15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases

<p>Abstract</p> <p>Background</p> <p>The Infiltration of leukocytes across the brain endothelium is a hallmark of various neuroinflammatory disorders. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules (CAMs) on activated vascular endothelial cells which increases the adhesion and infiltration of leukocytes. TNFα is one of the major proinflammatory cytokines that causes endothelial dysfunction by various mechanisms including activation of transcription factor NF-κB, a key transcription factor that regulates expression of CAMs. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone superfamily of ligand-activated transcriptional factors. 15-deoxy-δ 12, 14-prostaglandin J2 (15d-PGJ2) is a well recognized natural ligand of PPARγ and possesses anti-inflammatory properties both <it>in vitro </it>and <it>in vivo</it>. This study aims to elucidate the mechanism of 15-PGJ2 on the adhesion of mononuclear cells to activated endothelial cells.</p> <p>Methods</p> <p>To delineate the signaling pathway of 15d-PGJ2 mediated effects, we employed an <it>in vitro </it>adhesion assay model of endothelial-monocyte interaction. Expression of CAMs was examined using flow cytometry and real time PCR techniques. To define the mechanism of 15d-PGJ2, we explored the role of NF-κB by EMSA (Electrophoretic Mobility Shift Assay) gels, NF-κB reporter and p65-transcriptional activities by transient transfection in the brain-derived endothelial cell line (bEND.3).</p> <p>Results</p> <p>Using an <it>in vitro </it>adhesion assay model, we demonstrate that 15d-PGJ2 inhibits TNFα induced monocyte adhesion to endothelial cells, which is mediated by downregulation of endothelial cell adhesion molecules in a PPARγ independent manner. 15d-PGJ2 modulated the adhesion process by inhibiting the TNFα induced IKK-NF-κB pathway as evident from EMSA, NF-κB reporter and p65 mediated transcriptional activity results in bEND.3 cells.</p> <p>Conclusion</p> <p>These findings suggest that 15d-PGJ2 inhibits inflammation at multiple steps and thus is a potential therapeutic target for various inflammatory diseases.</p

Reduction of lipoxidative load by secretory phospholipase A2 inhibition protects against neurovascular injury following experimental stroke in rat

<p>Abstract</p> <p>Background</p> <p>In animal models, ischemia reperfusion (IR) injury triggers membrane lipid degradation and accumulation of lipoxidative exacerbations in neurovascular unit, leading to blood brain barrier (BBB) damage and neurologic deficits. In this study, we investigated whether impeding membrane lipid breakdown by inhibiting secretory phospholipase A2 (sPLA2) activity reduces BBB leakage, leading to neuroprotection and functional recovery.</p> <p>Methods</p> <p>Focal cerebral IR injury was induced by middle cerebral artery occlusion (MCAO) in adult male rats. A sPLA2 inhibitor, 7,7-dimethyleicosadienoic acid (DEDA), was administered following IR injury. DEDA-treated animals were compared with vehicle-treated in terms of BBB leakage, edema, infarct volume, and neurological deficit. Membrane lipid degradation and the expression/activity of sPLA2 were also assessed. The role of one of the sPLA2 products, arachidonic acid (AA), on the morphology of the differentiated neuronal cell PC12 was examined by light microscopy.</p> <p>Results</p> <p>Treatment with DEDA after IR injury not only reduced BBB leakage but also decreased infarct volume and improved neurologic function. The treatment attenuated both the activity of sPLA2 and the levels of sPLA2-derived oxidized products. The metabolites of lipid oxidation/peroxidation, including the protein carbonyl, were reduced as well. The treatment also restored the levels of glutathione, indicating attenuation of oxidative stress. I<it>n vitro </it>treatment of PC12 cells with DEDA did not restore the AA-mediated inhibition of neurite formation and the levels of glutathione, indicating that effect of DEDA is up stream to AA release.</p> <p>Conclusion</p> <p>sPLA2-derived oxidative products contribute to significant neurovascular damage, and treatment with sPLA2 inhibitor DEDA ameliorates secondary injury by reducing exacerbations from lipoxidative stress.</p

N-acetyl-L-cysteine ameliorates the inflammatory disease process in experimental autoimmune encephalomyelitis in Lewis rats

We report that N-acetyl-L-cysteine (NAC) treatment blocked induction of TNF-α, IL-1β, IFN-γ and iNOS in the CNS and attenuated clinical disease in the myelin basic protein induced model of experimental allergic encephalomyelitis (EAE) in Lewis rats. Infiltration of mononuclear cells into the CNS and induction of inflammatory cytokines and iNOS in multiple sclerosis (MS) and EAE have been implicated in subsequent disease progression and pathogenesis. To understand the mechanism of efficacy of NAC against EAE, we examined its effect on the production of cytokines and the infiltration of inflammatory cells into the CNS. NAC treatment attenuated the transmigration of mononuclear cells thereby lessening the neuroinflammatory disease. Splenocytes from NAC-treated EAE animals showed reduced IFN-γ production, a Th1 cytokine and increased IL-10 production, an anti-inflammatory cytokine. Further, splenocytes from NAC-treated EAE animals also showed decreased nitrite production when stimulated in vitro by LPS. These observations indicate that NAC treatment may be of therapeutic value in MS against the inflammatory disease process associated with the infiltration of activated mononuclear cells into the CNS

The effect of ammonium on the potassium content of unstriated muscle and its relation to the contraction produced on withdrawal of chemical substances from around the muscle

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A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases

In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol·HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-γ induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-γ caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-γ-induced iNOS, TNF-α, and IL-1β gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IκB phosphorylation, NF-κB DNA binding activity, and NF-κB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-α, and IL-1β expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI

5-Aminoimidazole-4-Carboxamide-1-β-4-Ribofuranoside Inhibits Proinflammatory Response in Glial Cells: A Possible Role of AMP-Activated Protein Kinase

AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis and metabolic stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR) inhibited lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines (tumor necrosis factor α, interleukin-1β, and interleukin-6) and inducible nitric oxide synthase in primary rat astrocytes, microglia, and peritoneal macrophages. AICAR attenuates the LPS-induced activation of nuclear factor κB via downregulation of IκB kinase α/β activity. It also inhibits nuclear translocation of CCAAT/enhancer-binding protein (C/EBP) transcription factor by inhibiting the expression of C/EBP-δ in brain glial cells. The dominant negative form of AMPKα2 (D157A) and its antisense documents a possible role of AMPK in the regulation of the cellular proinflammatory process. AICAR also inhibited the production of inflammatory mediators in serum and their expression in CNS of rats injected with a sublethal dose of LPS by intraperitoneal injection. These observations in cultured cells as well as in the animal model suggest that AICAR may be of therapeutic value in treating inflammatory diseases

Administration of S-nitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact

<p>Abstract</p> <p>Background</p> <p>Traumatic brain injury (TBI) is a major cause of preventable death and serious morbidity in young adults. This complex pathological condition is characterized by significant blood brain barrier (BBB) leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. Following other brain injuries, nitric oxide modulators such as S-nitrosoglutathione (GSNO) maintain not only redox balance but also inhibit the mechanisms of secondary injury. Therefore, we tested whether GSNO shows efficacy in a rat model of experimental TBI.</p> <p>Methods</p> <p>TBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO (50 μg/kg body weight) was administered at two hours after CCI. GSNO-treated injured animals (CCI+GSNO group) were compared with vehicle-treated injured animals (CCI+VEH group) in terms of tissue morphology, BBB leakage, edema, inflammation, cell death, and neurological deficit.</p> <p>Results</p> <p>Treatment of the TBI animals with GSNO reduced BBB disruption as evidenced by decreased Evan's blue extravasation across brain, infiltration/activation of macrophages (ED1 positive cells), and reduced expression of ICAM-1 and MMP-9. The GSNO treatment also restored CCI-mediated reduced expression of BBB integrity proteins ZO-1 and occludin. GSNO-mediated improvements in tissue histology shown by reduction of lesion size and decreased loss of both myelin (measured by LFB staining) and neurons (assayed by TUNEL) further support the efficacy of GSNO therapy. GSNO-mediated reduced expression of iNOS in macrophages as well as decreased neuronal cell death may be responsible for the histological improvement and reduced exacerbations. In addition to these biochemical and histological improvements, GSNO-treated injured animals recovered neurobehavioral functions as evaluated by the rotarod task and neurological score measurements.</p> <p>Conclusion</p> <p>GSNO is a promising candidate to be evaluated in humans after brain trauma because it not only protects the traumatic penumbra from secondary injury and improves overall tissue structure but also maintains the integrity of BBB and reduces neurologic deficits following CCI in a rat model of experimental TBI.</p

Inhibition of NF-κB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia

BACKGROUND: Stroke is one of the leading causes of death worldwide and a major cause of morbidity and mortality in the United States of America. Brain ischemia-reperfusion (IR) triggers a complex series of biochemical events including inflammation. Leukotrienes derived from 5-lipoxygenase (5-LOX) cause inflammation and are thus involved in the pathobiology of stroke injury. METHODS: To test the neuroprotective efficacy of 5-LOX inhibition in a rat model of focal cerebral IR, ischemic animals were either pre- or post-treated with a potent selective 5-LOX inhibitor, (N- [3-[3-(-fluorophenoxy) phenyl]-1-methyl-2-propenyl]-N-hydroxyurea (BW-B 70C). They were evaluated at 24 h after reperfusion for brain infarction, neurological deficit score, and the expression of 5-LOX. Furthermore, the mechanism and the anti-inflammatory potential of BW-B 70C in the regulation of nuclear factor kappa B (NF-κB) and inflammatory inducible nitric oxide synthase (iNOS) were investigated both in vivo and in vitro. RESULTS AND DISCUSSION: Both pre- and post-treatment with BW-B 70C reduced infarctions and improved neurological deficit scores. Immunohistochemical study of brain sections showed IR-mediated increased expression of 5-LOX in the neurons and microglia. BW-B 70C down-regulated 5-LOX and inhibited iNOS expression by preventing NF-κB activation. Two other structurally different 5-LOX inhibitors were also administered post IR: caffeic acid and 2, 3, 5-trimethyl-6- [12-hydroxy-5, 10-dodecadiynyl]-1, 4-benzoquinone (AA-861). As with BW-B 70C, they provided remarkable neuroprotection. Furthermore, in vitro, BW-B 70C inhibited lipopolysaccharide (LPS) mediated nitric oxide production, iNOS induction and NF-κB activation in the BV2 microglial cell line. Treating rat primary microglia with BW-B70C confirmed blockage of LPS-mediated translocation of the p65 subunit of NF-κB from cytosol to nucleus. CONCLUSION: The study demonstrates the neuroprotective potential of 5-LOX inhibition through down-regulation of NF-κB in a rat model of experimental stroke

Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells. We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells. AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL