostone on oxidative stress- and light irradiation-induced damage in photoreceptor and retinal pigment epithelial cultures.
The study used the mouse retinal cone-cell line 661W to investigate the effects of unoprostone and its major metabolite, unoprostone-free acid (M1), on oxidative stress- or light irradiation-induced cell death, and a human retinal pigment epithelial cell line (ARPE-19), was used to investigate the effects on light-induced disruption of phagocytotic function in a latex bead assay. Additionally, we examined whether the effects of unoprostone and M1 were mediated by BK channels using iberiotoxin, a selective inhibitor of BK channels.
Unoprostone and M1 protected against light- or H2O2-induced cell death in 661W cells, and against light-induced phagocytotic dysfunction in ARPE-19 cells. Additionally, iberiotoxin inhibited the protective effects of unoprostone and M1.
These findings indicate that unoprostone has protective effects on oxidative stress- and light irradiation-induced damage in vitro and that these effects are mediated by activation of BK channels. This confirms that unoprostone represents a promising therapeutic agent for the treatment of RP and other retinal diseases.
PMCID: PMC3250378  PMID: 22219651
17.  An Inducer of VGF Protects Cells against ER Stress-Induced Cell Death and Prolongs Survival in the Mutant SOD1 Animal Models of Familial ALS 
PLoS ONE  2010;5(12):e15307.
Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and recent evidence has suggested that endoplasmic reticulum (ER) stress signaling is involved in the pathogenesis of ALS. Here we identified a small molecule, SUN N8075, which has a marked protective effect on ER stress-induced cell death, in an in vitro cell-based screening, and its protective mechanism was mediated by an induction of VGF nerve growth factor inducible (VGF): VGF knockdown with siRNA completely abolished the protective effect of SUN N8075 against ER-induced cell death, and overexpression of VGF inhibited ER-stress-induced cell death. VGF level was lower in the spinal cords of sporadic ALS patients than in the control patients. Furthermore, SUN N8075 slowed disease progression and prolonged survival in mutant SOD1 transgenic mouse and rat models of ALS, preventing the decrease of VGF expression in the spinal cords of ALS mice. These data suggest that VGF plays a critical role in motor neuron survival and may be a potential new therapeutic target for ALS, and SUN N8075 may become a potential therapeutic candidate for treatment of ALS.
PMCID: PMC3000345  PMID: 21151573
18.  Phosphodiesterase-III Inhibitor Prevents Hemorrhagic Transformation Induced by Focal Cerebral Ischemia in Mice Treated with tPA 
PLoS ONE  2010;5(12):e15178.
The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA.
PMCID: PMC2997776  PMID: 21151895
19.  10-Hydroxy-2-decenoic Acid, a Major Fatty Acid from Royal Jelly, Inhibits VEGF-induced Angiogenesis in Human Umbilical Vein Endothelial Cells 
Vascular endothelial growth factor (VEGF) is reported to be a potent pro-angiogenic factor that plays a pivotal role in both physiological and pathological angiogenesis. Royal jelly (RJ) is a honeybee product containing various proteins, sugars, lipids, vitamins and free amino acids. 10-Hydroxy-2-decenoic acid (10HDA), a major fatty acid component of RJ, is known to have various pharmacological effects; its antitumor activity being especially noteworthy. However, the mechanism underlying this effect is unclear. We examined the effect of 10HDA on VEGF-induced proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Our findings showed that, 10HDA at 20 µM or more significantly inhibited such proliferation, migration and tube formation. Similarly, 10 µM GM6001, a matrix metalloprotease inhibitor, prevented VEGF-induced migration and tube formation. These findings indicate that 10HDA exerts an inhibitory effect on VEGF-induced angiogenesis, partly by inhibiting both cell proliferation and migration. Further experiments will be needed to clarify the detailed mechanism.
PMCID: PMC2781774  PMID: 18955252
HUVECs; migration; proliferation; tube formation
20.  Proteomic approach with LCMS-IT-TOF identified an increase of Rab33B after transient focal cerebral ischemia in mice 
Several proteins are known to be markedly expressed in the brain during cerebral ischemia; however, the changes in protein profiles within the ischemic brain after an ischemic insult have not been fully elucidated. We studied the changes in the ischemic brain proteome after focal cerebral ischemia, induced by middle cerebral artery occlusion (MCAO) in mice.
LCMS-IT-TOF mass spectrometry was used to detect the changes in ischemic brain protein patterns after MCAO. We evaluated the protein expression detected in the ischemic area, by western blotting and immunohistochemistry.
Nine unique proteins were identified from the ischemic area at 10 h after ischemic insult. Among these proteins, we focused on Rab33b, a member of RAS oncogene family and we found that Rab33b was up-regulated in the ischemic striatum and the number of Rab33B-positive cells increased in a time-dependent manner. Rab33B colocalized with Iba-1 positive microglia in the ischemic area.
These findings suggest that LCMS-IT-TOF is useful for identifying changes in proteins after cerebral ischemia and that Rab33B is partially related to the pathogenesis of transient cerebral ischemia in mice.
PMCID: PMC3002892  PMID: 21092243
21.  Laxative effects of agarwood on low-fiber diet-induced constipation in rats 
Agarwood (Aquilaria sinensis), well known as incense in Southeast Asia, has been used as a digestive in traditional medicine. We investigated the laxative effects of an ethanol extract of agarwood leaves (EEA) in a rat model of low-fiber diet-induced constipation.
A set of rats was bred on a normal diet while another set was placed on a low-fiber diet to induce constipation. The laxative effect of agarwood was then investigated on both sets of rats.
Pretreatment of normal rats with single dose of EEA (600 mg/kg, p.o.) significantly increased frequency and weight of stools. Also, treatments with EEA (300 and 600 mg/kg, p.o.) for 14 days caused a significant increase in stool frequency and weight. Feeding of the animals with a low-fiber diet resulted in a decrease in stool weight, frequency, and water content and also delayed carmine egestion. A single treatment with EEA (600 mg/kg) or senna (150 and 300 mg/kg) significantly increased stool frequency, weight, and water content and also accelerated carmine egestion in the model rats. Once daily administrations of EEA (150 mg/kg), for 14 days, caused a significant increase in water content of stools. The higher doses of EEA (300 and 600 mg/kg) significantly increased frequency, weight, and water content of the stools while accelerating carmine egestion in the constipated rats. Senna (150 and 300 mg/kg) produced similar effect as the higher doses of EEA but, in addition, induced severe diarrhea.
These findings indicate that EEA has a laxative effect, without causing diarrhea, in a rat model of low-fiber diet-induced constipation. These findings suggest that EEA may be highly effective on constipation as a complementary medicine in humans suffering from life style-induced constipation.
PMCID: PMC2995776  PMID: 21078136
22.  Diacylglycerol Kinase β Knockout Mice Exhibit Lithium-Sensitive Behavioral Abnormalities 
PLoS ONE  2010;5(10):e13447.
Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DGKβ is widely distributed in the central nervous system, such as the olfactory bulb, cerebral cortex, striatum, and hippocampus. Recent studies reported that the splice variant at the COOH-terminal of DGKβ was related to bipolar disorder, but its detailed mechanism is still unknown.
Methodology/Principal Findings
In the present study, we performed behavioral tests using DGKβ knockout (KO) mice to investigate the effects of DGKβ deficits on psychomotor behavior. DGKβ KO mice exhibited some behavioral abnormalities, such as hyperactivity, reduced anxiety, and reduced depression. Additionally, hyperactivity and reduced anxiety were attenuated by the administration of the mood stabilizer, lithium, but not haloperidol, diazepam, or imipramine. Moreover, DGKβ KO mice showed impairment in Akt-glycogen synthesis kinase (GSK) 3β signaling and cortical spine formation.
These findings suggest that DGKβ KO mice exhibit lithium-sensitive behavioral abnormalities that are, at least in part, due to the impairment of Akt-GSK3β signaling and cortical spine formation.
PMCID: PMC2956634  PMID: 20976192
23.  Protective effects of a gastrointestinal agent containing Korean red ginseng on gastric ulcer models in mice 
Korean red ginseng (KRG) is a ginseng that has been cultivated and aged for 4-6 years or more, and goes through an extensive cleaning, steaming and drying process. KRG contains more than 30 kinds of saponin components and has been reported as having various biological properties, such as anti-fatigue action, immune restoration, and neurovegetative effect. The purpose of this study was to assess the effects of a KRG-containing drug (KRGCD) on gastric ulcer models in mice.
Stomach ulcers were induced by oral ingestion of hydrochloride (HCl)/ethanol or indomethacin. Treatment with KRGCD (30, 100, and 300 mg/kg, p.o.) occurred 1 hr before the ulcer induction. Effect of KRGCD on anti-oxidant activity and gastric mucosal blood flow with a laser Doppler flowmeter in mice stomach tissue was evaluated.
KRGCD (100 and 300 mg/kg, p.o.) significantly decreased ethanol- and indomethacin-induced gastric ulcer compared with the vehicle-treated (control) group. KRGCD (100 and 300 mg/kg) also decreased the level of thiobarbituric acid reactive substance (TBARS) and increased gastric mucosal blood flow compared with the control group.
These results suggest that the gastroprotective effects of KRGCD on mice ulcer models can be attributed to its ameliorating effect on oxidative damage and improving effect of gastric mucosal blood flow.
PMCID: PMC2936409  PMID: 20718962
24.  Essential Role of Neuron-Enriched Diacylglycerol Kinase (DGK), DGKβ in Neurite Spine Formation, Contributing to Cognitive Function 
PLoS ONE  2010;5(7):e11602.
Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKβ is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated.
Methodology/Principal Findings
We, therefore, developed DGKβ KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKβ. In addition, overexpression of DGKβ in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKβ, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKβ but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed.
These results demonstrate that membrane-localized DGKβ regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory.
PMCID: PMC2904696  PMID: 20657643
25.  Anti-inflammatory effect of bee pollen ethanol extract from Cistus sp. of Spanish on carrageenan-induced rat hind paw edema 
Bee pollen, a honeybee product, is the feed for honeybees prepared themselves by pollens collecting from plants and has been consumed as a perfect food in Europe, because it is nutritionally well balanced. In this study, we aimed to investigate the anti-inflammatory effect of bee pollen from Cistus sp. of Spanish origin by a method of carrageenan-induced paw edema in rats, and to investigate the mechanism of anti-inflammatory action and also to elucidate components involved in bee pollen extracted with ethanol.
The bee pollen bulk, its water extract and its ethanol extract were administered orally to rats. One hour later, paw edema was produced by injecting of 1% solution of carrageenan, and paw volume was measured before and after carrageenan injection up to 5 h. The ethanol extract and water extract were measured COX-1 and COX-2 inhibitory activities using COX inhibitor screening assay kit, and were compared for the inhibition of NO production in LPS-stimulated RAW 264.7 cells. The constituents of bee pollen were purified from the ethanol extract subjected to silica gel or LH-20 column chromatography. Each column chromatography fractions were further purified by repeated ODS or silica gel column chromatography.
The bee pollen bulk mildly suppressed the carrageenan-induced paw edema and the water extract showed almost no inhibitory activity, but the ethanol extract showed relatively strong inhibition of paw edema. The ethanol extract inhibited the NO production and COX-2 but not COX-1 activity, but the water extract did not affect the NO production or COX activities. Flavonoids were isolated and purified from the ethanol extract of bee pollen, and identified at least five flavonoids and their glycosides.
It is suggested that the ethanol extract of bee pollen show a potent anti-inflammatory activity and its effect acts via the inhibition of NO production, besides the inhibitory activity of COX-2. Some flavonoids included in bee pollen may partly participate in some of the anti-inflammatory action. The bee pollen would be beneficial not only as a dietary supplement but also as a functional food.
PMCID: PMC2906419  PMID: 20573205

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