RESUMEN
BACKGROUND Cerebral ischemia-reperfusion injury (CIRI) remains a serious health problem. Centella asiatica formulations are used to treat central nervous system disorders. In the present study, asiaticoside, an extract of the plant Centella asiatica, was investigated in CIRI in vivo and vitro. MATERIAL AND METHODS We made a CIRI model in vivo in SD rats treated by middle cerebral artery occlusion, and a cell model of ischemia-reperfusion injury was made in PC12 cells treated by deprivation of oxygen and glucose/restoration. CIRI in vivo was assessed by scores of neurological functions, encephaledema, and cerebral infarction area. Inflammation level and oxidative stress level were detected by the appropriate kits. TUNEL assay was performed for assessment of cell apoptosis and Western blot analysis was performed to assess protein expression levels. CCK8 assay was performed for evaluation of cell survival and flow cytometer was used to detect cell apoptosis in vitro. RESULTS Nervous function injury, brain edema, cell apoptosis, infarct size, apoptosis-related protein expressions, and protein expressions of the NOD2/MAPK/NF-kappaB signaling pathway in the CIRI model were all reversed by asiaticoside in rats. The cell apoptosis, inflammation level, and oxidative stress level in the model of cerebral ischemia-reperfusion injury were reduced by asiaticoside. The effects of asiaticoside on CIRI were reversed by NOD 2 agonists. CONCLUSIONS Asiaticoside showed a protective effect against cerebral ischemia-reperfusion injury via the NOD2/MAPK/NF-kappaB signaling pathway. These findings are vital for future research on use of asiaticoside in CIRI, providing a new avenue for alleviating CIRI.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Transducción de Señal , Triterpenos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/patología , Edema Encefálico/fisiopatología , Infarto Encefálico/tratamiento farmacológico , Infarto Encefálico/patología , Infarto Encefálico/fisiopatología , Supervivencia Celular/efectos de los fármacos , Inflamación/patología , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/fisiopatología , Triterpenos/farmacologíaRESUMEN
The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/farmacología , Adyuvantes Inmunológicos/farmacología , Ácido Diaminopimélico/análogos & derivados , Microglía/efectos de los fármacos , Proteína Adaptadora de Señalización NOD1/agonistas , Proteína Adaptadora de Señalización NOD2/agonistas , Fenotipo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Forma de la Célula , Extensiones de la Superficie Celular/efectos de los fármacos , Células Cultivadas , Ácido Diaminopimélico/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microglía/citología , Microglía/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Toll-like receptors (TLRs) and nuclear-binding domain (NOD)-like receptors (NLRs) are sensors of bacterial cell wall components to trigger an immune response. The TLR4 agonist lipopolysaccharide (LPS) is a strong immune activator leading to sickness and depressed mood. NOD agonists are less active but can prime immune cells to augment LPS-induced cytokine production. Since the impact of NOD and TLR co-activation in vivo has been little studied, the effects of the NOD1 agonist FK565 and the NOD2 agonist muramyl dipeptide (MDP), alone and in combination with LPS, on immune activation, brain function and sickness behavior were investigated in male C57BL/6N mice. Intraperitoneal injection of FK565 (0.001 or 0.003mg/kg) or MDP (1 or 3mg/kg) 4h before LPS (0.1 or 0.83mg/kg) significantly aggravated and prolonged the LPS-evoked sickness behavior as deduced from a decrease in locomotion, exploration, food intake and temperature. When given alone, FK565 and MDP had only minor effects. The exacerbation of sickness behavior induced by FK565 or MDP in combination with LPS was paralleled by enhanced plasma protein and cerebral mRNA levels of proinflammatory cytokines (IFN-γ, IL-1ß, IL-6, TNF-α) as well as enhanced plasma levels of kynurenine. Immunohistochemical visualization of c-Fos in the brain revealed that NOD2 synergism with TLR4 resulted in increased activation of cerebral nuclei relevant to sickness. These data show that NOD1 or NOD2 synergizes with TLR4 in exacerbating the immune, sickness and brain responses to peripheral immune stimulation. Our findings demonstrate that the known interactions of NLRs and TLRs at the immune cell level extend to interactions affecting brain function and behavior.
Asunto(s)
Encéfalo/inmunología , Conducta de Enfermedad/fisiología , Proteína Adaptadora de Señalización NOD1/fisiología , Proteína Adaptadora de Señalización NOD2/fisiología , Receptor Toll-Like 4/fisiología , Acetilmuramil-Alanil-Isoglutamina/farmacología , Adyuvantes Inmunológicos/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Corticosterona/sangre , Citocinas/sangre , Citocinas/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Conducta de Enfermedad/efectos de los fármacos , Quinurenina/sangre , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Proteína Adaptadora de Señalización NOD1/agonistas , Proteína Adaptadora de Señalización NOD2/agonistas , Oligopéptidos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Receptor Toll-Like 4/agonistas , Triptófano/sangreRESUMEN
The precise regulation of odontoblast differentiation and osteoclastogenic cytokine expression in human dental pulp cells (HDPCs) is crucial for the pathology of bacteria-related pulpitis. Although the up-regulation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been reported in inflamed human dental pulps, the role of NOD2 in the differentiation of HDPCs remains unclear. Here, we show the involvement of NOD2 in odontoblast differentiation together with osteoclastogenic cytokine expression in HDPCs. Treatment with muramyl dipeptide (MDP), a known NOD2-agonist, significantly inhibited odontoblast differentiation of HDPCs, as revealed by reduced ALP activity, osteoblast/odontoblast marker expression, and mineralized nodule formation. Importantly, the forced down-regulation of NOD2 by small interfering RNA (siRNA) recovered MDP-down-regulated odontoblast differentiation. MDP-elicited suppression of odontoblast differentiation resulted from the increased expression of MKP-1 protein and the subsequent decline of MAPKs phosphorylation, which is a prerequisite for odontoblast differentiation. Furthermore, we found that MDP treatment elevated the expression of osteoclastogenic cytokines in HDPCs, which was also reversed by NOD2 silencing. Analysis of these data, taken together, suggests that the regulation of NOD2 expression upon MDP challenge might serve as an intrinsic mechanism that underlies the hindered dentin formation and accelerated dentin resorption in bacterial infection-mediated pulpitis.