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Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes.

Daniele S, Zappelli E, Martini C - J Neuroinflammation (2015)

Bottom Line: TDZ had no effect on cell proliferation, but it decreased pro-inflammatory mediator release and modulated trophic and transcription factor mRNA expression.These effects mainly involved an activation of 5-HT1A and an antagonism at 5-HT2A/C serotonin receptors.Fluoxetine, used in parallel, showed similar final effects nevertheless it activates different receptors/intracellular pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Pisa, Via Bonanno Pisano, 6, Pisa, 56126, PI, Italy. simona.daniele@for.unipi.it.

ABSTRACT

Background: In the central nervous system, glial cells provide metabolic and trophic support to neurons and respond to protracted stress and insults by up-regulating inflammatory processes. Reactive astrocytes and microglia are associated with the pathophysiology of neuronal injury, neurodegenerative diseases and major depression, in both animal models and human brains. Several studies have reported clear anti-inflammatory effects of anti-depressant treatment on astrocytes, especially in models of neurological disorders. Trazodone (TDZ) is a triazolopyridine derivative that is structurally unrelated to other major classes of antidepressants. Although the molecular mechanisms of TDZ in neurons have been investigated, it is unclear whether astrocytes are also a TDZ target.

Methods: The effects of TDZ on human astrocytes were investigated in physiological conditions and following inflammatory insult with lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α). Astrocytes were assessed for their responses to pro-inflammatory mediators and cytokines, and the receptors and signalling pathways involved in TDZ-mediated effects were evaluated.

Results: TDZ had no effect on cell proliferation, but it decreased pro-inflammatory mediator release and modulated trophic and transcription factor mRNA expression. Following TDZ treatment, the AKT pathway was activated, whereas extracellular signal-regulated kinase and c-Jun NH2-terminal kinase were inhibited. Most importantly, a 72-h TDZ pre-treatment before inflammatory insult completely reversed the anti-proliferative effects induced by LPS-TNF-α. The expression or the activity of inflammatory mediators, including interleukin-6, c-Jun NH2-terminal kinase and nuclear factor κB, were also reduced. Furthermore, TDZ affected astrocyte metabolic support to neurons by counteracting the inflammation-mediated lactate decrease. Finally, TDZ protected neuronal-like cells against neurotoxicity mediated by activated astrocytes. These effects mainly involved an activation of 5-HT1A and an antagonism at 5-HT2A/C serotonin receptors. Fluoxetine, used in parallel, showed similar final effects nevertheless it activates different receptors/intracellular pathways.

Conclusions: Altogether, our results demonstrated that TDZ directly acts on astrocytes by regulating intracellular signalling pathways and increasing specific astrocyte-derived neurotrophic factor expression and lactate release. TDZ may contribute to neuronal support by normalizing trophic and metabolic support during neuroinflammation, which is associated with neurological diseases, including major depression.

No MeSH data available.


Related in: MedlinePlus

TDZ modulation of the ERK1/2 pathway. a, b Astrocytes were treated with medium alone (basal), or with the indicated concentrations of TDZ, or FLUOX (10 μM) or 5-HT (10 μM)for 30 min (a) or 24 h (b). Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit as described in the Methods section. The data are expressed as phosphorylated/total ERK1/2 ratio. The data are the mean ± SEM of three independent experiments performed in triplicate. c Cells were pre-incubated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist), or 250 nM clonidine (α-adrenergic receptor agonist), or 100 μM histamine (H1 histamine receptor agonist). After 15 min, cells were incubated with TDZ (10 μM) for an additional 30 min. d Human astrocytes were pre-treated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 10 nM GR 127935 (5-HT1B/DR antagonist), or 5 nM RS 127445 (5-HT2BR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist) or 100 nM SR 57227 (5-HT3R agonist). After 15 min, cells were incubated with FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. e Human astrocytes were pre-treated with 200 ng/ml PTX (Gαi/o inhibitor), 1 μM H89 (PKA inhibitor), or 1 μM bisindolylmaleimide (PKC inhibitor), or 500 nM wortmannin (PI3K inhibitor); then, cells were incubated with TDZ (10 μM) or FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. The significance of the differences was determined using a one-way ANOVA-Tukey HSD post hoc test: *P < 0.05, **P < 0.01 ***P < 0.01 vs. basal; ##P < 0.01, ###P < 0.001 vs cells stimulated with TDZ or FLUOX
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Fig10: TDZ modulation of the ERK1/2 pathway. a, b Astrocytes were treated with medium alone (basal), or with the indicated concentrations of TDZ, or FLUOX (10 μM) or 5-HT (10 μM)for 30 min (a) or 24 h (b). Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit as described in the Methods section. The data are expressed as phosphorylated/total ERK1/2 ratio. The data are the mean ± SEM of three independent experiments performed in triplicate. c Cells were pre-incubated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist), or 250 nM clonidine (α-adrenergic receptor agonist), or 100 μM histamine (H1 histamine receptor agonist). After 15 min, cells were incubated with TDZ (10 μM) for an additional 30 min. d Human astrocytes were pre-treated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 10 nM GR 127935 (5-HT1B/DR antagonist), or 5 nM RS 127445 (5-HT2BR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist) or 100 nM SR 57227 (5-HT3R agonist). After 15 min, cells were incubated with FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. e Human astrocytes were pre-treated with 200 ng/ml PTX (Gαi/o inhibitor), 1 μM H89 (PKA inhibitor), or 1 μM bisindolylmaleimide (PKC inhibitor), or 500 nM wortmannin (PI3K inhibitor); then, cells were incubated with TDZ (10 μM) or FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. The significance of the differences was determined using a one-way ANOVA-Tukey HSD post hoc test: *P < 0.05, **P < 0.01 ***P < 0.01 vs. basal; ##P < 0.01, ###P < 0.001 vs cells stimulated with TDZ or FLUOX

Mentions: Challenging cells with FLUOX significantly increased p-ERK (Fig. 10a), consistent with previous reports on the SSRI [64]. Surprisingly, TDZ significantly decreased p-ERK/T-ERK ratio in a concentration-dependent manner after 30 min (Fig. 10a). The concentration-dependence was lost in longer treatment (24 h, Fig. 10b), and TDZ significantly decreased basal ERK activation at 1 nM and 10 μM. These data were confirmed by western blot experiments (Additional file 2: Figure S2).Fig. 10


Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes.

Daniele S, Zappelli E, Martini C - J Neuroinflammation (2015)

TDZ modulation of the ERK1/2 pathway. a, b Astrocytes were treated with medium alone (basal), or with the indicated concentrations of TDZ, or FLUOX (10 μM) or 5-HT (10 μM)for 30 min (a) or 24 h (b). Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit as described in the Methods section. The data are expressed as phosphorylated/total ERK1/2 ratio. The data are the mean ± SEM of three independent experiments performed in triplicate. c Cells were pre-incubated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist), or 250 nM clonidine (α-adrenergic receptor agonist), or 100 μM histamine (H1 histamine receptor agonist). After 15 min, cells were incubated with TDZ (10 μM) for an additional 30 min. d Human astrocytes were pre-treated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 10 nM GR 127935 (5-HT1B/DR antagonist), or 5 nM RS 127445 (5-HT2BR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist) or 100 nM SR 57227 (5-HT3R agonist). After 15 min, cells were incubated with FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. e Human astrocytes were pre-treated with 200 ng/ml PTX (Gαi/o inhibitor), 1 μM H89 (PKA inhibitor), or 1 μM bisindolylmaleimide (PKC inhibitor), or 500 nM wortmannin (PI3K inhibitor); then, cells were incubated with TDZ (10 μM) or FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. The significance of the differences was determined using a one-way ANOVA-Tukey HSD post hoc test: *P < 0.05, **P < 0.01 ***P < 0.01 vs. basal; ##P < 0.01, ###P < 0.001 vs cells stimulated with TDZ or FLUOX
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Fig10: TDZ modulation of the ERK1/2 pathway. a, b Astrocytes were treated with medium alone (basal), or with the indicated concentrations of TDZ, or FLUOX (10 μM) or 5-HT (10 μM)for 30 min (a) or 24 h (b). Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit as described in the Methods section. The data are expressed as phosphorylated/total ERK1/2 ratio. The data are the mean ± SEM of three independent experiments performed in triplicate. c Cells were pre-incubated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist), or 250 nM clonidine (α-adrenergic receptor agonist), or 100 μM histamine (H1 histamine receptor agonist). After 15 min, cells were incubated with TDZ (10 μM) for an additional 30 min. d Human astrocytes were pre-treated with medium alone (basal), or 15 nM (S)-WAY 100135 (5-HT1AR antagonist), or 10 nM GR 127935 (5-HT1B/DR antagonist), or 5 nM RS 127445 (5-HT2BR antagonist), or 30 nM (R)-DOI (5-HT2A/CR agonist) or 100 nM SR 57227 (5-HT3R agonist). After 15 min, cells were incubated with FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. e Human astrocytes were pre-treated with 200 ng/ml PTX (Gαi/o inhibitor), 1 μM H89 (PKA inhibitor), or 1 μM bisindolylmaleimide (PKC inhibitor), or 500 nM wortmannin (PI3K inhibitor); then, cells were incubated with TDZ (10 μM) or FLUOX (10 μM) for an additional 30 min. Following incubation, the levels of phosphorylated and total ERK1/2 were evaluated using an ELISA kit. The significance of the differences was determined using a one-way ANOVA-Tukey HSD post hoc test: *P < 0.05, **P < 0.01 ***P < 0.01 vs. basal; ##P < 0.01, ###P < 0.001 vs cells stimulated with TDZ or FLUOX
Mentions: Challenging cells with FLUOX significantly increased p-ERK (Fig. 10a), consistent with previous reports on the SSRI [64]. Surprisingly, TDZ significantly decreased p-ERK/T-ERK ratio in a concentration-dependent manner after 30 min (Fig. 10a). The concentration-dependence was lost in longer treatment (24 h, Fig. 10b), and TDZ significantly decreased basal ERK activation at 1 nM and 10 μM. These data were confirmed by western blot experiments (Additional file 2: Figure S2).Fig. 10

Bottom Line: TDZ had no effect on cell proliferation, but it decreased pro-inflammatory mediator release and modulated trophic and transcription factor mRNA expression.These effects mainly involved an activation of 5-HT1A and an antagonism at 5-HT2A/C serotonin receptors.Fluoxetine, used in parallel, showed similar final effects nevertheless it activates different receptors/intracellular pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Pisa, Via Bonanno Pisano, 6, Pisa, 56126, PI, Italy. simona.daniele@for.unipi.it.

ABSTRACT

Background: In the central nervous system, glial cells provide metabolic and trophic support to neurons and respond to protracted stress and insults by up-regulating inflammatory processes. Reactive astrocytes and microglia are associated with the pathophysiology of neuronal injury, neurodegenerative diseases and major depression, in both animal models and human brains. Several studies have reported clear anti-inflammatory effects of anti-depressant treatment on astrocytes, especially in models of neurological disorders. Trazodone (TDZ) is a triazolopyridine derivative that is structurally unrelated to other major classes of antidepressants. Although the molecular mechanisms of TDZ in neurons have been investigated, it is unclear whether astrocytes are also a TDZ target.

Methods: The effects of TDZ on human astrocytes were investigated in physiological conditions and following inflammatory insult with lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α). Astrocytes were assessed for their responses to pro-inflammatory mediators and cytokines, and the receptors and signalling pathways involved in TDZ-mediated effects were evaluated.

Results: TDZ had no effect on cell proliferation, but it decreased pro-inflammatory mediator release and modulated trophic and transcription factor mRNA expression. Following TDZ treatment, the AKT pathway was activated, whereas extracellular signal-regulated kinase and c-Jun NH2-terminal kinase were inhibited. Most importantly, a 72-h TDZ pre-treatment before inflammatory insult completely reversed the anti-proliferative effects induced by LPS-TNF-α. The expression or the activity of inflammatory mediators, including interleukin-6, c-Jun NH2-terminal kinase and nuclear factor κB, were also reduced. Furthermore, TDZ affected astrocyte metabolic support to neurons by counteracting the inflammation-mediated lactate decrease. Finally, TDZ protected neuronal-like cells against neurotoxicity mediated by activated astrocytes. These effects mainly involved an activation of 5-HT1A and an antagonism at 5-HT2A/C serotonin receptors. Fluoxetine, used in parallel, showed similar final effects nevertheless it activates different receptors/intracellular pathways.

Conclusions: Altogether, our results demonstrated that TDZ directly acts on astrocytes by regulating intracellular signalling pathways and increasing specific astrocyte-derived neurotrophic factor expression and lactate release. TDZ may contribute to neuronal support by normalizing trophic and metabolic support during neuroinflammation, which is associated with neurological diseases, including major depression.

No MeSH data available.


Related in: MedlinePlus