Synthesis of 4-(3-oxo-3-phenylpropyl)morpholin-4-ium chloride analogues and their inhibitory activities of nitric oxide production in lipopolysaccharide-induced BV2 cells.

Based on our previous report that 3-morpholino-1-phenylpropan-1-one 2, one of the fluoxetine's simplified morpholino analogue, inhibited nitric oxide (NO) production, in this paper, various substituted benzene analogues with morpholine hydrochloride of 2 were synthesized and their inhibitory effects on NO production in lipopolysaccharide (LPS)-induced BV2 cells were tested. Among the synthesized compounds, 2-trifluoromethyl analogue 16n (IC50 = 8.6 µM) showed a significantly higher inhibitory activity than that of the parent compound 2a (IC50 > 50 µM) and suppressed NO production dose-dependently without cytotoxicity. Compound 16n also inhibited iNOS expression in LPS-induced BV2 cells at 2, 10 and 20 µM concentrations. These results suggest that compound 16n inhibited NO production by suppressing the expression of iNOS and can be used as a lead structure for developing new inhibitor of NO production.

Fetuin-B regulates vascular plaque rupture via TGF-ß receptor-mediated Smad pathway in vascular smooth muscle cells.

Fetuin-B is a serum protein linked to the regulation of physiological or pathophysiological events such as fertility, energy metabolism, and liver disease. Recently, fetuin-B has been reported to be involved in the modulation of the rupture of atherosclerotic plaques associated with acute myocardial infarction. However, the exact mechanism involved in the modulation of atherosclerotic plaque rupture event by fetuin-B is not fully elucidated yet. In the present study, we investigated whether fetuin-B could influence atherosclerotic plaque rupture through vascular smooth muscle cells (VSMCs). Immunoprecipitation assay using membrane proteins from VSMCs revealed that fetuin-B tightly bound to transforming growth factor-ß receptor (TGF-ßR). Fetuin-B treatment elevated TGF-ßR signals (e.g., phosphorylation of Smad2 and Smad3) in VSMCs. Fetuin-B also stimulated nuclear translocation of phosphorylated Smads. Phosphorylation of Smad and its nuclear translocation by treatment with fetuin-B were inhibited in VSMCs by treatment with SB431542, a selective inhibitor of TGF-ßR. Fetuin-B enhanced expression levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in VSMCs through its epigenetic modification including recruitments of both histone deacetylase 1 and RNA polymerase II. These epigenetic alterations in VSMCs were also inhibited by treatment with SB431542. In vivo administration of fetuin-B protein increased expression levels of PAI-1 and MMP-2 in the vascular plaque. However, these increases in expression were inhibited by the administration of SB43154. These results indicate that fetuin-B may modulate vascular plaque rupture by promoting expression of PAI-1 and MMP-2 in VSMCs via TGF-ßR-mediated Smad pathway.

Ursolic Acid Suppresses Cholesterol Biosynthesis and Exerts Anti-Cancer Effects in Hepatocellular Carcinoma Cells.

Abnormally upregulated cholesterol and lipid metabolism, observed commonly in multiple cancer types, contributes to cancer development and progression through the activation of oncogenic growth signaling pathways. Although accumulating evidence has shown the preventive and therapeutic benefits of cholesterol-lowering drugs for cancer management, the development of cholesterol-lowering drugs is needed for treatment of cancer as well as metabolism-related chronic diseases. Ursolic acid (UA), a natural pentacyclic terpenoid, suppresses cancer growth and metastasis, but the precise underlying molecular mechanism for its anti-cancer effects is poorly understood. Here, using sterol regulatory element (SRE)-luciferase assay-based screening on a library of 502 natural compounds, this study found that UA activates sterol regulatory element-binding protein 2 (SREBP2). The expression of cholesterol biosynthesis-related genes and enzymes increased in UA-treated hepatocellular carcinoma (HCC) cells. The UA increased cell cycle arrest and apoptotic death in HCC cells and reduced the activation of oncogenic growth signaling factors, all of which was significantly reversed by cholesterol supplementation. As cholesterol supplementation successfully reversed UA-induced attenuation of growth in HCC cells, it indicated that UA suppresses HCC cells growth through its cholesterol-lowering effect. Overall, these results suggested that UA is a promising cholesterol-lowering nutraceutical for the prevention and treatment of patients with HCC and cholesterol-related chronic diseases.

IDH1R132H Causes Resistance to HDAC Inhibitors by Increasing NANOG in Glioblastoma Cells.

The R132H mutation in isocitrate dehydrogenase 1 (IDH1R132H) is commonly observed and associated with better survival in glioblastoma multiforme (GBM), a malignant brain tumor. However, the functional role of IDH1R132H as a molecular target for GBM treatment is not completely understood. In this study, we found that the overexpression of IDH1R132H suppresses cell growth, cell cycle progression and motility in U87MG glioblastoma cells. Based on cell viability and apoptosis assays, we found that IDH1R132H-overexpressing U87MG and U373MG cells are resistant to the anti-cancer effect of histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA), vorinostat (SAHA), and valproic acid. Octyl-(R)-2-hydroxyglutarate (Octyl-2HG), which is a membrane-permeable precursor form of the oncometabolite (R)-2-hydroxyglutarate (R-2HG) produced in IDH1-mutant tumor cells, significantly increased HDACi resistance in glioblastoma cells. Mechanistically, IDH1R132H and Octyl-2HG enhanced the promoter activation of NANOG via increased H3K4-3Me, consequently increasing NANOG mRNA and protein expression. Indeed, HDACi resistance was attenuated in IDH1R132H-expressing glioblastoma cells by the suppression of NANOG using small interfering RNAs. Furthermore, we found that AGI-5198, a selective inhibitor of IDH1R132H, significantly attenuates HDACi resistance and NANOG expression IDH1R132H-expressing glioblastoma cells. These results suggested that IDH1R132H is a potential molecular target for HDACi-based therapy for GBM.

Corrigendum to: Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells.

[This corrects the article on p. 679 in vol. 22, PMID: 30402028.].

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation.

The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

Zerumbone, a Tropical Ginger Sesquiterpene of Zingiber officinale Roscoe, Attenuates α-MSH-Induced Melanogenesis in B16F10 Cells.

Zerumbone (ZER), an active constituent of the Zingiberaceae family, has been shown to exhibit several biological activities, such as anti-inflammatory, anti-allergic, anti-microbial, and anti-cancer; however, it has not been studied for anti-melanogenic properties. In the present study, we demonstrate that ZER and Zingiber officinale (ZO) extract significantly attenuate melanin accumulation in α-melanocyte-stimulating hormone (α-MSH)-stimulated mouse melanogenic B16F10 cells. Further, to elucidate the molecular mechanism by which ZER suppresses melanin accumulation, we analyzed the expression of melanogenesis-associated transcription factor, microphthalmia-associated transcription factor (MITF), and its target genes, such as tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2), in B16F10 cells that are stimulated by α-MSH. Here, we found that ZER inhibits the MITF-mediated expression of melanogenic genes upon α-MSH stimulation. Additionally, cells treated with different concentrations of zerumbone and ZO showed increased extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, which are involved in the degradation mechanism of MITF. Pharmacological inhibition of ERK1/2 using U0126 sufficiently reversed the anti-melanogenic effect of ZER, suggesting that increased phosphorylation of ERK1/2 is required for its anti-melanogenic activity. Taken together, these results suggest that ZER and ZO extract can be used as active ingredients in skin-whitening cosmetics because of their anti-melanogenic effect.

Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders that share behavioral features, the results of numerous studies have suggested that the underlying causes of ASDs are multifactorial. Behavioral and/or neurobiological analyses of ASDs have been performed extensively using a valid model of prenatal exposure to valproic acid (VPA). Abnormal synapse formation resulting from altered neurite outgrowth in neural progenitor cells (NPCs) during embryonic brain development has been observed in both the VPA model and ASD subjects. Although several mechanisms have been suggested, the actual mechanism underlying enhanced neurite outgrowth remains unclear. In this study, we found that VPA enhanced the expression of brain-derived neurotrophic factor (BDNF), particularly mature BDNF (mBDNF), through dual mechanisms. VPA increased the mRNA and protein expression of BDNF by suppressing the nuclear expression of methyl-CpG-binding protein 2 (MeCP2), which is a transcriptional repressor of BDNF. In addition, VPA promoted the expression and activity of the tissue plasminogen activator (tPA), which induces BDNF maturation through proteolytic cleavage. Trichostatin A and sodium butyrate also enhanced tPA activity, but tPA activity was not induced by valpromide, which is a VPA analog that does not induce histone acetylation, indicating that histone acetylation activity was required for tPA regulation. VPA-mediated regulation of BDNF, MeCP2, and tPA was not observed in astrocytes or neurons. Therefore, these results suggested that VPA-induced mBDNF upregulation was associated with the dysregulation of MeCP2 and tPA in developing cortical NPCs.

A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways.

AIM: To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro. METHODS: Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence microscopy, respectively. RESULTS: Pretreatment with MCAP (0.1, 1, 10 µmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 µmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1ß, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). CONCLUSION: MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells.

Scoparone Inhibits LPS-Simulated Inflammatory Response by Suppressing IRF3 and ERK in BV-2 Microglial Cells.

Microglia activation and the release of various inflammatory cytokines are largely related to neurological diseases, including Parkinson's, Alzheimer's, and other brain diseases. The suppression of microglial cells using natural bioactive compounds has become increasingly important for brain therapy owing to the expected beneficial effect of lower toxicity. Scoparone (6,7-dimethoxycoumarin), a major bioactive compound found in various plant parts, including the inner shell of chestnut (Castanea crenata), was evaluated on lipopolysaccharide (LPS)-activated BV-2 microglia cells. The results indicated that scoparone suppresses the LPS-stimulated increase of neuroinflammatory responses and inhibited the pro-inflammatory cytokine production in the BV-2 microglial cells. A mechanistic study showed that scoparone specifically inhibited the LPS-stimulated activation via a major regulation of IRF-3 and a regulation of ERK, whereby the phosphorylation in the BV-2 microglial cells is blocked. These data suggest that scoparone has anti-neuroinflammatory effects in LPS-activated BV-2 microglial cells, and could possibly be used in the development of novel drugs for the prevention and treatment of neuroinflammatory diseases.

ß-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia.

BACKGROUND: ß-Lapachone (ß-LAP) is a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia sp.), and it has been used for treatment of rheumatoid arthritis, infection, and cancer. In the present study, we investigated whether ß-LAP has anti-inflammatory effects under in vitro and in vivo neuroinflammatory conditions. METHODS: The effects of ß-LAP on the expression of inducible nitric oxide synthase (iNOS), cytokines, and matrix metalloproteinases (MMPs) were examined in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia by ELISA, reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. Microglial activation and the expression levels of proinflammatory molecules were measured in the LPS-injected mouse brain by immunohistochemistry and RT-PCR analysis. The detailed molecular mechanism underlying the anti-inflammatory effects of ß-LAP was analyzed by electrophoretic mobility shift assay, reporter gene assay, Western blot, and RT-PCR analysis. RESULTS: ß-LAP inhibited the expression of iNOS, proinflammatory cytokines, and MMPs (MMP-3, MMP-8, MMP-9) at mRNA and protein levels in LPS-stimulated microglia. On the other hand, ß-LAP upregulated the expressions of anti-inflammatory molecules such as IL-10, heme oxygenase-1 (HO-1), and the tissue inhibitor of metalloproteinase-2 (TIMP-2). The anti-inflammatory effect of ß-LAP was confirmed in an LPS-induced systemic inflammation mouse model. Thus, ß-LAP inhibited microglial activation and the expressions of iNOS, proinflammatory cytokines, and MMPs in the LPS-injected mouse brain. Further mechanistic studies revealed that ß-LAP exerts anti-inflammatory effects by inhibiting MAPKs, PI3K/AKT, and NF-κB/AP-1 signaling pathways in LPS-stimulated microglia. ß-LAP also inhibited reactive oxygen species (ROS) production by suppressing the expression and/or phosphorylation of NADPH oxidase subunit proteins, such as p47(phox) and gp91(phox). The anti-oxidant effects of ß-LAP appeared to be related with the increase of HO-1 and NQO1 via the Nrf2/anti-oxidant response element (ARE) pathway and/or the PKA pathway. CONCLUSIONS: The strong anti-inflammatory/anti-oxidant effects of ß-LAP may provide preventive therapeutic potential for various neuroinflammatory disorders.

Chronic exposure to ethanol of male mice before mating produces attention deficit hyperactivity disorder-like phenotype along with epigenetic dysregulation of dopamine transporter expression in mouse offspring.

Preconception exposure to EtOH through the paternal route may affect neurobehavioral and developmental features of offspring. This study investigates the effects of paternal exposure to EtOH before conception on the hyperactivity, inattention, and impulsivity behavior of male offspring in mice. Sire mice were treated with EtOH in a concentration range approximating human binge drinking (0-4 g/kg/day EtOH) for 7 weeks and mated with untreated females mice to produce offspring. EtOH exposure to sire mice induced attention deficit hyperactivity disorder (ADHD)-like hyperactive, inattentive, and impulsive behaviors in offspring. As a mechanistic link, both protein and mRNA expression of dopamine transporter (DAT), a key determinant of ADHD-like phenotypes in experimental animals and humans, were significantly decreased by paternal EtOH exposure in cerebral cortex and striatum of offspring mice along with increased methylation of a CpG region of the DAT gene promoter. The increase in methylation of DAT gene promoter was also observed in the sperm of sire mice, suggesting germline changes in the epigenetic methylation signature of DAT gene by EtOH exposure. In addition, the expression of two key regulators of methylation-dependent epigenetic regulation of functional gene expression, namely, MeCP2 and DNMT1, was markedly decreased in offspring cortex and striatum sired by EtOH-exposed mice. These results suggest that preconceptional exposure to EtOH through the paternal route induces behavioral changes in offspring, possibly via epigenetic changes in gene expression, which is essential for the regulation of ADHD-like behaviors.

Valproic acid induces astrocyte-dependent neurite outgrowth from cultured rat primary cortical neuron via modulation of tPA/PAI-1 activity.

Tissue plasminogen activator (tPA) is expressed in several regions of brain and plays regulatory roles such as neurite outgrowth, synaptic plasticity and long term potentiation. The activity of tPA is regulated by an endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1), which is expressed mainly in astrocytes. Valproic acid (VPA), a histone deacetylase inhibitor that is used for the treatment of epilepsy and bipolar disorders, promotes neurite extension, neuronal growth and has neuroprotective effect in neurodegenerative diseases. In this study, we examined whether the neurite extension effects of VPA is mediated by modulating tPA/PAI-1 system. VPA dose-dependently increased tPA activity and decreased PAI-1 activity in rat primary astrocytes but not in neurons. PAI-1 protein level secreted into the culture medium but not tPA per se was decreased by VPA. In co-culture system or in neuronal culture stimulated with astrocyte conditioned media but not in pure neuronal cell culture, VPA induced neurite outgrowth via increased tPA activity due to the decreased PAI-1 activity in astrocytes. The decrease in PAI-1 activity and increased neurite extension was regulated via JNK mediated post-transcriptional pathway. The essential role of tPA/PAI-1 system in the regulation of VPA-mediated neurite extension was further demonstrated by experiments using astrocyte conditioned media obtained from tPA or PAI-1 knockout mice. Regulation of PAI-1 activity in astrocyte by VPA may affect both physiological and pathological processes in brain by upregulating tPA activity.

Synergistic activation of lipopolysaccharide-stimulated glial cells by propofol.

Despite the extensive use of propofol in general anesthetic procedures, the effects of propofol on glial cell were not completely understood. In lipopolysaccharide (LPS)-stimulated rat primary astrocytes and BV2 microglial cell lines, co-treatment of propofol synergistically induced inflammatory activation as evidenced by the increased production of NO, ROS and expression of iNOS, MMP-9 and several cytokines. Propofol augmented the activation of JNK and p38 MAPKs induced by LPS and the synergistic activation of glial cells by propofol was prevented by pretreatment of JNK and p38 inhibitors. When we treated BV2 cell culture supernatants treated with LPS plus propofol on cultured rat primary neuron, it induced a significant neuronal cell death. The results suggest that the repeated use of propofol in immunologically challenged situation may induce glial activation in brain.

Activation of microglial cells via protease-activated receptor 2 mediates neuronal cell death in cultured rat primary neuron.

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2-PKC-MAPK pathway-induced microglial activation was NF-kappaB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-alpha and IL-1beta. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X(L), and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N(G)-nitro-l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.

2-(5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl)-N-(2-hydroxyethyl)-2-oxoacetamide (CDMPO) has anti-inflammatory properties in microglial cells and prevents neuronal and behavioral deficits in MPTP mouse model of Parkinson's disease.

Parkinson's disease (PD) is characterized by the selective loss of nigrostriatal dopamine neurons associated with microglial activation. Inhibition of the inflammatory response elicited by activated microglia could be an effective strategy to alleviate the progression of PD. Here, we synthesized 2-(5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3-yl)-N-(2-hydroxyethyl)-2-oxoacetamide (CDMPO) and studied its protective anti-inflammatory mechanisms following lipopolysaccharide (LPS)-induced neuroinflammation in vitro and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in vivo. CDMPO and its parent compound, rimonabant, significantly attenuated nitric oxide (NO) production in LPS-stimulated primary microglia and BV2 cells. Furthermore, CDMPO significantly inhibited the release of proinflammatory cytokines and prostaglandin E2 (PGE2) by activated BV2 cells, also suppressed expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Mechanistically, CDMPO attenuated LPS-induced activation of nuclear factor-kappa B (NF-κB), inhibitor of kappa B alpha (IκBα), and p38 phosphorylation in BV2 cells. MPTP intoxication of mice results in glial activation, tyrosine hydroxylase (TH) depletion, and significant behavioral deficits. Prophylactic treatment with CDMPO decreased proinflammatory molecules via NF-κB and p38 mitogen-activated protein kinase signaling, resulting in protection of dopaminergic neurons and improved behavioral impairments. These results suggest that CDMPO is a promising neuroprotective agent for the prevention and treatment of microglia-mediated neuroinflammatory conditions and may be useful for behavioral improvement in PD phenotype.

Anti­inflammatory role of Prunus persica L. Batsch methanol extract on lipopolysaccharide­stimulated glial cells.

Glial cells are the resident immune cells of the central nervous system. Reactive glial cells release inflammatory mediators that induce neurotoxicity or aggravate neurodegeneration. Regulation of glial activation is crucial for the initiation and progression of neuropathological conditions. Constituents of the peach tree (Prunus persica L. Batsch), which has a global distribution, have been found to exert therapeutic effects in pathological conditions, such as rashes, eczema and allergies. However, the therapeutic potential of its aerial parts (leaves, fruits and twigs) remains to be elucidated. The present study aimed to evaluate the anti­inflammatory role of P. persica methanol extract (PPB) on lipopolysaccharide (LPS)­stimulated glial cells. High­performance liquid chromatography coupled with tandem mass spectrometry analysis showed that PPB contained chlorogenic acid and catechin, which have antioxidant properties. Western blot and reverse transcription polymerase chain reaction results indicated that PPB reduced the transcription of various proinflammatory enzymes (nitric oxide synthase and cyclooxygenase­2) and cytokines [tumor necrosis factor­α, interleukin (IL)­1ß and IL­6] in LPS­stimulated BV2 cells. In addition, PPB inhibited the activation of NF­κB and various mitogen­activated protein kinases required for proinflammatory mediator transcription. Finally, nitrite measurement and immunocytochemistry results indicated that PPB also suppressed nitrite production and NF­κB translocation in LPS­stimulated primary astrocytes. Thus, PPB may be used as a potential therapeutic agent for neurodegenerative diseases and neurotoxicity via the suppression of glial cell activation.

Association between sphingosine-1-phosphate-induced signal transduction via mitogen-activated protein kinase pathways and keloid formation.

We conducted this experimental study to analyze the relationship between sphingosine-1-phosphate (S1P)-induced mitogen-activated protein (MAP) kinase pathways and keloid formation. We collected samples of the normal tissue and the keloid tissue from 10 normal healthy individuals and 12 patients with keloid scars, respectively. Then, we compared the level of sphingosine-1-phosphate receptor (S1PR1/S1PR2) mRNA/protein expression between the normal tissue and the keloid tissue. Moreover, we also compared the level of S1PR protein expression, that of S1P-induced COL1A1 (collagen Type I, α-1 chain) expression, that of S1P-induced JNK/ERK phosphorylation, that of S1P-induced COL1A1 expression following the treatment with 30 µM PD98059 (ERK inhibitor) or 30 µM SP600125 (JNK inhibitor) and that of S1P-induced COL1A1 expression following the treatment with W146 (S1PR1 inhibitor) or JTE013 (S1PR2 inhibitor) between the normal fibroblasts and the keloid fibroblasts. We found that the level of S1PR1/S1PR2 mRNA/protein expression was significantly higher in the keloid tissue as compared with the normal tissue. Our results also showed that the level of S1P-induced COL1A1 expression and that of S1P-induced JNK/ERK phosphorylation were significantly higher in the keloid fibroblasts as compared with the normal ones (P < 0.05). Furthermore, there were significant decreases in the level of S1P-induced COL1A1 expression when the keloid fibroblasts were treated with 30 µM SP600125 or 30 µM PD98059 and that of S1P-induced COL1A1 expression when the treated with 100 nM W146 or 100 nM JTE013 (P < 0.05). Our results indicate that S1P-induced signal transduction is associated with increased collagen synthesis via S1PR-mediated signaling pathways in the keloid tissue.

Various approaches for measurement of synaptic vesicle endocytosis at the central nerve terminal.

At the presynaptic terminal, neurotransmitters are stored in synaptic vesicles (SVs), which are released and recycled via exo- and endocytosis. SV endocytosis is crucial for sustaining synaptic transmission by maintaining the SV pool. Many studies have shown that presynaptic dysfunction, particularly impairment of SV endocytosis, is related to neurological disorders. Notably, the presynaptic terminal is considered to be a sensitive structure because certain presynaptic dysfunctions, manifested as impaired SV endocytosis or ultrastructural changes in the presynaptic terminal, can be observed before there is a biochemical or pathological evidence of a neurological disorder. Therefore, monitoring and assessing the presynaptic function by SV endocytosis facilitates the development of early markers for neurological disorders. In this study, we reviewed the current methods for assessing and visualizing SV endocytosis at the central nerve terminal.

Quercetin, a Plant Polyphenol, Has Potential for the Prevention of Bone Destruction in Rheumatoid Arthritis.

We investigated the immune-regulatory function of quercetin, in interleukin (IL)-17-produced osteoclastogenesis in rheumatoid arthritis (RA). RA fibroblasts-like synoviocytes (RA-FLS) were stimulated with IL-17, and the mRNA expression and secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. CD14+ monocytes (osteoclast precursors) were stimulated with IL-17, RANKL, with/without quercetin, and tartrate-resistant acid phosphatase activity was evaluated to assess osteoclast differentiation. Osteoclast differentiation was investigated after coculturing IL-17-stimulated RA-FLS and Th17 cells with monocytes. CD4+ T cells were cocultured with quercetin under Th17-inducing conditions, and their differentiation to Th17 cells and Treg cells was determined by flow cytometry analysis. We found that IL-17 stimulated RA-FLS to produce RANKL and quercetin decreased the IL-17-induced RANKL protein levels. Quercetin decreased the IL-17-produced activation of mammalian target of rapamycin, extracellular signal-regulated kinase and inhibitor of kappa B-alpha. When monocytes were stimulated with IL-17, macrophage colony-stimulating factor or RANKL, mature osteoclasts were formed, and quercetin decreased this osteoclastogenesis. When monocytes were cultured with IL-17-prestimulated RA-FLS or Th17 cells, osteoclasts were produced, and quercetin decreased this osteoclast differentiation. In Th17-differentiation conditions, quercetin suppressed Th17 cell and the production of IL-17, but quercetin did not affect Treg cells. Quercetin inhibits IL-17-stimulated RANKL production in RA-FLS and IL-17-stimulated osteoclast formation. Quercetin reduces Th17 differentiation. Quercetin could be an additional therapeutic option for bone destructive processes in RA.