ABSTRACT
Inhibition of phosphodiesterase 4 (PDE4) suppressed the inflammatory responses in the brain. However, the underlying mechanisms are poorly understood. Roflupram (ROF) is a novel PDE4 inhibitor. In the present study, we found that ROF enhanced the level of microtubule-associated protein 1 light chain 3 II (LC3-II) and decreased p62 in microglial BV-2 cells. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by Lysotracker red and acridine orange staining. In addition, immunofluorescence indicated a significant increase in punctate LC3. Moreover, ß amyloid 25-35 (Aß25-35) or lipopolysaccharide (LPS) with ATP was used to activate inflammasome. We found that both LPS plus ATP and Aß25-35 enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1ß in BV-2 cells. Interestingly, these effects were blocked by the treatment of ROF. Consistently, knocking down the expression of PDE4B in primary microglial cells led to enhanced level of LC-3 II and decreased activation of inflammasome. What's more, Hoechst staining showed that ROF decreased the apoptosis of neuronal N2a cells in conditioned media from microglia. Our data also showed that ROF dose-dependently enhanced autophagy, reduced the activation of inflammasome and suppressed the production of IL-1ß in mice injected with LPS. These effects were reversed by inhibition of microglial autophagy. These results put together demonstrate that ROF inhibits inflammasome activities and reduces the release of IL-1ß by inducing autophagy. Therefore, ROF could be used as a potential therapeutic compound for the intervention of inflammation-associated diseases in the brain.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Autophagy/drug effects , Benzene Derivatives/pharmacology , Furans/pharmacology , Inflammasomes/drug effects , Microglia/drug effects , Phosphodiesterase 4 Inhibitors/pharmacology , Adenine/analogs & derivatives , Adenine/pharmacology , Amyloid beta-Peptides/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Autophagy-Related Protein 7/antagonists & inhibitors , Autophagy-Related Protein 7/genetics , Benzene Derivatives/chemistry , Cells, Cultured , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Drug Evaluation, Preclinical , Female , Furans/chemistry , Gene Expression Regulation/drug effects , Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Molecular Structure , Peptide Fragments/pharmacology , Phosphodiesterase 4 Inhibitors/chemistry , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Random Allocation , Sequestosome-1 Protein/biosynthesis , Sequestosome-1 Protein/geneticsABSTRACT
OBJECTIVE: To investigate the effects of vortioxetine on cAMP/CREB/BDNF signal pathway. METHODS: Forty Kunming mice were randomized into control group and chronic unpredictable mild stress (CUMS) group. After establishment of depressive models verified by sucrose preference test, the mice in CUMS group were divided into model group, fluoxetine group and vortioxetine group. The antidepressive effect of vortioxetine was analyzed by tail suspension test, forced swim test and open field test. The levels of cAMP were detected using a commercial ELISA kit, and the expressions of pCREB and brain-derived neurotrophic factor (BDNF) were evaluated with Western blotting. RESULTS: Vortioxetine significantly shortened the immobility time of the depressive mice in tail suspension test and forced swim test without affecting the locomotor activity of the mice in open fields, suggesting the antidepressive effect of against depression in mice. Vortioxetine significantly increased the levels of cAMP and promoted the expression of pCREB and BDNF in the hippocampus of the mice (P<0.01). CONCLUSION: Vortioxetine improves the behaviors of mice with depression possibly by affecting the cAMP/CREB/BDNF signal pathway.
Subject(s)
Antidepressive Agents/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP/metabolism , Depression/drug therapy , Depression/metabolism , Hippocampus/drug effects , Vortioxetine/pharmacology , Animals , Disease Models, Animal , Hippocampus/metabolism , Immobilization , Locomotion , Mice , Random Allocation , Signal Transduction/drug effects , SwimmingABSTRACT
The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.
Subject(s)
Fluoxetine/pharmacology , Neurons/metabolism , Neuroprotective Agents/pharmacology , Signal Transduction , Animals , Apoptosis , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Corticosterone/toxicity , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Forkhead Box Protein O3/metabolism , Neurons/drug effects , PC12 Cells , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Rats , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
In this study, we evaluated a simple and sensitive method for determination of a novel phosphodiesterase-4 (PDE4) inhibitor, chlorbipram, in mouse plasma and brain using ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). Separation was achieved using an Acquity UPLC BEH C18 column (50mm×2.1mm, particle size 1.7µm) with a gradient mobile phase consisting of water and methanol at a flow rate of 0.25ml/min. Detection was performed in the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive ion mode. The liquid-liquid extraction method with ethyl acetate was used for both pretreatment of plasma and brain homogenates. The calibration curves of chlorbipram showed good linearity over the concentration range of 0.5-200ng/ml (R(2)>0.994) for mouse plasma and over the range of 0.25-100ng/ml (R(2)>0.994) for mouse brain homogenate. The extraction recovery was in the range of 78.3-84.8% for chlorbipram and the internal standard (IS) ZXI14 in two different biological matrices. The intra- and inter-day precision values were less than 13.0% and the accuracy ranged from 97.8% to 106.0% for quality control samples. No noteworthy matrix effects and instability were observed for chlorbipram. This validated method was successfully applied to a pharmacokinetic study of chlorbipram in mice after intravenous administration. The results show that this novel drug crosses the blood-brain barrier and provides the basis for further studies on chlorbipram.
