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ABSTRACT: We investigated the interaction between proinflammatory and inflammatory responses caused by Staphylococcus aureus-derived lipoteichoic acid (LTA) in primary cultured microglial cells and BV-2 microglia. LTA induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels increase in a concentration- and time-dependent manner. Meanwhile, LTA also increased nitric oxide (NO) and PGE2 production in microglia. Administration of TLR2 antagonist effectively inhibited LTA-induced NO, iNOS, and COX-2 expression. Moreover, treatment of cells with LTA caused a time-dependent activation of ERK, p38, JNK, as well as AKT. We also found that LTA-induced iNOS and COX-2 up-regulation were attenuated by p38, JNK, and PI3-kinase inhibitors. On the other hand, LTA-enhanced HO-1 expression was attenuated by p38 and PI3-kinase inhibitors. Treatment of cells with NF-κB and AP-1 inhibitors antagonized LTA-induced iNOS and COX-2 expression. However, only NF-κB inhibitors reduced LTA-induced HO-1 expression in microglia. Furthermore, stimulation of cells with LTA also activated IκBα phosphorylation, p65 phosphorylation at Ser(536), and c-Jun phosphorylation. Moreover, LTA-induced increases of κB-DNA and AP-1-DNA binding activity were inhibited by p38, JNK, and PI3-kinase inhibitors. HO-1 activator CoPP IX dramatically reversed LTA-induced iNOS expression. Our results provided mechanisms linking LTA and inflammation/anti-inflammation, and indicated that LTA plays a regulatory role in microglia activation.
Toxicology and Applied Pharmacology 03/2013; · 4.45 Impact Factor
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ABSTRACT: Malignant gliomas are associated with high morbidity and mortality because they are highly invasive into surrounding brain tissue, making complete surgical resection impossible. Osteopontin is abundantly expressed in the brain and is involved in cell adhesion, migration, and invasion. The aim of the present study was to investigate the mechanisms of glioma cell migration. Migration and invasion activity were determined by transwell and wound-healing assays. Gene and protein expressions were analyzed by reverse transcription-PCR, real time-PCR, and Western blotting. Nrf2-DNA binding activity was determined by electrophoretic mobility shift assay. Establishment of migration-prone sublines were performed to select highly migratory glioma. An intracranial xenograft mouse model was used for the in vivo study. Application of recombinant human osteopontin enhanced the migration of glioma cells. Expression of heme oxygenase (HO)-1 mRNA and protein also increased in response to osteopontin stimulation. Osteopontin-induced increase in cell migration was antagonized by HO-1 inhibitor or HO-1 small interfering (si)RNA. Osteopontin-mediated HO-1 expression was reduced by treatment with MEK/ERK and phosphatidylinositol 3-kinase/Akt inhibitors, as well as siRNA against Nrf2. Furthermore, osteopontin stimulated Nrf2 accumulation in the nucleus and increased Nrf2-DNA binding activity. In migration-prone sublines, cells with greater migration ability had higher osteopontin and HO-1 expression, and zinc protoporphyrin IX treatment could effectively reduce the enhanced migration ability. In an intracranial xenograft mouse model, transplantation of migration-prone subline cells exhibited higher cell migration than parental tumor cells. These results indicate that osteopontin activates Nrf2 signaling, resulting in enhanced HO-1 expression and cell migration in glioma cells.
Neuro-Oncology 10/2012; 14(11):1367-78. · 5.72 Impact Factor
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ABSTRACT: Desipramine is known principally as a tricyclic antidepressant drug used to promote recovery of depressed patients. It has also been used in a number of other psychiatric and medical conditions. The present study is the first to investigate the neuroprotective effect of desipramine.
Mes23.5 dopaminergic cells were used to examine neuroprotective effect of desipramine. Western blot, reverse transcription-PCR, MTT assay, siRNA transfection and electrophoretic mobility shift assay (EMSA) were carried out to assess the effects of desipramine. Desipramine induces endogenous anti-oxidative enzyme, heme oxygenase-1 (HO-1) protein and mRNA expression in concentration- and time-dependent manners. A different type of antidepressant SSRI (selective serotonin reuptake inhibitor), fluoxetine also shows similar effects of desipramine on HO-1 expression. Moreover, desipramine induces HO-1 expression through activation of ERK and JNK signaling pathways. Desipramine also increases NF-E2-related factor-2 (Nrf2) accumulation in the nucleus and enhances Nrf2-DNA binding activity. Moreover, desipramine-mediated increase of HO-1 expression is reduced by transfection with siRNA against Nrf2. On the other hand, pretreatment of desipramine protects neuronal cells against rotenone- and 6-hydroxydopamine (6-OHDA)-induced neuronal death. Furthermore, inhibition of HO-1 activity by a HO-1 pharmacological inhibitor, ZnPP IX, attenuates the neuroprotective effect of desipramine. Otherwise, activation of HO-1 activity by HO-1 activator and inducer protect 6-OHDA-induced neuronal death.
These findings suggest that desipramine-increased HO-1 expression is mediated by Nrf2 activation through the ERK and JNK signaling pathways. Our results also suggest that desipramine provides a novel effect of neuroprotection, and neurodegenerative process might play an important role in depression disorder.
PLoS ONE 01/2012; 7(11):e50138. · 4.09 Impact Factor
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ABSTRACT: Our previous report has shown that berberine effectively inhibits LPS- and IFN-γ-induced neuroinflammation in microglia cells. Recently, we also reported that HO-1 (Heme oxygenase-1) may be a therapeutic target to regulate neuroinflammation in microglia cells. The present study examined the ability of berberine, the major constituents of Chinese herb Rhizoma coptidis, to induce expression of HO-1, and analyzed its signaling mechanism in rat brain astrocytes. HO-1 is known as an antioxidant enzyme which helps to protect against cellular damage and maintains tissue homeostasis. Here, we found that berberine increased HO-1 mRNA and protein expression concentration- and time-dependently. In addition, berberine-induced HO-1 expression was attenuated by PI 3-kinase (phosphatidylinositol 3-kinase) inhibitors LY294002 and wortmannin, and an AKT inhibitor. Treatment of astrocytes with berberine also induced p85 (PI 3-kinase) and AKT phospholation, and increased AKT kinase activity. Berberine also increased NF-E2-related factor-2 (Nrf2) accumulation in the nucleus and increased Nrf2-DNA binding activity as determined by the EMSA (electrophoretic mobility shift assay). Moreover, berberine-induced increase of Nrf2-DNA binding activity was reduced by PI 3-kinase and AKT inhibitors. Berberine-increased HO-1-luciferase activity was also inhibited by co-transfection with dominant-negative (DN) mutants of p85 and AKT. Moreover, berberine-mediated increase of HO-1 transcriptional activity and protein expression were reduced by transfection with siRNA againt Nrf2. These findings suggest that berberine-increased HO-1 expression is mediated by Nrf2 activation through the PI 3-kinase/AKT pathway in astrocytes. Thus, berberine may be useful as a therapeutic agent for the treatment of neuroinflammation-associated disorders.
International immunopharmacology 11/2011; 12(1):94-100. · 2.21 Impact Factor
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ABSTRACT: In this study, we investigated the signaling pathways involved in inflammatory production caused by peptidoglycan (PGN), a cell wall component of the gram-positive bacterium, in BV-2 microglia. PGN caused a concentration- and time-dependent increase in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein levels. In addition, PGN also induced IL-1 beta, TNF-alpha and IL-6 mRNA up-regulation in a concentration-dependent manner. Moreover, PGN also increased Toll-like receptor 2 (TLR2) expression in BV-2 microglia. Administration of TLR2 neutralizing antibody effectively inhibited PGN-induced iNOS and COX-2 expression. On the other hand, PGN-induced iNOS and COX-2 up-regulation were attenuated by PI3-kinase inhibitors (LY294002 and wortmannin), and an AKT inhibitor. Treatment of BV-2 microglia with PGN caused a time-dependent activation of PI3-kinase (p85) and AKT. PGN-induced PI3-kinase/AKT activation, iNOS and COX-2 expression were also inhibited by MyD88 inhibitory peptide. Treatment of cells with NF-kappaB inhibitor (pyrrolidine dithiocarbamate), I kappaB alpha phosphorylation inhibitor (Bay 117082), or I kappaB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone) inhibited PGN-induced iNOS and COX-2 expression. Furthermore, stimulation of cells with PGN also activated IKK alpha/beta, I kappaB alpha phosphorylation, I kappaB alpha degradation, p65 phosphorylation at Ser(536), and increased kappaB-luciferase activity. PGN-induced IKK alpha/beta phosphorylation, I kappaB alpha phosphorylation, and I kappaB alpha degradation were further inhibited by pre-treatment with PI3-kinase inhibitors. Moreover, PGN-mediated increase of kappaB-luciferase activity was also inhibited by pre-transfection with dominant-negative mutants of p85, AKT, IKK alpha or IKK beta. Our data demonstrate that PGN-induced iNOS, COX-2 and proinflammatory cytokine expression was mediated through the TLR2/MyD88/PI3-kinase/AKT pathway, which in turn initiates IKK alpha/beta and NF-kappaB activation in BV-2 microglia.
International immunopharmacology 05/2010; 10(8):883-91. · 2.21 Impact Factor
