EP2-PKA signaling is suppressed by triptolide in lipopolysaccharide-induced microglia activation
© Zhang et al.; licensee BioMed Central. 2015
Received: 29 September 2014
Accepted: 2 March 2015
Published: 14 March 2015
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.
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.
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.
KeywordsTriptolide EP2 Microglia Neuroinflammation Nitric oxide
Microglia, the immune-like cells of the brain, play an important role in inflammatory responses in the central nervous system (CNS) . The modulation of microglial activation and their production of pro-inflammatory mediators and cytokines could also be a promising strategy to alleviate the progression of neurodegenerative disorders [2-5]. The cyclooxygenase-2 (COX-2) pathway mediates the main inflammatory responses in microglia and is thus a very attractive target for researchers and drug developers [6,7]. However, the deleterious cardiovascular and cerebrovascular side effect of sustained COX-2 inhibition has led to the investigation of its downstream targets . Among these targets, prostaglandin E2 (PGE2) signaling via its EP2 receptor subtype appears to be a major mediator of inflammatory and anaphylactic reactions within both the periphery and brain . EP2 upregulation by lipopolysaccharide (LPS) contributes to cerebral oxidative damage and secondary neurotoxicity, effects that are usually accompanied by the induction of NO synthase (NOS) and cyclooxygenase (COX) activities . EP2 receptor not only serves as a downstream target of COX2 but also acts to influence COX2 and iNOS expression. It was reported that iNOS and COX2 induction was completely absent in EP2 KO microglia after LPS treatment, indicating that EP2 was necessary for the induction of iNOS and COX2 after LPS stimulation . Moreover, LPS-activated microglia-mediated neurotoxicity was completely abolished in cultures lacking microglial EP2, indicating that microglial EP2 was critical to LPS-activated microglia-mediated neurotoxicity in vitro .
Triptolide is the major active component of Tripterygium extracts and possesses potent anti-inflammatory and immunosuppressive properties . Our group demonstrated for the first time that triptolide possesses potent neuroprotective properties both in vitro and in vivo . In an LPS-challenged inflammation model of Parkinson’s disease (PD), intraperitoneal injection with triptolide for 24 days significantly improved the behavior of PD rats, decreased DAergic neuron death, and increased DA levels in the striatum . Further study in primary cultured rat microglia indicated that triptolide inhibits LPS-induced microglial activation and suppresses COX-2 expression and PGE2 release . In the current study, we investigated the main pathway of triptolide in LPS-induced inflammatory responses in primary rat microglial cells. We found that triptolide suppressed LPS-induced nitric oxide (NO) production and inducible NO synthase (iNOS) synthesis in primary rat microglial cells, which in turn protects neuronal cells from microglia-conditioned medium-induced cell injury. Moreover, triptolide inhibits LPS-stimulated NO production in microglia via a signaling mechanism involving EP2 and PKA.
Triptolide was generously provided by Professor Peng-FeiTu (School of Pharmaceutical Sciences, Peking University, Beijing, China). This white crystalline drug has a melting point of 226°C to 240°C and, for this study, was 98% pure, as evaluated by reverse-phase high pressure liquid chromatography. The following materials were used for these studies: LPS (Sigma-Aldrich, St. Louis, MO, USA), a rabbit polyclonal antibody to iNOS (Abcam, Cambridge, UK), a rabbit polyclonal antibody to EP2 (Cayman, MI, USA), a mouse monoclonal antibody to CD11b (Millipore, Billerica, MA, USA), a mouse monoclonal antibody to GAPDH (Sigma-Aldrich), butaprost (Cayman, MI, USA), AH6809 (Cayman, MI, USA), 6-Bnz-cAMP (BIOLOG Life Science Institute, Bremen, Germany), 8-pCPT-2-O-Me-cAMP (Millipore, Billerica, MA, USA), KT5720 (Life Technologies, Carlsbad, CA, USA), FBS (Hyclone, Logan, UT, USA), streptomycin and penicillin (Life Technologies), 0.2-ml syringe filters, 96- and 24-well tissue culture plates, 100-mm diameter dishes (Corning, NY, USA), Dulbecco’s modified Eagle’s medium (DMEM), and DMEM/F-12 (Life Technologies).
The immortalized murine BV2 microglial cell line was purchased from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). MN9D cells were kindly provided by Dr. Bastian Hengerer (Novartis Institute for BioMedical Research, Basel, Switzerland). The two cell lines were maintained in 5% CO2 at 37°C in DMEM/F-12 supplemented with 10 % fetal bovine serum (FBS), 1% streptomycin, and penicillin. The primary rat microglial cells were prepared as described previously . Briefly, cerebral cortices of 0- to 1-day-old SD rats, devoid of meninges and blood vessels, were dissociated by mild mechanical trituration. The isolated cells were seeded in 75-cm2 culture flasks (1.5 brains in a flask) in DMEM/F-12 containing 10% FBS, 1% penicillin, and streptomycin. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Fourteen days later, the microglia were separated from astrocytes by shaking the flasks at 180 rpm for 2 h. The purity of the enriched microglia was >95%, as identified by CD11b (dilution, 1:800) immunocytochemical staining. All of the results are expressed as the mean ± standard deviation (SD) of at least three independent experiments performed in duplicate. One-way analysis of variance (ANOVA) and Newman-Keuls multiple comparison tests were used to compare the groups. The differences were considered to be significant at P < 0.05. The experimental procedures in this study were approved by the Committee on Animal Care and Usage (Capital Medical University), and efforts were engaged to minimize the number of animal usage and suffering.
To investigate the inhibitory effect of triptolide on LPS-induced NO and iNOS synthesis, primary rat microglial cells were pretreated for 30 min with different concentrations of triptolide and subsequently stimulated with LPS. As shown in Figure 1A,B, triptolide dose dependently inhibited LPS-induced NO synthesis at concentrations from 12.5 to 50 nM. In BV2 cells, pretreatment with triptolide led to a dose-dependent inhibition on LPS-induced NO release by 6% (P < 0.05) at 12.5 nM, 22% (P < 0.01) at 25 nM, and 42% (P < 0.001) at 50 nM (Figure 1A). In primary microglial cells, pretreatment with triptolide led to a dose-dependent inhibition on LPS-induced NO release by 30% (P < 0.05) at 12.5 nM, 41% (P < 0.01) at 25 nM, and 61% (P < 0.001) at 50 nM (Figure 1B). As further illustrated in Figure 1C,D, the inhibitory effect of triptolide on NO synthesis was due to a dose-dependent inhibition of iNOS synthesis. iNOS synthesis was inhibited at a concentration of 25 nM and decreased significantly at a concentration of 50 nM triptolide. These results were consistent with a previous study in which Shen et al. reported that 30 nM triptolide inhibits NO production in LPS-activated macrophages .
We confirm the neuroprotective effect of triptolide in SH-SY5Y cells. The neuroblastoma cell line SH-SY5Y is often used in the cellular model of PD due to its dopaminergic ability [30,31]. As shown in Figure 2B, conditioned media from LPS-stimulated primary microglial cells significantly (P < 0.01) increased cell death of SH-SY5Y cells. Cell viability decreased to 76% of cells treated with medium from untreated microglia. This result is consistent with that reported by Liu et al. , Munch et al. , and Tseng et al. . In contrast, the conditioned media from primary microglia cells pretreated with triptolide prior to LPS stimulation showed little neurotoxicity on SH-SY5Y cells, suggesting that triptolide suppresses microglia-mediated neurotoxicity.
To further investigate the role of NO release from microglia in toxicity of microglia to neuronal cells, we treated microglial cells with peroxynitrite (ONOO-) decomposition catalysts FeTMPyP [5,10,15,20-tetrakis(n-methyl-4′-pyridyl) porphinato iron (III) chloride]. NO reacts with superoxide (O2−), producing the highly reactive and toxic peroxynitrite (ONOO-) . In the present study, we observed FeTMPyP at 10 μM blocked the LPS-induced microglial neurotoxicity in SH-SY5Y cells (Figure 2B), indicating peroxynitrite is a main mediator for the neurotoxicity of LPS-induced microglia.
The above findings that triptolide reduced the amount of proinflammatory factors in microglia and protected neurons from NO stimulation may help establish the pharmacological function of triptolide in neurodegenerative disorders.
To investigate the effect of triptolide on LPS-regulated EP2 expression, microglia were pretreated for 30 min with triptolide (50 nM) and subsequently stimulated with LPS. As shown in Figure 4B, 50 nM triptolide almost completely inhibited LPS-induced EP2 expression. To confirm the triptolide inhibition of EP2 expression, we next monitored the protein level changes of EP2 by Western blot (Figure 4C,D). Although the changes in EP2 protein levels were not as dramatic as were observed for mRNA levels, triptolide did inhibit EP2 protein expression.
EP2 activation leads to increased levels of cytoplasmic cAMP, which then initiates multiple downstream events via the PKA or Epac pathways. To differentiate between these two pathways, we added the PKA-specific activator 6-Bnz-cAMP (6Bnz) or the Epac-specific activator 8-pCPT-2-O-Me-cAMP (8-CPT) to triptolide-treated microglia. We found that 6Bnz (10 μM) but not 8-CPT (10 μM) increased NO production in triptolide-LPS treated microglia (Figure 5B), indicating that EP2-PKA was mainly involved in the triptolide inhibition. This specificity was most likely due to the nature of LPS stimulation. Peters-Golden et al. reported that in rat alveolar macrophages, the down-modulation of LPS-induced TNF-α by PGE2 is dependent on cAMP-PKA activation. This was concluded given that the selective PKA activating cAMP analog 6Bnz but not the Epac-1 activating analog 8-CPT inhibited TNF-α production . Their recent work also indicates that EP2-PKA, rather than Epac-1, is involved in the enhancement of LPS-induced NO . This result suggests that the EP2-PKA pathway is very important for the anti-inflammatory effect of triptolide in microglia. More studies are needed to clarify whether the EP2-PKA signaling pathway is the dominant mediator of harmful EP2-mediated effects in microglia.
We further observe this effect in BV2 cells, and found 6Bnz (10 μM) increased NO production in triptolide-LPS treated microglia (Figure 5C). To confirm that PKA is indeed responsible for the inhibition of NO generation by triptolide, BV2 cells were pre-treated with the PKA inhibitor KT5720, and the PKA-specific activator 6Bnz was added before triptolide treatment. We found that 6Bnz (10 μM) increased NO production in triptolide-LPS treated microglia, KT5720 attenuated NO production to an extent similar to that observed with triptolide treatment (Figure 5C), suggesting that PKA is responsible for the triptolide inhibition.
The direct target of triptolide in the regulation of EP2-PKA pathway remains unknown. Liu et al. report that triptolide directly binds to human XPB, a subunit of the transcription factor TFIIH, leading to the inhibition of RNA polymerase II-mediated transcription in tumor cells . However, these authors used higher concentrations of triptolide (1 to 100 μM) to inhibit tumor cell proliferation. We used lower concentrations of triptolide (no more than 50 nM) to suppress inflammation and to protect neurons in the brain. Therefore, different targets may exist for triptolide with respect to the modulation of the inflammatory response in the CNS. Recently, Shen et al. reported that triptolide inhibited TAK1 kinase activity by interfering with the formation of the TAK1-TAB1 complex, and that the binding affinity of triptolide to TAB1 was highly correlated with the inhibitory activity of triptolide against the MAPK pathway activation in macrophages . Although macrophages and microglia are both tissue-resident immune cells during inflammation and the effective triptolide concentration was similar in both cell types, it remains to be investigated whether TAB1 is the target of triptolide in microglia.
The present study demonstrates that triptolide inhibits LPS-stimulated NO production in microglia via a signaling mechanism involving EP2 and PKA. The finding that triptolide reduces the proinflammatory factors in microglia and protects neurons from inflammatory stimulation may help establish the pharmacological function of triptolide in neurodegenerative disorders. Moreover, the observation of inflammatory EP2 signaling in primary microglia also provides important evidence with respect to EP2 in the regulation of innate immunity in the CNS.
inducible NO synthase
- PGE2 :
central nervous system
G protein coupled receptor
protein kinase A
exchange proteins that are directly activated by cAMP
mitogen-activated protein kinase
Dulbecco’s modified Eagle’s medium
fetal bovine serum
polymerase chain reaction
This study was supported by the Chinese National Basic Research Program (2011CB504100), the National Natural Science Foundation of China (81030062), the Projects of Beijing Municipality (TJSHG201310025006), and the Beijing Higher Education Young Elite Teacher Project (YETP1667).
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