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EP2-PKA signaling is suppressed by triptolide in lipopolysaccharide-induced microglia activation.

Zhang T, Gong X, Hu G, Wang X - J Neuroinflammation (2015)

Bottom Line: We found that triptolide inhibited LPS-induced NO and iNOS synthesis in microglial cells, which in turn protected neurons.Additionally, by further treating triptolide-treated microglia with the downstream PKA-specific activator 6-Bnz-cAMP or the Epac-specific activator 8-pCPT-2-O-Me-cAMP, we found that 6-Bnz-cAMP but not 8-pCPT-2-O-Me-cAMP increased NO production in triptolide-LPS treated microglia.These results indicate that the EP2-PKA pathway is very important for triptolide's effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Capital Medical University, Beijing Institute for Brain Disorders and Key Laboratory for Neurodegenerative Disorder, Ministry of Education, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing, 100069, China. zhang.t@ccmu.edu.cn.

ABSTRACT

Background: Microglia are key players for the inflammatory responses in the central nervous system. Suppression of microglial activation and the resulting production of proinflammatory molecules are considered a promising strategy to alleviate the progression of neurodegenerative disorders. Triptolide was demonstrated as a potent anti-inflammatory compound both in vitro and in vivo. The present study explored potential signal pathways of triptolide in the lipopolysaccharide (LPS)-induced inflammatory response using primary rat microglial cells.

Findings: Microglial cells were pretreated with triptolide and stimulated with LPS. To investigate the anti-inflammatory effect of triptolide, we used Griess reagent and Western blot for NO release and iNOS expression, respectively. Moreover, we applied microglia-conditioned medium to neuronal cells and used the MTS assay to test cell viability. We found that triptolide inhibited LPS-induced NO and iNOS synthesis in microglial cells, which in turn protected neurons. To evaluate the involvement of the EP2 pathway, we used real-time PCR and Western blot to determine EP2 expression. We found that LPS induced a large increase in EP2 expression in microglia, and triptolide almost completely inhibited LPS-induced EP2 expression. Using the selective EP2 agonist butaprost and the EP2 antagonist AH6809, we determined that triptolide inhibited LPS-stimulated NO production in microglia mainly through the EP2 pathway. Additionally, by further treating triptolide-treated microglia with the downstream PKA-specific activator 6-Bnz-cAMP or the Epac-specific activator 8-pCPT-2-O-Me-cAMP, we found that 6-Bnz-cAMP but not 8-pCPT-2-O-Me-cAMP increased NO production in triptolide-LPS treated microglia. These results indicate that the EP2-PKA pathway is very important for triptolide's effects.

Conclusions: Triptolide inhibits LPS-stimulated NO production in microglia via a signaling mechanism involving EP2 and PKA. This finding may help establish the pharmacological function of triptolide in neurodegenerative disorders. Moreover, the observation of inflammatory EP2 signaling in primary microglia provides important evidence that EP2 regulates innate immunity in the central nervous system.

No MeSH data available.


Related in: MedlinePlus

Triptolide protects neuronal cells from the toxicity of LPS-stimulated microglia-conditioned medium. (A) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to MN9D cells. MN9D cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), as well as triptolide alone (50 nM) treated microglia for 24 h. Cell viability was assessed by MTS assay. (B) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to SH-SY5Y cells. SH-SY5Y cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), triptolide alone (50 nM) treated microglia, as well as LPS-stimulated microglia with FeTMPyP (10 μM) for 72 h. Cell viability was assessed by MTS assay. (C) Triptolide did not change primary rat microglial cell viability. Primary rat microglial cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. (D) Triptolide did not change MN9D cell viability. MN9D cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. **P < 0.01. #P < 0.05 and ##P < 0.01.
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Fig2: Triptolide protects neuronal cells from the toxicity of LPS-stimulated microglia-conditioned medium. (A) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to MN9D cells. MN9D cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), as well as triptolide alone (50 nM) treated microglia for 24 h. Cell viability was assessed by MTS assay. (B) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to SH-SY5Y cells. SH-SY5Y cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), triptolide alone (50 nM) treated microglia, as well as LPS-stimulated microglia with FeTMPyP (10 μM) for 72 h. Cell viability was assessed by MTS assay. (C) Triptolide did not change primary rat microglial cell viability. Primary rat microglial cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. (D) Triptolide did not change MN9D cell viability. MN9D cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. **P < 0.01. #P < 0.05 and ##P < 0.01.

Mentions: Activated microglia are capable of releasing neurotoxic molecules, such as proinflammatory cytokines and toxic oxygen and nitrogen species [24,25]. Accumulating evidence shows that activated microglia can damage or kill neurons in vitro by generating nitric oxide (NO) [26-29]. As microglia secreted increased amount of NO in response to LPS stimulation and triptolide inhibited this effect, we reasoned that triptolide may also reduce the neuronal toxicity of LPS-stimulated microglia-conditioned medium. MN9D cell, a fusion of neuroblastoma with mice embryonic ventral mesencephalic cell, was treated with the conditioned medium for 24 h, and an MTS assay was used to assess cell viability. The medium from microglia treated with only triptolide (50 nM) exhibited no toxicity. However, a 24 h exposure to medium from LPS-stimulated microglia caused a significant decrease of MN9D cell viability to 85% of that observed for MN9D cell exposed to medium from untreated microglia. In contrast, there was no significant change of MN9D cell viability between cells exposed to medium from microglia treated with triptolide (50 nM) followed by LPS and unstimulated microglia (Figure 2A). Since triptolide alone did not change the cell viability of MN9D cells or primary rat microglial cells (Figure 2C,D), the results indicated that triptolide-treated microglia attenuated the toxicity of the LPS-stimulated microglia-conditioned medium to MN9D cells.Figure 2


EP2-PKA signaling is suppressed by triptolide in lipopolysaccharide-induced microglia activation.

Zhang T, Gong X, Hu G, Wang X - J Neuroinflammation (2015)

Triptolide protects neuronal cells from the toxicity of LPS-stimulated microglia-conditioned medium. (A) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to MN9D cells. MN9D cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), as well as triptolide alone (50 nM) treated microglia for 24 h. Cell viability was assessed by MTS assay. (B) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to SH-SY5Y cells. SH-SY5Y cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), triptolide alone (50 nM) treated microglia, as well as LPS-stimulated microglia with FeTMPyP (10 μM) for 72 h. Cell viability was assessed by MTS assay. (C) Triptolide did not change primary rat microglial cell viability. Primary rat microglial cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. (D) Triptolide did not change MN9D cell viability. MN9D cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. **P < 0.01. #P < 0.05 and ##P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Triptolide protects neuronal cells from the toxicity of LPS-stimulated microglia-conditioned medium. (A) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to MN9D cells. MN9D cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), as well as triptolide alone (50 nM) treated microglia for 24 h. Cell viability was assessed by MTS assay. (B) Triptolide attenuated the toxicity of conditioned medium from LPS-stimulated microglia to SH-SY5Y cells. SH-SY5Y cells treated with conditioned medium from LPS-unstimulated microglia, LPS-stimulated microglia with or without triptolide (50 nM), triptolide alone (50 nM) treated microglia, as well as LPS-stimulated microglia with FeTMPyP (10 μM) for 72 h. Cell viability was assessed by MTS assay. (C) Triptolide did not change primary rat microglial cell viability. Primary rat microglial cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. (D) Triptolide did not change MN9D cell viability. MN9D cells were treated with triptolide (0, 6.25, 12.5, 25, 50 nM) for 24 h. Cell viability was assessed by MTS assay. **P < 0.01. #P < 0.05 and ##P < 0.01.
Mentions: Activated microglia are capable of releasing neurotoxic molecules, such as proinflammatory cytokines and toxic oxygen and nitrogen species [24,25]. Accumulating evidence shows that activated microglia can damage or kill neurons in vitro by generating nitric oxide (NO) [26-29]. As microglia secreted increased amount of NO in response to LPS stimulation and triptolide inhibited this effect, we reasoned that triptolide may also reduce the neuronal toxicity of LPS-stimulated microglia-conditioned medium. MN9D cell, a fusion of neuroblastoma with mice embryonic ventral mesencephalic cell, was treated with the conditioned medium for 24 h, and an MTS assay was used to assess cell viability. The medium from microglia treated with only triptolide (50 nM) exhibited no toxicity. However, a 24 h exposure to medium from LPS-stimulated microglia caused a significant decrease of MN9D cell viability to 85% of that observed for MN9D cell exposed to medium from untreated microglia. In contrast, there was no significant change of MN9D cell viability between cells exposed to medium from microglia treated with triptolide (50 nM) followed by LPS and unstimulated microglia (Figure 2A). Since triptolide alone did not change the cell viability of MN9D cells or primary rat microglial cells (Figure 2C,D), the results indicated that triptolide-treated microglia attenuated the toxicity of the LPS-stimulated microglia-conditioned medium to MN9D cells.Figure 2

Bottom Line: We found that triptolide inhibited LPS-induced NO and iNOS synthesis in microglial cells, which in turn protected neurons.Additionally, by further treating triptolide-treated microglia with the downstream PKA-specific activator 6-Bnz-cAMP or the Epac-specific activator 8-pCPT-2-O-Me-cAMP, we found that 6-Bnz-cAMP but not 8-pCPT-2-O-Me-cAMP increased NO production in triptolide-LPS treated microglia.These results indicate that the EP2-PKA pathway is very important for triptolide's effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Capital Medical University, Beijing Institute for Brain Disorders and Key Laboratory for Neurodegenerative Disorder, Ministry of Education, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing, 100069, China. zhang.t@ccmu.edu.cn.

ABSTRACT

Background: Microglia are key players for the inflammatory responses in the central nervous system. Suppression of microglial activation and the resulting production of proinflammatory molecules are considered a promising strategy to alleviate the progression of neurodegenerative disorders. Triptolide was demonstrated as a potent anti-inflammatory compound both in vitro and in vivo. The present study explored potential signal pathways of triptolide in the lipopolysaccharide (LPS)-induced inflammatory response using primary rat microglial cells.

Findings: Microglial cells were pretreated with triptolide and stimulated with LPS. To investigate the anti-inflammatory effect of triptolide, we used Griess reagent and Western blot for NO release and iNOS expression, respectively. Moreover, we applied microglia-conditioned medium to neuronal cells and used the MTS assay to test cell viability. We found that triptolide inhibited LPS-induced NO and iNOS synthesis in microglial cells, which in turn protected neurons. To evaluate the involvement of the EP2 pathway, we used real-time PCR and Western blot to determine EP2 expression. We found that LPS induced a large increase in EP2 expression in microglia, and triptolide almost completely inhibited LPS-induced EP2 expression. Using the selective EP2 agonist butaprost and the EP2 antagonist AH6809, we determined that triptolide inhibited LPS-stimulated NO production in microglia mainly through the EP2 pathway. Additionally, by further treating triptolide-treated microglia with the downstream PKA-specific activator 6-Bnz-cAMP or the Epac-specific activator 8-pCPT-2-O-Me-cAMP, we found that 6-Bnz-cAMP but not 8-pCPT-2-O-Me-cAMP increased NO production in triptolide-LPS treated microglia. These results indicate that the EP2-PKA pathway is very important for triptolide's effects.

Conclusions: Triptolide inhibits LPS-stimulated NO production in microglia via a signaling mechanism involving EP2 and PKA. This finding may help establish the pharmacological function of triptolide in neurodegenerative disorders. Moreover, the observation of inflammatory EP2 signaling in primary microglia provides important evidence that EP2 regulates innate immunity in the central nervous system.

No MeSH data available.


Related in: MedlinePlus