Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia.
J Neuroinflammation
; 10: 23, 2013 Feb 06.
Article
en En
| MEDLINE
| ID: mdl-23388509
ABSTRACT
BACKGROUND:
Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury.METHODS:
One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining.RESULTS:
TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1ß and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1ß, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1ß and iNOS on microglia post-hypoxia.CONCLUSION:
Activated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Encéfalo
/
Hipoxia Encefálica
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Microglía
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Mediadores de Inflamación
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Receptor Toll-Like 4
Límite:
Animals
Idioma:
En
Revista:
J Neuroinflammation
Asunto de la revista:
NEUROLOGIA
Año:
2013
Tipo del documento:
Article
País de afiliación:
China