ABSTRACT
BACKGROUND: Microglial activation plays an important role in neurodegenerative diseases through production of nitric oxide (NO) and several pro-inflammatory cytokines. Lipoxins (LXs) and aspirin-triggered LXs (ATLs) are considered to act as 'braking signals' in inflammation. In the present study, we investigated the effect of aspirin-triggered LXA4 (ATL) on infiammatory responses induced by lipopolysaccharide (LPS) in murine microglial BV-2 cells. METHODS: BV-2 cells were treated with ATL prior to LPS exposure, and the effects of such treatment production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) were analysed by Griess reaction, ELISA, western blotting and quantitative RT-PCR. Moreover, we investigated the effects of ATL on LPS-induced nuclear factor-κB (NF-κB) activation, phosphorylation of mitogen-activated protein kinases (MAPKs) and activator protein-1 (AP-1) activation. RESULTS: ATL inhibited LPS-induced production of NO, IL-1ß and TNF-α in a concentration-dependent manner. mRNA expressions for iNOS, IL-1ß and TNF-α in response to LPS were also decreased by ATL. These effects were inhibited by Boc-2 (a LXA4 receptor antagonist). ATL significantly reduced nuclear translocation of NF-κB p65, degradation of the inhibitor IκB-α, and phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in BV-2 cells activated with LPS. Furthermore, the DNA binding activity of NF-κB and AP-1 was blocked by ATL. CONCLUSIONS: This study indicates that ATL inhibits NO and pro-inflammatory cytokine production at least in part via NF-κB, ERK, p38 MAPK and AP-1 signaling pathways in LPS-activated microglia. Therefore, ATL may have therapeutic potential for various neurodegenerative diseases.
Subject(s)
Aspirin/pharmacology , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Lipoxins/pharmacology , Microglia , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Line , Enzyme Activation , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Lipoxins/metabolism , Mice , Microglia/cytology , Microglia/drug effects , Microglia/metabolism , Mitogen-Activated Protein Kinases/genetics , NF-kappa B/genetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE: The neuroprotective effect of erythropoietin (EPO) against 1-methyl-4-phenylpyridinium (MPP(+))-induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated. METHODS: PC12 cells impaired by MPP(+) were used as the cell model of Parkinson's disease. Methyl thiazolyl tetrazolium (MTT) was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was used to analyze the apoptosis ratio of PC12 cells. The expression of Bcl-2 and Bax in PC12 cells were examined by Western blot, and the reactive oxygen species (ROS), the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer. RESULTS: Treatment of PC12 cells with MPP(+) caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP(+) significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL. CONCLUSION: The inhibitive effect of EPO on the MPP(+)-induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson's disease.
Subject(s)
1-Methyl-4-phenylpyridinium/toxicity , Erythropoietin/pharmacology , Herbicides/toxicity , Neuroprotective Agents/pharmacology , PC12 Cells/drug effects , Analysis of Variance , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Interactions , Flow Cytometry/methods , In Situ Nick-End Labeling/methods , Membrane Potential, Mitochondrial/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Reactive Oxygen Species/metabolism , Tetrazolium Salts , Thiazoles , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Objective To observe the activating effect of ciliary neurotrophic factor (CNTF) on astrocyte in vitro. Methods Astrocytes cultured purely from newborn rats. Cerebral cortex was raised in normal and serum deprivation condition with different concentrations (in ng/ml: 0, 2, 20, or 200) of CNTF. After cultured for 24 h, the shape and the cell cycle of astrocytes were examined by immunocytochemistry and flow cytometer, respectively. Results The immunoactivity of glial fibrillary acidic protein (GFAP) and the nuclear size of astrocytes were increased when CNTF was applied, whether cells were cultured in medium with or without serum. CNTF promoted astrocytes to enter the cell cycle in medium with serum, but had no this effect in medium without serum. Conclusion In medium without serum, astrocytes could differentiate into activated state cells with CNTF application, but could not proliferate; in medium with serum, astrocytes could proliferate with aid of CNTF.
