RESUMO
Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKß and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKß. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-ß (IFN-ß) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation.
Assuntos
Encéfalo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Frutosedifosfatos/farmacologia , Molécula 1 de Adesão Intercelular/biossíntese , Fármacos Neuroprotetores/farmacologia , Receptor 4 Toll-Like/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Frutosedifosfatos/administração & dosagem , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Células U937RESUMO
BACKGROUND AND PURPOSE: Prostaglandin E2 (PGE2) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells. EXPERIMENTAL APPROACH: bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE2 with or without agonists/antagonists of PGE2 receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry. KEY RESULTS: PGE2 significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE2 effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N6-Bnz-cAMP). PGE2-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11-7082 and MG-132) attenuated the induction of ICAM-1 by PGE2. PGE2, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti. CONCLUSION AND IMPLICATIONS: Our findings suggest that PGE2 induces ICAM-1 expression via EP4 receptor and Epac/Akt/NF-κB signalling pathway in bEnd.3 brain endothelial cells, supporting its pathophysiological role in brain inflammation.
Assuntos
Encéfalo/irrigação sanguínea , Dinoprostona/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Receptores de Prostaglandina E Subtipo EP4/agonistas , Sistemas do Segundo Mensageiro , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , AMP Cíclico/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Fructose-1,6-bisphosphate (FBP), a glycolytic intermediate, has neuroprotective effects in various brain injury models. However, its effects on blood-brain barrier (BBB) are largely unknown. In this study, we investigated the effects of FBP on lipopolysaccharide (LPS)-induced BBB dysfunction in in vitro BBB model comprising co-culture of mouse brain endothelial cell line, bEnd.3 and mouse primary astrocyte and explored its action mechanism therein involved. LPS induced the impairment of endothelial permeability and transendothelial electrical resistance (TEER). The functional changes were confirmed by alterations in immunostaining for junctional proteins occludin, ZO-1 and VE-cadherin, such as the loss of cortical staining pattern and appearance of intercellular gaps in endothelial cells. Co-administration of FBP alleviated the deleterious effects of LPS on BBB permeability and TEER in a dose dependent manner. And also FBP inhibited the LPS-induced changes in the distribution of endothelial junctional proteins, resulting in the better preservation of monolayer integrity. FBP suppressed the production of reactive oxygen species (ROS) but did not affect cyclooxygenase-2 expression and prostaglandin E2 production in endothelial cells stimulated with LPS. Taken together, these data suggest that FBP could ameliorate LPS-induced BBB dysfunction through the maintenance of junctional integrity, which might be mediated by downregulation of ROS production.
Assuntos
Astrócitos/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Frutosedifosfatos/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos ICR , Ratos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
AIMS: Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with neuroprotective action in various brain injury models. However, the mechanism underlying the neuroprotection of FBP has not been fully defined. In this study, we investigated whether FBP inhibits endotoxin-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in microglial cells and explored the possible mechanisms of the effects of FBP. MAIN METHODS: Murine microglial cell line BV2 and primary cultured murine microglial cells were used. NO production and iNOS expression were determined by Griess reaction, RT-PCR and Western blot. Luciferase assay using iNOS promoter-luciferase (iNOS-Luc) construct was adopted for measuring transcriptional activity. KEY FINDINGS: FBP dose-dependently suppressed lipopolysaccharide (LPS)-induced NO production, along with reducing the expression of iNOS at both the protein and mRNA level in primary cultured murine microglia and BV2 cells. FBP significantly inhibited iNOS promoter activity but stabilized iNOS mRNA. Among transcription factors known to be related to iNOS expression, activator protein (AP-1) activation was significantly blocked by FBP. FBP suppressed LPS-induced phosphorylation of three MAPK subtypes-p38 MAPK, JNK and ERK. FBP inhibited LPS-induced production of reactive oxygen species (ROS) and decreased intracellular GSSG/GSH ratio. SIGNIFICANCE: Our findings suggest that FBP attenuates the LPS-induced iNOS expression through inhibition of JNK and p38 MAPK, which might be related to ROS downregulation.