Omega-3 polyunsaturated fatty acids protect human hepatoma cells from developing steatosis through FFA4 (GPR120).

Protective effect of omega-3 polyunsaturated fatty acids (n-3 PUFA) on non-alcoholic fatty liver disease has been demonstrated. FFA4 (also known as GPR120; a G protein-coupled receptor) has been suggested to be a target of n-3 PUFA. FFA4 expression in hepatocytes has also been reported from liver biopsies in child fatty liver patients. In order to assess the functional role of FFA4 in hepatic steatosis, we used an in vitro model of liver X receptor (LXR)-mediated hepatocellular steatosis. FFA4 expression was confirmed in Hep3B and HepG2 human hepatoma cells. T0901317 (a specific LXR activator) induced lipid accumulation and docosahexaenoic acid (DHA; a representative n-3 PUFA) inhibited lipid accumulation. This DHA-induced inhibition was blunted by treatment of AH7614 (a FFA4 antagonist) and by transfection of FFA4 siRNA. SREBP-1c (a key transcription factor of lipogenesis) was induced by treatment with T0901317, and SREBP-1c induction was also inhibited by DHA at mRNA and protein levels. DHA-induced suppression of SREBP-1c expression was also blunted by FFA4-knockdown. Furthermore, DHA inhibited T0901317-induced lipid accumulation in primary hepatocytes from wild type mice, but not in those from FFA4 deficient mice. In addition, DHA-induced activations of Gq/11 proteins, CaMKK, and AMPK were found to be signaling components of the steatosis protective pathway. The results of this study suggest that n-3 PUFA protect hepatic steatosis by activating FFA4 in hepatocytes, and its signaling cascade sequentially involves FFA4, Gq/11 proteins, CaMKK, AMPK, and SREBP-1c suppression.

Chrysin Attenuates VCAM-1 Expression and Monocyte Adhesion in Lipopolysaccharide-Stimulated Brain Endothelial Cells by Preventing NF-κB Signaling.

Adhesion of leukocytes to endothelial cells plays an important role in neuroinflammation. Therefore, suppression of the expression of adhesion molecules in brain endothelial cells may inhibit neuroinflammation. Chrysin (5,7-dihydroxyflavone) is a flavonoid component of propolis, blue passion flowers, and fruits. In the present study, we examined the effects of chrysin on lipopolysaccharide (LPS)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in mouse cerebral vascular endothelial (bEnd.3) cells. In bEnd.3 cells, LPS increased mRNA expression of VCAM-1 in a time-dependent manner, and chrysin significantly decreased LPS-induced mRNA expression of VCAM-1. Chrysin also reduced VCAM-1 protein expression in a concentration-dependent manner. Furthermore, chrysin blocked adhesion of monocytes to bEnd.3 cells exposed to LPS. Nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase, which are all activated by LPS, were significantly inhibited by chrysin. These results indicate that chrysin inhibits the expression of VCAM-1 in brain endothelial cells by inhibiting NF-κB translocation and MAPK signaling, resulting in the attenuation of leukocyte adhesion to endothelial cells. The anti-inflammatory effects of chrysin suggest a possible therapeutic application of this agent to neurodegenerative diseases, such as multiple sclerosis, septic encephalopathy, and allergic encephalomyelitis.

Benzo[a]pyrene-7,8-diol-9,10-epoxide inhibits gap junction intercellular communication via phosphorylation of tumor progression locus 2 in WB-F344 rat liver epithelial cells.

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), a major metabolite of benzo[a]pyrene, has been reported to function as a human carcinogen. However, the molecular mechanism of how B[a]PDE regulates signaling pathways during tumor promotion remains unclear. In this study, we investigated the effects of B[a]PDE on the regulation of gap junction intercellular communication (GJIC), one of the major carcinogenic processes, and its main regulatory signaling pathways using WB-F344 rat liver epithelial (WB-F344 RLE) cells. Treatment of benzo[a]pyrene or B[a]PDE resulted in GJIC inhibition, and B[a]PDE was more active at lower concentrations than benzo[a]pyrene in the suppression of GJIC. This suggests that B[a]PDE is a stronger GJIC inhibitor. B[a]PDE at 1 µM reversibly inhibited GJIC in WB-F344 RLE cells, which was attributable to hyperphosphorylation of connexin43 (Cx43) via phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). We found that B[a]PDE induced phosphorylation of tumor progression locus 2 (Tpl2), a direct upstream regulator of MEK. Tpl2 inhibitor recovered B[a]PDE-induced GJIC inhibition and attenuated B[a]PDE-induced MEK/ERK phosphorylation in WB-F344 RLE cells. Collectively, our results suggest that B[a]PDE suppresses GJIC by activating Tpl2 and subsequently the MEK/ERK pathway and Cx43 phosphorylation in WB-F344 RLE cells. These results outline the potential importance of Tpl2 as a novel therapeutic target for B[a]PDE-induced GJIC inhibition during cancer promotion.

The Na+/H+ exchanger-1 inhibitor cariporide prevents glutamate-induced necrotic neuronal death by inhibiting mitochondrial Ca2+ overload.

In the brain, Na+/H+ exchanger-1 (NHE-1) activation has a significant impact on ischemic injury, and, in recent studies, NHE-1 inhibition has been found to protect neurons from ischemic injury. This protective effect has been ascribed to the prevention of apoptosis, but neuronal cell death following ischemia is a consequence of both necrotic and apoptotic cell death. Here, we evaluated the ability of the potent NHE-1 inhibitor cariporide to prevent necrotic cell death in an in vitro model of excitotoxic neuronal death. Cariporide (100 nM) was found to reduce both glutamate-induced necrotic and apoptotic neuronal cell death. Ca2+ concentrations were observed to peak twice in cytosol and mitochondria in cultured neuronal cells after glutamate exposure, and cariporide was found to reduce the second Ca2+ concentration increase, but not the first. Furthermore, glutamate-mediated mitochondrial death pathways involving loss of mitochondrial membrane potential and reactive oxygen species (ROS) accumulation were found to be attenuated by cariporide. In addition, cariporide effectively prevented necrosis following exposure to glutamate and ameliorated the mitochondrial Ca2+ and ROS production increases implicated in necrotic cell death. These results suggest that NHE-1 participates in the necrotic cell death process and that its inhibition offers a means of preventing both necrosis and apoptosis.

Involvement of the adenosine A1 receptor in the hypnotic effect of rosmarinic acid.

Insomnia, the most common sleep disorder, is characterized by a longer sleep latency, greater sleep fragmentation, and consequent excessive daytime fatigue. Due to the various side effects of prescribed hypnotics, demand for new drugs is still high. Recent studies have suggested the adenosine receptor (AR) as a potential therapeutic target for insomnia, however, clinically useful hypnotics targeting AR are not yet available. In the present study, we evaluated the hypnotic effect of rosmarinic acid, a phenolic compound widely found in medicinal plants, through pentobarbital-induced sleep test, electroencephalography/electromyography (EEG/EMG), and immunohistochemistry in mice. The underlying mechanisms were assessed by pharmacological approach using 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and SCH5826, antagonists for A1R and A2AR, respectively. Receptor-binding assay and functional agonism were also performed. Our study provides a new evidence that rosmarinic acid has a direct binding activity (Ki = 14.21 ± 0.3 µM) and agonistic activity for A1R. We also found that rosmarinic acid significantly decreased sleep fragmentation and onset latency to NREM sleep, and these effects were abolished by DPCPX. The results from c-Fos immunostaining showed that rosmarinic acid decreased the neuronal activity in wake-promoting brain regions, such as the basal forebrain and the lateral hypothalamus, while increasing the neuronal activity in the ventrolateral preoptic nucleus, a sleep-promoting region; all these effects were significantly inhibited by DPCPX. Taken together, this study suggests that rosmarinic acid possesses novel activity as an A1R agonist and thereby exerts a hypnotic effect, and thus it may serve as a potential therapeutic agent for insomnia through targeting A1R.

Aß1-40-Induced Platelet Adhesion Is Ameliorated by Rosmarinic Acid through Inhibition of NADPH Oxidase/PKC-δ/Integrin αIIbß3 Signaling.

In platelets, oxidative stress reportedly increases platelet adhesion to vessels, thus promoting the vascular pathology of various neurodegenerative diseases, including Alzheimer's disease (AD). Recently, it has been shown that ß-amyloid (Aß) can increase oxidative stress in platelets; however, the underlying mechanism remains elusive. In the present study, we aimed to elucidate the signaling pathway of platelet adhesion induced by Aß1-40, the major form of circulating Aß, through Western blotting, immunofluorescence confocal microscopy, and fluorescence-activated cell sorting analysis. Additionally, we examined whether rosmarinic acid (RA), a natural polyphenol antioxidant, can modulate these processes. Our results show that Aß1-40-induced platelet adhesion is mediated through NADPH oxidase/ROS/PKC-δ/integrin αIIbß3 signaling, and these signaling pathways are significantly inhibited by RA. Collectively, these results suggest that RA may have beneficial effects on platelet-associated vascular pathology in AD.

Yuzu and Hesperidin Ameliorate Blood-Brain Barrier Disruption during Hypoxia via Antioxidant Activity.

Yuzu and its main component, hesperidin (HSP), have several health benefits owing to their anti-inflammatory and antioxidant properties. We examined the effects of yuzu and HSP on blood-brain barrier (BBB) dysfunction during ischemia/hypoxia in an in vivo animal model and an in vitro BBB endothelial cell model, and also investigated the underlying mechanisms. In an in vitro BBB endothelial cell model, BBB permeability was determined by measurement of Evans blue extravasation in vivo and in vitro. The expression of tight junction proteins, such as claudin-5 and zonula occludens-1 (ZO-1), was detected by immunochemistry and western blotting, and the reactive oxygen species (ROS) level was measured by 2'7'-dichlorofluorescein diacetate intensity. Yuzu and HSP significantly ameliorated the increase in BBB permeability and the disruption of claudin-5 and ZO-1 in both in vivo and in vitro models. In bEnd.3 cells, yuzu and HSP were shown to inhibit the disruption of claudin-5 and ZO-1 during hypoxia, and the protective effects of yuzu and HSP on claudin-5 degradation seemed to be mediated by Forkhead box O 3a (FoxO3a) and matrix metalloproteinase (MMP)-3/9. In addition, well-known antioxidants, trolox and N-acetyl cysteine, significantly attenuated the BBB permeability increase, disruption of claudin-5 and ZO-1, and FoxO3a activation during hypoxia, suggesting that ROS are important mediators of BBB dysfunction during hypoxia. Collectively, these results indicate that yuzu and HSP protect the BBB against dysfunction via maintaining integrity of claudin-5 and ZO-1, and these effects of yuzu and HSP appear to be a facet of their antioxidant properties. Our findings may contribute to therapeutic strategies for BBB-associated neurodegenerative diseases.

Downregulated Platelet miR-1233-5p in Patients with Alzheimer's Pathologic Change with Mild Cognitive Impairment is Associated with Aß-Induced Platelet Activation via P-Selectin.

MicroRNAs (miRNAs) have been proposed as a promising biomarker for various diseases including Alzheimer's disease (AD). More attention has recently been focused on the diagnosis and treatment at earlier stage of mild cognitive impairment (MCI) for preventing its progression to AD. To identify potential pathologic markers for Aß(+)MCI (Alzheimer's pathologic change with MCI), we investigated miRNA expression profiles in the platelets from patients with Aß(+)MCI, in comparison with those from Aß(-)MCI (Non-Alzheimer's pathologic change with MCI) and CNI (cognitively normal individuals). We found that let-7i-5p, miR-125a, miR-1233-5p, and miR-6787-5p were significantly downregulated, while miR-6880-5p expression was upregulated. Of these, only miR-1233-5p was significantly downregulated by Aß treatment in both human platelets and their precursor megakaryocytes (MEG-01 cells). We explored the role of miRNAs by using miRNA mimics or inhibitors, and found that the diminished level of miR-1233-5p was associated with Aß-induced increase in the expression of P-selectin and cell adhesion to fibronectin. Our results further indicated that Aß-induced increase in platelet/MEG adhesion to fibronectin is likely mediated via P-selectin. In conclusion, this study suggests the downregulation of platelet-derived miR-1233-5p as a pathologic marker for Aß(+)MCI.

Impact of Sleep Disorder as a Risk Factor for Dementia in Men and Women.

Sleep is an essential physiological process, especially for proper brain function through the formation of new pathways and processing information and cognition. Therefore, when sleep is insufficient, this can result in pathophysiologic conditions. Sleep deficiency is a risk factor for various conditions, including dementia, diabetes, and obesity. Recent studies have shown that there are differences in the prevalence of sleep disorders between genders. Insomnia, the most common type of sleep disorder, has been reported to have a higher incidence in females than in males. However, sex/gender differences in other sleep disorder subtypes are not thoroughly understood. Currently, increasing evidence suggests that gender issues should be considered important when prescribing medicine. Therefore, an investigation of the gender-dependent differences in sleep disorders is required. In this review, we first describe sex/gender differences not only in the prevalence of sleep disorders by category but in the efficacy of sleep medications. In addition, we summarize sex/gender differences in the impact of sleep disorders on incident dementia. This may help understand gender-dependent pathogenesis of sleep disorders and develop therapeutic strategies in men and women.

Differential regulation of the hydrogen-peroxide-induced inhibition of gap-junction intercellular communication by resveratrol and butylated hydroxyanisole.

The present study was performed to evaluate the effects of two different phenolic antioxidants, resveratrol (3,5,4'-trihydroxystilbene) and butylated hydroxyanisole (BHA), on the hydrogen peroxide (H2O2)-induced inhibition of gap-junction intercellular communication (GJIC) in WB-F344 rat liver epithelial cells (WB-F344). Resveratrol is a naturally occurring polyphenolic antioxidant; on the other hand, BHA is a synthetic phenolic compound. We found that only resveratrol protects WB-F344 cells from H2O2-induced inhibition of GJIC, and BHA has no effect. The extracellular-signal-regulated protein kinase 1/2 (ERK1/2)-connexin 43 (Cx43) signaling pathway is crucial for the regulation of GJIC in rat liver epithelial cells, and resveratrol, but not BHA, blocked the H2O2-induced phosphorylation of Cx43, a critical regulator of GJIC, and ERK1/2 in WB-F344 cells. Resveratrol appears to attenuate the H2O2-mediated ERK1/2-Cx43 signaling pathway and consequently reverses H2O2-mediated inhibition of GJIC. DPPH and ABTS radical-scavenging assays revealed that the protective effect of resveratrol on the H2O2-mediated inhibition of GJIC was not mediated through its free radical-scavenging activity.

Formulation And Evaluation Of Nanostructured Lipid Carriers (NLCs) Of 20(S)-Protopanaxadiol (PPD) By Box-Behnken Design.

BACKGROUND: 20(S)-Protopanaxadiol (PPD) has a higher anti-wrinkle effect than the other glycone forms of ginsenosides. However, as PPD has low solubility in water and a high molecular weight, it cannot easily penetrate the stratum corneum layer, which is the rate-limiting step of topical skin delivery. Thus, the objective was to enhance the topical skin deposition of PPD using an optimized nanostructured lipid carriers (NLC) formulation. NLC formulations were optimized using a Box-Behnken design. MATERIALS AND METHODS: NLC formulations were optimized using a Box-Behnken design, where the amount of PDD (X1), volume of the liquid lipid (X2), and amount of surfactant (X3) were set as the independent variables, while the particle size (Y1), polydispersity index (PDI) (Y2), and entrapment efficiency (EE) (Y3) were dependent factors. An in vitro deposition study was performed using Strat-M® and human cadaver skin, while in vivo skin irritation effect of the NLC formulation was evaluated in humans. RESULTS: An NLC was successfully prepared based on the optimized formulation determined using the Box-Behnken design. The particle size, PDI, and EE of the NLC showed less than 5% difference from the predicted values. The in vitro deposition of PPD after the application of the NLC formulation on a Strat-M® artificial membrane and human cadaver skin was significantly higher than that of the controls. Moreover, NLC formulations with and without PDD were not skin irritants in a human study. CONCLUSION: An NLC formulation for the topical delivery of PPD was successfully optimized using the Box-Behnken design, and could be further developed to enhance the topical skin deposition of PPD.

Vitamin D deficiency is associated with disease activity in patients with Crohn's disease.

BACKGROUND/AIMS: Previous data suggest that vitamin D has a significant role in inflammatory bowel disease (IBD). We investigated the incidence of vitamin D deficiency in Korean patients with IBD and the correlation between serum vitamin D level and disease activity. METHODS: We retrospectively analyzed the medical records of patients with IBD whose serum vitamin D levels were checked. Deficiency of 25-hydroxyvitamin D was defined as <20 ng/mL. Disease activity was evaluated using the partial Mayo score for ulcerative colitis (≥2 defined as active disease) and Harvey-Bradshaw index for Crohn's disease (≥4 defined as active disease). RESULTS: We enrolled 87 patients with IBD (UC, 45; CD, 42). Among them, 65.5% (57/87) were men, with a mean age of 44.9±15.1 years (range, 18-75 years). The mean duration of disease was 4.7±4.8 years (range, 0.1-17.1 years). Vitamin D deficiency was found in 73.6% (64/87) of patients with IBD. Patients with IBD (mean vitamin D level, 16.3±9.0 ng/mL) showed lower vitamin D level than the healthy control group (mean vitamin D level, 20.4±7.0 ng/mL), with no statistically significant difference (P=0.136). Disease activity was inversely correlated with vitamin D deficiency in patients with CD (P=0.007). However, no correlation was observed in patients with UC (P=0.134). CONCLUSIONS: Approximately 75% of Korean patients with IBD showed vitamin D deficiency state. Vitamin D deficiency is associated with disease activity, particularly in patients with CD.

Gallic acid, a metabolite of the antioxidant propyl gallate, inhibits gap junctional intercellular communication via phosphorylation of connexin 43 and extracellular-signal-regulated kinase1/2 in rat liver epithelial cells.

Propyl gallate and its metabolite, gallic acid, are widely used as antioxidants in the food industry, but they have been shown to exhibit liver toxicity and enhance carcinogenesis. In the present study, we investigated the possible undesirable effects of propyl gallate and gallic acid on gap junctional intercellular communication (GJIC), inhibition of which is closely linked to carcinogenesis. Gallic acid and propyl gallate exhibited dose-dependent free-radical-scavenging activities as determined by 1,1-diphenyl-2-picrylhydrazyl- or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-radical-scavenging assays, and the free-radical-scavenging activity of gallic acid was stronger than that of propyl gallate. However, using WB-F344 rat liver epithelial cells, gallic acid inhibited GJIC in a dose-dependent manner, while propyl gallate had no significant effect compared with untreated controls. The gallic-acid-induced inhibition of GJIC was reversible, with a recovery of nearly 65% after 120 min. Gallic acid induced the phosphorylation of connexin 43 (Cx43) and phosphorylation of extracellular-signal-regulated kinase1/2 (ERK1/2). The gallic-acid-induced inhibition of GJIC was attenuated by treatment with mitogen-activated protein kinase kinase inhibitors (U0126 and PD098059). U0126 blocked the gallic-acid-induced phosphorylation of Cx43 and ERK1/2, indicating that the gallic-acid-induced inhibition of GJIC is mediated by phosphorylation of Cx43 via activation of ERK1/2. In addition, gallic-acid-induced inhibition of GJIC was protected by ascorbic acid and quercetin, which might represent a simple example of the different effects of natural antioxidants in carcinogenesis.

Monitoring of algicidal bacterium, Alteromonas sp. strain A14 in its application to natural Cochlodinium polykrikoides blooming seawater using fluorescence in situ hybridization.

The red tide of dinoflagellate, Cochlodinium polykrikoides has frequently occurred in coastal waters, causing severe damage to fisheries. In the present study, the algicidal bacterium Alteromonas sp. A14 isolated from the southern coast of Korea was applied to a red tide of C. polykrikoides in a laboratory experiment. In the experiment, the abundance of the strain A14 was monitored using fluorescence in situ hybridization. Inoculation of the A14 at a final cell density of 9.0 x 10(5) cells/ml caused a significant decrease in C. polykrikoides abundance from 1,830 to 700 cells/ml during 2 days, while abundances of harmless diatoms rapidly increased from 3 days. Abundances of both A14 and other bacteria increased to 1 day. After 1 day, with flagellate abundance increased, bacterial abundance decreased. Finally, algicidal bacterial abundance decreased to 3.5 x 10(4) cells/ml. In the biological control of harmful algal blooms, in addition to decrease in target algal abundance and not occurrence of other harmful blooms, decrease in abundance of utilized organism is also important. This study emphasizes the importance of monitoring the inoculated bacterium when applying bacterium to natural seawater.

Linalool Ameliorates Memory Loss and Behavioral Impairment Induced by REM-Sleep Deprivation through the Serotonergic Pathway.

Rapid eye movement (REM) sleep has an essential role in the process of learning and memory in the hippocampus. It has been reported that linalool, a major component of Lavandula angustifolia, has antioxidant, anti-inflammatory, and neuroprotective effects, along with other effects. However, the effect of linalool on the cognitive impairment and behavioral alterations that are induced by REM-sleep deprivation has not yet been elucidated. Several studies have reported that REM-sleep deprivation-induced memory deficits provide a well-known model of behavioral alterations. In the present study, we examined whether linalool elicited an anti-stress effect, reversing the behavioral alterations observed following REM-sleep deprivation in mice. Furthermore, we investigated the underlying mechanism of the effect of linalool. Spatial memory and learning memory were assessed through Y maze and passive avoidance tests, respectively, and the forced swimming test was used to evaluate anti-stress activity. The mechanisms through which linalool improves memory loss and behavioral alterations in sleep-deprived mice appeared to be through an increase in the serotonin levels. Linalool significantly ameliorated the spatial and learning memory deficits, and stress activity observed in sleep-deprived animals. Moreover, linalool led to serotonin release, and cortisol level reduction. Our findings suggest that linalool has beneficial effects on the memory loss and behavioral alterations induced by REM-sleep deprivation through the regulation of serotonin levels.

Anti-allergic effects of sesquiterpene lactones from Saussurea costus (Falc.) Lipsch. determined using in vivo and in vitro experiments.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea costus (Falc.) Lipsch. root has been used in Asian traditional medicine for the treatment of asthma, rheumatism, and other conditions. S. costus extracts were shown to alleviate house dust mite-induced atopic-like dermatitis in Nc/Nga mice; besides, sesquiterpene lactones were isolated from S. costus extracts. AIMS OF THE STUDY: We aimed to investigate the effects of sesquiterpene lactones (alantolactone, costunolide, and dehydrocostuslactone) in allergic asthma using female Balb/c mice and rat RBL-2H3 mast cells. MATERIALS AND METHODS: Antigen-induced degranulation was assessed by measuring ß-hexosaminidase activity in vitro. In addition, a murine ovalbumin-induced allergic asthma model was used to test the in vivo efficacy of sesquiterpene lactones. RESULTS: Sesquiterpene lactones inhibited antigen-induced degranulation, wherein dehydrocostuslactone > costunolide > alantolactone in potency. Administration of sesquiterpene lactones decreased the number of immune cells, particularly eosinophils, and reduced the expression and secretion of Th2 cytokines (IL-4 and IL-13) in the bronchoalveolar lavage fluid and lung tissues of mice with ovalbumin-induced allergic asthma. Histological studies showed that sesquiterpene lactones reduced inflammation and mucin production in the lungs. Similar to the in vitro study, dehydrocostuslactone showed the highest potency, followed by costunolide and alantolactone. CONCLUSION: These findings provide evidence that sesquiterpene lactones might be potential anti-allergic therapeutics.

Suppressive Effect of 4-Hydroxy-2-(4-Hydroxyphenethyl) Isoindoline-1,3-Dione on Ovalbumin-Induced Allergic Asthma.

4-Hydroxy-2-(4-hydroxyphenethyl)isoindoline-1,3-dione (PD1) is a synthetic phthalimide derivative of a marine compound. PD1 has peroxisome proliferator-activated receptor (PPAR) γ agonistic and anti-inflammatory effects. This study aimed to investigate the effect of PD1 on allergic asthma using rat basophilic leukemia (RBL)-2H3 mast cells and an ovalbumin (OVA)-induced asthma mouse model. In vitro, PD1 suppressed ß-hexosaminidase activity in RBL-2H3 cells. In the OVA-induced allergic asthma mouse model, increased inflammatory cells and elevated Th2 and Th1 cytokine levels were observed in bronchoalveolar lavage fluid (BALF) and lung tissue. PD1 administration decreased the numbers of inflammatory cells, especially eosinophils, and reduced the mRNA and protein levels of the Th2 cytokines including interleukin (IL)-4 and IL-13, in BALF and lung tissue. The severity of inflammation and mucin secretion in the lungs of PD1-treated mice was also less. These findings indicate that PD1 could be a potential compound for anti-allergic therapy.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity.

The Na⁺/H⁺ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H⁺ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-ß (PKC-ß) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 µM glutamate or 20 mM NH4Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-ß, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for 〉3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH4Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-ß, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-ß/ERK1/2/p90RSK pathway in neuronal cells.

Anxiolytic effects of the aqueous extract of Uncaria rhynchophylla.

The purpose of this study was to characterize the putative anxiolytic-like effects of the aqueous extract of hooks with stem of Uncaria rhynchophylla using the elevated plus maze (EPM) and the hole-board apparatus in rats and mice. Control rats were treated with an equal volume of saline, and positive control rats with buspirone (1 mg/kg). Single or repeated treatments of the aqueous extract of Uncaria rhynchophylla (200 mg/kg/day, p.o.) for 7 days significantly increased the time-spent and entries into open arms of the EPM, and reduced the time-spent and entries into the closed arms versus saline controls (P<0.05). However, no changes in spontaneous locomotor activity or myorelaxant effects were observed versus saline controls. In the hole-board test, repeated treatment with the aqueous extract of Uncaria rhynchophylla (100 or 200 mg/kg/day, p.o.) significantly increased the number of head-dips (P<0.05). In addition, the anxiolytic-like effects of Uncaria rhynchophylla extract as assessed using the EPM test were abolished by WAY 100635 (0.3 mg/kg, i.p.), a 5-HT(1A) receptor antagonist. These results suggest that Uncaria rhynchophylla is an effective anxiolytic agent, and acts via the serotonergic nervous system.

Allium cepa Extract and Quercetin Protect Neuronal Cells from Oxidative Stress via PKC-ε Inactivation/ERK1/2 Activation.

Oxidative stress plays an important role in the pathophysiology of various neurologic disorders. Allium cepa extract (ACE) and their main flavonoid component quercetin (QCT) possess antioxidant activities and protect neurons from oxidative stress. We investigated the underlying molecular mechanisms, particularly those linked to the antioxidant effects of the ACE. Primary cortical neuronal cells derived from mouse embryos were preincubated with ACE or QCT for 30 min and exposed to L-buthionine sulfoximine for 4~24 h. We found that ACE and QCT significantly decreased neuronal death and the ROS increase induced by L-buthionine-S, R-sulfoximine (BSO) in a concentration-dependent manner. Furthermore, ACE and QCT activated extracellular signal-regulated kinase 1/2 (ERK1/2), leading to downregulation of protein kinase C-ε (PKC-ε) in BSO-stimulated neuronal cells. In addition, ACE and QCT decreased the phosphorylated levels of p38 mitogen-activated protein kinase. Our results provide new insight into the protective mechanism of ACE and QCT against oxidative stress in neuronal cells. The results suggest that the inactivation of PKC-ε induced by phosphorylating ERK1/2 is responsible for the neuroprotective effect of ACE and QCT against BSO-induced oxidative stress.