RESUMEN
Epidemiological studies suggest that intake of omega-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease which exhibit inflammatory pathology. We therefore investigated the anti-inflammatory effects of eicosapentaenoic acid (EPA) on interleukin (IL)-1beta-stimulated C6 glioma cells. In the present study, EPA inhibited pro-inflammatory cytokine IL-6 production, a characteristic of certain neurodegenerative disorders, in IL-1beta-stimulated C6 glioma cells in dose-dependent fashion. EPA down-regulated the expression of IL-6 at mRNA level, indicating that the effect of EPA occurs at the transcriptional level. In addition, peroxisome proliferator-activated receptor (PPAR) gamma antagonists abolished the inhibitory effect of EPA on IL-1beta-induced IL-6 production, whereas PPARalpha antagonist did not block the inhibitory effect of EPA. EPA might thus contribute to the regulation of pro-inflammatory cytokine production in astrocytes through interaction with PPARgamma. Among the PPARgamma ligands tested in this study, ciglitazone, a synthetic agonist of PPARgamma, effectively inhibited IL-6 production, but while neither rosiglitazone nor 15-deoxy-Delta(12,14)-prostaglandin J2 did. These findings indicate that the coordination of PPAR gamma ligands is important in inhibiting the production of IL-6 in C6 glioma cells.
Asunto(s)
Ácido Eicosapentaenoico/farmacología , Glioma/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/antagonistas & inhibidores , PPAR gamma/metabolismo , Anilidas/farmacología , Animales , Benzamidas/farmacología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-6/farmacología , Ácidos Grasos Insaturados/farmacología , Glioma/tratamiento farmacológico , Indoles/farmacología , Interleucina-1beta/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , PPAR gamma/efectos de los fármacos , PPAR gamma/genética , Piridinas/farmacología , ARN/efectos de los fármacos , ARN/genética , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Tiazolidinedionas/farmacología , Células Tumorales CultivadasRESUMEN
Excessive accumulation of phospholipids results in phospholipidosis (PL), which may interfere with cellular functions, leading to acute or chronic disease or even death. Electron-microscopic detection of cytoplasmic lamellar bodies is often used as a diagnostic criterion of PL, but a faster, more convenient procedure is required for high-throughput assay of the PL-inducing potential of candidate drugs. We have developed a 96-well microplate cell-culture method for detecting PL, using a phosphatidylcholine-conjugated dye (NBD-PC) and a fluoro-microplate reader. The fluorescence intensity due to NBD-PC was normalized to that of Hoechst33342, used as an indicator of cell number, to obtain the amount of NBD-PC taken up per living cell. To select a suitable cell type, we examined the PL-detection sensitivity of five cell lines, as well as human and rat primary hepatocyte cultures, with five cationic amphiphilic drugs (CAD) as PL inducers and a negative control compound. The cell lines CHO-K1 and CHL/IU gave the best results. The NBD-PC uptake per CHO-K1 cell showed a high correlation with the pathological score of PL for 24 compounds, including PL-positive and negative compounds. This high-throughput screening assay for PL-inducing potential (HTS-PL assay) offers high sensitivity and accuracy, and it allows simultaneous determination of cytotoxicity.