| 1. IntroductionDepression is a highly prevalent and severely debilitating disorder for which current treatments are inadequate and the pathogenesis of which is poorly understood. 40 years ago, the monoamine hypothesis of depression was first put forward which proposes that the underlying biological or neuroanatomical basis for depression is a deficiency of central noradrenergic and/or serotonergic systems. To date, immuno-logic abnormalities in depression have been described for two decades and proinflammatory cytokines may be involved in the pathophysiology of depression, which was called "cytokine hypothesis of depression".Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication. The relationship between cytokines and depression was observed from clinical patients and experimental animals. For example, patients with depression showed high levels of proinflammatory cytokines in serum. And cytokine therapy, like IFN-a, could induce depressive symptoms when it was administrated in patients with hepatitis C or various forms of cancer. In addition, rodents treated with LPS could also exhibit sickness-like behavior.Increasing evidence indicates that microglial activation play an important role in the pathogenesis of depression. Microglia, the resident macrophages of the central nervous system (CNS), are now recognized as the primary component of the brain immune system. Once activated, microglia change their morphology, proliferate and upregulate surface molecules. In addition, activated microglia have the capability of producing proinflammatory cytokines and neurotoxic mediators such as nitric oxide (NO), prostaglandin (PG) E2, and superoxide anion. Several lines of evidence have demonstrated that excessive proinflammatory cytokines are involved in the pathophysiology of depression. For example, proinflammatory cytokines, including interferon(IFN)-y, interleukin-6(IL-6), and tumor necrosis factor-alpha (TNF-α), have been shown to increase the expression of indoleamine-2,3-dioxygenase (IDO) enzyme in both central and peripheral immune-competent cell types. IDO, the first rate-limiting tryptophan(TRP)-degrading enzyme, could break down tryptophan, the primary amino acid precursor of serotonin, into kynurenine (KYN). The breakdown of TRP is believed to contribute to reduced serotonin availability which has been shown to play an important role in the induction of depressive symptoms. Moreover, proinflammatory cytokines have been found to interact with many of the pathophysiological domains that characterize depression, including neurotransmitter metabolism, neuroendocrine function, synaptic plasticity and behavior. In addition, proinflammatory mediators like NO, have been shown to exert a negative effect on brain neurogenesis and participate in stress-induced depression. Thus, controlling microglial activation and neuroinflammatory processes may prove to be a therapeutic benefit in the treatment of depression.Of note, proinflammatory cytokines and their signaling pathways can also contribute to pathogenesis of depression. Mitogen activated protein kinase (MAPK) pathways, including p38 and extracellular signal-regulated kinases (ERK) 1/2, which mediate the effects of cytokines on cell proliferation/differentiation and apoptosis, as well as gene expression of inflammatory mediators, have been found to have a negative effect on both monoamine synthesis and reuptake, leading to reduced monamine availability. In addition, nuclear factor-kappaB (NF-κB), one of relevant inflammatory signaling molecules, also plays an important role in depression. NF-κB, once activated in immune cells, results in the release of inflammatory mediators that promote inflammation. Proinflammatory cytokines, in turn, can access the brain, induce inflammatory signaling pathways including NF-κB, and ultimately contribute to altered monoamine metabolism, increased excitotoxicity, and decreased production of relevant trophic factors.Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is commonly prescribed for treating major depression due to its tolerability and safety. It is well known that the therapeutic action of fluoxetine is ascribed to selective inhibition of presynaptic serotonin reuptake. Recently, it has been reported that fluoxetine has anti-inflammatory effects in animal models of peripheral inflammation. For example, in septic shock and allergic asthma animal models, fluoxetine was found to suppress the number of inflammation-related cells and TNF-a release from monocytes. In addition, fluoxetine was found to have anti-inflammatory effects in a carrageenan-induced paw inflammation model.Although the anti-inflammatory effects of fluoxetine in the peripheral tissues have been well documented, its effects on cells in the CNS, specifically the microglia have remained elusive. Indeed, the detailed molecular mechanisms underlying the inhibitory effects of fluoxetine on microglial cells are not fully understood. Therefore, in this study, we sought to investigate the pharmacological effect of fluoxetine on microglia activated by lipopolysaccharide (LPS). Along with this, we also examined its molecular mechanism regulating microglial production of proinflammtory mediators such as TNF-a, IL-6 and NO. In this connection, two putative pathways including MAPK and NF-κB activation that may be involved in the anti-inflammatory effects of fluoxetine were also investigated.2. Materials and methods2.1 BV2 microglial cell cultureBV2 cells were maintained in Dulbecco’s modified Eagle medium with 10% fetal bovine serum in a 5% CO2 incubator. In all experiments, cells were treated with the indicated concentrations of fluoxetine in the absence or presence of LPS (100ng/ml) in serum-free DMEM.2.2. Primary microglial cell culturePrimary microglia were prepared from the whole brains of 1-2 days old BALB/c mouse as described previously. Briefly, glial cells were cultured for 14 days in DMEM/F12 supplemented with 10% FBS. Isolated microglial cells were plated into 24-well plates at a density of 2×105 cells/well. The purity of microglia cultures was assessed using CD11b antibody; more than 90% of cells were stained positively. Cells were cultured for 2 days before drug treatment.2.3. Cell viability assay Cell viability was determined by the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. BV2 cells were plated into 96-well culture plates at a density of 5×104 cells/ml with 200μl culture medium per well. Following treatment with different concentrations of fluoxetine,20μl MTT solution (5 mg/ml) was added to each well and incubated at 37℃for 4 h. The medium was aspirated and 200μl dimethyl sulfoxide was added. The absorbance value was measured using a multiwell spectrophotometer at 490 nm.2.4. Enzyme-linked immunosorbent assay (ELISA)BV2 cells were treated with different concentrations of fluoxetine in the presence or absence of LPS. The supernatant of the BV2 cells was collected at 24 h after drug stimulation. The levels of cytokines including TNF-a and IL-6 in culture medium were measured using commercially available enzyme-linked immunosorbent assay kits according to manufacturer’s instructions. Briefly, serial dilutions of protein standards and samples were added to 96-well ELISA plates, followed by biotinylated anti-TNF-a or IL-6 antibody. After rinsing with wash buffer, the prepared solution of avidin, horseradish peroxidase -conjugated complex was added followed by addition of substrate solution. The reaction was stopped by the stopping solution. The optical density was detected at 450 nm in a microplate reader. The concentration of each sample was calculated from the linear equation derived from the standard curve of known concentrations of the cytokine.2.5. NO release assayAccumulation of nitrite in the culture medium for 24 h stimulation was measured by a Griess reaction. Nitrite was taken as a measure of NO production. The supernatant collected was mixed with an equal volume of Griess reagent in a 96-well plate and incubated at room temperature for 10 min. Absorbance was measured at 550 nm in a microplate reader. Sodium nitrite, diluted in culture medium at concentrations ranging from 10 to 100μM, was used to prepare a standard curve.2.6. Reverse transcription-polymerase chain reactions (RT-PCR)Total RNA was extracted from induced cell cultures using the Trizol reagent according to the manufacturer’s instructions. RNA concentration was determined by a spectrophotometer at 260 nm. Identical amounts of RNA (1μg) were reverse transcribed into cDNA by using a commercial RT-PCR kit according to the manufacturer’s instructions. cDNA was subsequently amplified by PCR with specific primers PCR was conducted by using the following conditions for 32 cycles: denaturation at 94℃for 30s, annealing at 60℃for 45s, and extension at 72℃for 30s. PCR products separated on a 1.2% agarose/TAE gel were visualized by staining with ethidium bromide. The densitometric analysis of the data was normalized toβ-actin. The intensity of bands was determined using the Image-Pro Plus 6.0 software.2.7. Western blotting analysisCells were rinsed with cold phosphate buffered saline (PBS) and lysed in cold lysis buffer. Cell lysates were incubated at 4℃for 20 min. The sample was centrifuged at 12,000 rpm for 10 min at 4℃, then the supernatant was collected and protein content was assayed colorimetrically.30μg total proteins were loaded onto a 12% SDS-PAGE gel, electrophoretically transferred to polyvinylidene difluoride membrane. The membranes were incubated with primary antibodies at 4℃overnight following incubation of secondary antibodies for 1h. The membranes were developed using an enhanced chemiluminescence (ECL) detection system. The intensity of bands was determined using the Image-Pro Plus 6.0 software.2.8. Immunofluorescence assayMicroglial cells were stained for CD11b, inducible nitric oxide synthase (iNOS), NF-κB p65 and phospho-p38 MAPK protein expression. Briefly, the cells were plated on glass coverslips overnight. After drug treatment they were fixed in 4% paraformaldehyde for 10 min. After permeabilisation with 0.3% Triton-X100 in PBS, the cells were blocked with 10% FBS in PBS. Then, the cells were incubated in the primary antibody overnight at 4℃. Following primary antibody incubation, cells were washed again and incubated in the appropriate fluorescent-conjugated secondary antibody for 1 h. The cells were counterstained by DAPI. Images were captured with a Nikon TE2000U microscope. To analyze CD11b staining in the primary microglial culture, at least three representative images were taken accordingly. A threshold for positive staining was determined for each image that included all cell bodies and processes, but excluded the background staining. The images were analyzed using Image-Pro Plus 6.0 software.2.9. Nuclear protein extraction and electrophoretic mobility shift assay (EMSA)Nuclear extracts from treated microglial cells were prepared as follows. After treatment, BV2 cells were treated with 1 ml of lysis buffer on ice for 4 min. After 10 min of centrifugation at 3000 rpm, the pellet was resuspended in 50μl of extraction buffer and incubated on ice for 30 min. After centrifugation at 14000 rpm for 5 min, the supernatant was harvested as the nuclear protein extract and stored at-70℃. Protein concentration was determined by using the BCA protein assay. The double-stranded DNA oligonucleotides containing the NF-κB consensus sequences were end-labeled using the digoxigenin (DIG) gel shift kit. Five micrograms of the nuclear proteins was incubated with DIG-labeled NF-κB oligonucleotides and EMSA was performed according to manufacturer’s instructions. Competition studies were performed by incubating samples, before the addition of the DIG-labeled NF-κB probe, in the absence and presence of 100-fold molar excess of unlabeled specific (5’-AGTTGAGGGGACTTTCCCAGGC-3’) and nonspecific (5’-GCAGAGCATATA AGGTGAGGTAGGA-3’) double-stranded oligonucleotides.2.10. Transient transfection and luciferase AssayTransfection of the NF-κB reporter gene into BV2 cells was performed using Lipofectamine 2000 according to the manufacturer’s instructions. The NF-κB reporter plasmid contained three copies of theκB-binding sequence fused to firefly luciferase gene. BV2 cells were transfected with 0.8μg of the reporter construct along with 0.04μg of Renilla luciferase plasmid. After 48 h, cells were harvested and a luciferase assay was performed using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions. To determine the effect of fluoxetine on LPS-induced NF-κB activity, cells were treated with 10μM fluoxetine in the absence or presence of LPS and incubated for 6 h prior to harvesting cells for luciferase assay. Luciferase activity was measured using a Monolight 2010 luminometer. Renilla luciferase activity was used as an internal control. The relative luciferase activity was then calculated by normalizing firefly luciferase activity to Renilla luciferase activity. 3. Results3.1. Fluoxetine inhibits LPS-induced microglial activationPrior to the investigation of the effects of fluoxetine on microglial cells, MTT assay was performed to determine its cytotoxicity to BV2 cells after 24 h incubation with the different concentrations of fluoxetine. Cell viability following treatment with fluoxetine at 0.1μM (94.51±4.29%), 1μM (96.00±12.47%), and 10μM (98.90±21.21%) was not significantly different from the control (99.99±4.38%). However, exposure of BV2 cells to fluoxetine at 50μM resulted in significantly fewer viable cells (65.78±3.9%) as compared to cells in the control.It has been previously reported that microglial activation is associated with marked increase in CD11b expression. Immuno- fluorescence analysis showed that, after 24 h incubation with LPS (100ng/ml), the expression of CD11b was evidently increased. Treatment with fluoxetine (10μM) attenuated LPS-mediated upregulation of CD11b in primary microglial cells.3.2. Effect of the fluoxetine on cytokine production in LPS-stimulated microglial cellsTo assess whether fluoxetine could inhibit production of LPS-induced pro-inflammatory cytokines including TNF-a and IL-6, BV2 cells were treated with LPS in the absence or presence of fluoxetine for 24 h. As shown in Fig.3, fluoxetine (10μM) alone had no effect on the production of TNF-a and IL-6 in BV2 cells. Stimulation of BV2 cells with LPS led to a significant increase in the TNF-a and IL-6 levels after 24 h. Treatment with fluoxetine significantly inhibited their production in BV2 cells in a dose-dependent manner. To elucidate the mechanism responsible for the inhibitory effect of fluoxetine on TNF-a and IL-6 production, we next examined the cytokine mRNA expression levels by RT-PCR. Consistent with the results obtained from the cytokine production, the LPS-stimulated mRNA levels of TNF-a and IL-6 were reduced by fluoxetine, suggesting that fluoxetine negatively regulated the production of TNF-a and IL-6 at the transcriptional level in the LPS-stimulated microglial cells.3.3. Fluoxetine decreased NO production in LPS-stimulated microglial cellsTo investigate the effects of fluoxetine on NO production in LPS-stimulated BV2 cells, cells were treated with LPS alone or with various concentrations of fluoxetine for 24 h. The levels of NO in the culture media were determined with the Griess assay. Fluoxetine significantly decreased the LPS-induced production of NO in BV2 cells in a dose-dependent manner. Next, to elucidate the mechanism responsible for the inhibitory effect of fluoxetine on NO production, we determined the iNOS mRNA and protein levels by RT-PCR and Western blot analysis. At a concentration of 10μM, fluoxetine effectively inhibited iNOS mRNA expression at 6 h after drug treatment in BV2 cells. The protein levels of iNOS were also repressed by fluoxetine at 24 h after drug treatment in BV2 cells. To confirm the above result, immunofluorescence assay was performed to observe intracellular iNOS levels using primary microglial cells. The result showed that iNOS immunofluorescence which was enhanced by LPS was also decreased by fluoxetine (10μM) at 24 h after LPS stimulation in primary microglial cells. These results showed that fluoxetine inhibited NO production through downregulation of iNOS mRNA and protein expression in LPS-stimulated microglial cells.3.4. Fluoxetine inhibits LPS-induced phosphorylation of p38 MAPKWe then assessed whether the repressive effect of fluoxetine on synthesis and release of proinflammatory mediators occurred via MAPK signaling pathway. BV2 cells were treated with 10μM fluoxetine in the presence or absence of LPS for 30 minutes. Fluoxetine did not inhibit LPS-induced phosphorylation levels of ERK1/2 and JNK MAPKs, while LPS-induced phosphorylation of p38 MAPK was markedly inhibited by fluoxetine in BV2 cells. To confirm the above result, immunofluorescence labeling for detection of intracellular phosphorylation of p38 MAPK was also carried out in primary microglia cells. The results showed that upon treatment with LPS, there was an increased immunofluorescence of phospho-p38 MAPK, which was also significantly reduced by fluoxetine in primary microglial cells. These findings indicate that fluoxetine is effective in the inhibition of p38 MAPK phosphorylation in LPS-stimulated microglial cells.3.5. Fluoxetine inhibits LPS-indcuced NF-κB activation in microglial cellsNF-κB has been shown to be one of the most important upstream modulators for pro-inflammatory cytokines and iNOS expression in microglia. Thus, we next determined whether the repressive effect of fluoxetine on gene expression occurred via blockade of NF-κB activity in microglial cells.First, we examined the influence of fluoxetine on the IκB-αand NF-κB activity by Western blot and immunofluorescence analysis. LPS treatment induced markedly degradation of IκB-α, which was reversed by fluoxetine in BV2 cells at 30 min after drug treatment. In addition, fluoxetine significantly suppressed LPS-induced phosphorylation levels of NF-κB p65 in BV2 cells. The above results were further confirmed by immunofluorescence analysis. NF-κB p65 was mainly localized in the cytoplasm, treatment with LPS induced NF-κB p65 translocation from cytoplasm to the nucleus. However, fluoxetine decreased the translocation of NF-κB p65 into the nuclei of BV2 cells.Based on the above results, the involvement of NF-κB activity in fluoxetine-induced suppression of pro-inflammatory cytokines and NO was further examined by EMS A and luciferase assay. Stimulation of BV2 cells with LPS resulted in strong NF-κB binding, which was significantly inhibited by fluoxetine. Specificity of protein binding to the DIG-labeled probe was also tested. When excess unlabeled NF-κB oligonucleotides was added to protein extracts from LPS-stimulated BV2 cells, binding to the DIG-labeled oligonucleotides was abolished. Further, addition of an unlabeled non-competitive oligonucleotides to protein extracts had no effect on NF-κB binding.In addition, fluoxetine also affected NF-κB-mediated transcription in BV2 cells. The NF-κB transcriptional activity was assayed by transfecting the BV2 cells with a plasmid containing three NF-κB binding sites and a luciferase reporter gene. The luciferase assay showed that fluoxetine repressed the LPS-stimulated NF-κB transcriptional activation in a dose-dependent manner. In sum, these results indicated the potential role of NF-κB in the possible mechanism of fluoxetine in suppressing proinflammatory cytokines and NO in activated microglia.In sum, the effect of fluoxetine on the inflammatory activation of microglia has been determined. This study has shown that fluoxetine inhibited LPS-induced production of inflammatory mediators such as TNF-a, IL-6 and NO, and their gene expression in microglia. In addition, the anti-inflammatory property of fluoxetine was mediated by the inhibition of p38 MAPK phosphorylation and NF-κB activation in LPS-stimulated microglial cells.These data suggest that therapeutic effects of fluoxetine are partially mediated by their anti-inflammatory actions on microglial activation. |
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