RESUMEN
Traumatic brain injury (TBI) is caused by physical damage to the brain and it induces blood-brain barrier (BBB) breakdown and inflammation. To diminish the sequelae of TBI, it is important to decrease haemorrhage and alleviate inflammation. In this study, we aimed to determine the effects of 2-carba-cyclic phosphatidic acid (2ccPA) on the repair mechanisms after a stab wound injury as a murine TBI model. The administration of 2ccPA suppressed serum immunoglobulin extravasation after the injury. To elucidate the effects of 2ccPA on inflammation resulting from TBI, we analysed the mRNA expression of inflammatory cytokines. We found that 2ccPA prevents a TBI-induced increase in the mRNA expression of Il-1ß, Il-6, Tnf-α and Tgf-ß1. In addition, 2ccPA reduces the elevation of Iba1 levels. These data suggest that 2ccPA attenuates the inflammation after a stab wound injury via the modulation of pro-inflammatory cytokines release from microglial cells. Therefore, we focused on the function of 2ccPA in microglial polarisation towards M1 or M2 phenotypes. The administration of 2ccPA decreased the number of M1 and increased the number of M2 type microglial cells, indicating that 2ccPA modulates the microglial polarisation and shifts them towards M2 phenotype. These data suggest that 2ccPA treatment suppresses the extent of BBB breakdown and inflammation after TBI.
Asunto(s)
Microglía/citología , Microglía/metabolismo , Ácidos Fosfatidicos/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/metabolismo , Femenino , Inmunohistoquímica , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-6/genética , Ratones , Microglía/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta1/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Vitronectin (VN), one of the extracellular matrix proteins, controls the maturation of cerebellar granule cells (CGCs) through the promotion of the initial differentiation stage progress. However, the receptors of VN in the initial differentiation stage of CGC precursors (CGCPs) have not been clarified. In this study, we characterized the receptor candidates for VN in CGCPs. First, we confirmed that αvß3 and αvß5 integrins, which are receptor candidates for VN, were co-localized with VN in the developing cerebellum and primary cultured CGCPs. Next, the knockdown (KD) of αv, ß3, and ß5 integrins with small interference RNA (siRNA) for each integrin reduced the ratio of Tuj1, a final differentiation marker, -positive CGCPs. We further studied whether αvß3 and αvß5 integrins control the initial differentiation stage. The KD of αv and ß5, but not ß3, integrins significantly increased the ratio of transient axonal glycoprotein 1 (TAG1), an initial differentiation marker, -positive CGCPs, whereas the KD of αv and ß3 integrins, not ß5 integrin, stimulated the proliferation of CGCPs. Overexpression of ß5 integrin stimulated the progress of the initial differentiation stage as well. To confirm the interaction between αvß5 integrin and VN, VN was added to ß5 integrin-KD CGCPs. The promotion of the progress of initial differentiation by VN was abrogated by ß5 integrin KD using small hairpin RNA (shRNA). Taken together, our results indicated that αvß5 integrin, as the very receptor of VN, is responsible for the progress of the initial differentiation stage in mouse CGCPs.
Asunto(s)
Cerebelo/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células-Madre Neurales/fisiología , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Cerebelo/crecimiento & desarrollo , Contactina 2/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Fosfopiruvato Hidratasa/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Tubulina (Proteína)/metabolismo , Vitronectina/metabolismoRESUMEN
The proliferation and differentiation of cerebellar granule cell precursors (GCPs) are highly regulated spatiotemporally during development. We focused on cyclic phosphatidic acid (cPA) as a lipid mediator with a cyclic phosphate group as a regulatory factor of GCPs. While its structure is similar to that of lysophosphatidic acid (LPA), its function is very unique. cPA is known to be present in the cerebellum at high levels, but its function has not been fully elucidated. In this study, we examined the role of cPA on the proliferation and differentiation of GCPs. A cell cycle analysis of GCPs revealed that cPA reduced the number of phospho-histone H3 (Phh3)-positive cells and bromodeoxy uridine (BrdU)-incorporated cells and increased an index of the cell cycle exit. We next analyzed the effect of cPA on GCP differentiation using Tuj1 as a neuronal marker of final differentiation. The results show that cPA increased the number of Tuj1-positive cells. Further analysis of the proliferation of GCPs showed that cPA suppressed Sonic hedgehog (Shh)-dependent proliferation, but did not suppress insulin-like growth factor-1 (IGF-1)-dependent proliferation. P2Y5 (LPA6), an LPA receptor, is highly expressed in GCPs. The knockdown of P2Y5 suppressed the inhibitory effect of cPA on the proliferation of GCPs, suggesting that P2Y5 is a candidate receptor for cPA. Thus, cPA suppresses the Shh-dependent proliferation of GCPs and promotes the differentiation of GCPs through P2Y5. These results demonstrate that cPA plays a critical role in the development of GCPs.
