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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

Effect of TDZ on NF-kB /CREB activation and on the autophagic pathway. a, b Human astrocytes were treated with medium alone or TDZ (1 μM) for 24 or 72 h, and then with LPS-TNF-α for an additional 24 h. NF-kB p65 protein levels were evaluated in cytoplasm and nuclei by western blot analysis. GAPDH and H3 were the loading controls. a Representative western blots. b Densitometric analysis of the immunoreactive bands was performed using ImageJ. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 % and are the mean values ± SEM of three different experiments. c Human astrocytes were treated with medium alone, or TDZ (1 μM) or FLUOX (10 μM) for 24 h or 72 h, and then with LPS-TNF-α for an additional 24 h. At the end of treatment, CREB activation was determined by ELISA, as described in the Methods section. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. d, e Human astrocytes were treated with medium alone, TDZ or FLUOX or Rapamycin (RAPA) for 24 or 72 h. Following incubation, the protein levels of LC3B were evaluated by western blot analysis. GAPDH was the loading control. d Representative western blots. e Densitometric analysis of the immunoreactive bands. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 %, and are the mean values ± SEM of three different experiments. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: **P < 0.01, ***P < 0.001 vs. control; ##P < 0.01, ###P < 0.001 vs. cells treated with LPS-TNF-α
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Fig6: Effect of TDZ on NF-kB /CREB activation and on the autophagic pathway. a, b Human astrocytes were treated with medium alone or TDZ (1 μM) for 24 or 72 h, and then with LPS-TNF-α for an additional 24 h. NF-kB p65 protein levels were evaluated in cytoplasm and nuclei by western blot analysis. GAPDH and H3 were the loading controls. a Representative western blots. b Densitometric analysis of the immunoreactive bands was performed using ImageJ. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 % and are the mean values ± SEM of three different experiments. c Human astrocytes were treated with medium alone, or TDZ (1 μM) or FLUOX (10 μM) for 24 h or 72 h, and then with LPS-TNF-α for an additional 24 h. At the end of treatment, CREB activation was determined by ELISA, as described in the Methods section. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. d, e Human astrocytes were treated with medium alone, TDZ or FLUOX or Rapamycin (RAPA) for 24 or 72 h. Following incubation, the protein levels of LC3B were evaluated by western blot analysis. GAPDH was the loading control. d Representative western blots. e Densitometric analysis of the immunoreactive bands. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 %, and are the mean values ± SEM of three different experiments. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: **P < 0.01, ***P < 0.001 vs. control; ##P < 0.01, ###P < 0.001 vs. cells treated with LPS-TNF-α

Mentions: Following stimuli that elicit the NF-kB pathway, its transcription factor subunits, p65 and p50, expose nuclear targeting signals and then translocate into the nucleus [50]. Consistent with real-time PCR data, LPS-TNF-α induced a significant p65 nuclear accumulation (Fig. 6a, b); astrocyte pre-treatment for 72 h with TDZ (1 μM) significantly counteracted LPS-TNF-α-mediated NF-kB activation (Fig. 6a, b).Fig. 6


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)

Effect of TDZ on NF-kB /CREB activation and on the autophagic pathway. a, b Human astrocytes were treated with medium alone or TDZ (1 μM) for 24 or 72 h, and then with LPS-TNF-α for an additional 24 h. NF-kB p65 protein levels were evaluated in cytoplasm and nuclei by western blot analysis. GAPDH and H3 were the loading controls. a Representative western blots. b Densitometric analysis of the immunoreactive bands was performed using ImageJ. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 % and are the mean values ± SEM of three different experiments. c Human astrocytes were treated with medium alone, or TDZ (1 μM) or FLUOX (10 μM) for 24 h or 72 h, and then with LPS-TNF-α for an additional 24 h. At the end of treatment, CREB activation was determined by ELISA, as described in the Methods section. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. d, e Human astrocytes were treated with medium alone, TDZ or FLUOX or Rapamycin (RAPA) for 24 or 72 h. Following incubation, the protein levels of LC3B were evaluated by western blot analysis. GAPDH was the loading control. d Representative western blots. e Densitometric analysis of the immunoreactive bands. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 %, and are the mean values ± SEM of three different experiments. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: **P < 0.01, ***P < 0.001 vs. control; ##P < 0.01, ###P < 0.001 vs. cells treated with LPS-TNF-α
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Fig6: Effect of TDZ on NF-kB /CREB activation and on the autophagic pathway. a, b Human astrocytes were treated with medium alone or TDZ (1 μM) for 24 or 72 h, and then with LPS-TNF-α for an additional 24 h. NF-kB p65 protein levels were evaluated in cytoplasm and nuclei by western blot analysis. GAPDH and H3 were the loading controls. a Representative western blots. b Densitometric analysis of the immunoreactive bands was performed using ImageJ. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 % and are the mean values ± SEM of three different experiments. c Human astrocytes were treated with medium alone, or TDZ (1 μM) or FLUOX (10 μM) for 24 h or 72 h, and then with LPS-TNF-α for an additional 24 h. At the end of treatment, CREB activation was determined by ELISA, as described in the Methods section. The data are expressed as percentages relative to untreated cells (control), which were set at 100 %, and represent the mean ± SEM of two independent experiments, each performed in duplicate. d, e Human astrocytes were treated with medium alone, TDZ or FLUOX or Rapamycin (RAPA) for 24 or 72 h. Following incubation, the protein levels of LC3B were evaluated by western blot analysis. GAPDH was the loading control. d Representative western blots. e Densitometric analysis of the immunoreactive bands. The data are expressed as the percentage of optical density of the immunoreactive band relative to that of the control, which was set at 100 %, and are the mean values ± SEM of three different experiments. Statistical significance was determined using a one-way ANOVA-Tukey HSD post hoc test: **P < 0.01, ***P < 0.001 vs. control; ##P < 0.01, ###P < 0.001 vs. cells treated with LPS-TNF-α
Mentions: Following stimuli that elicit the NF-kB pathway, its transcription factor subunits, p65 and p50, expose nuclear targeting signals and then translocate into the nucleus [50]. Consistent with real-time PCR data, LPS-TNF-α induced a significant p65 nuclear accumulation (Fig. 6a, b); astrocyte pre-treatment for 72 h with TDZ (1 μM) significantly counteracted LPS-TNF-α-mediated NF-kB activation (Fig. 6a, b).Fig. 6

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