JOURNAL OF
NEUROINFLAMMATION

Zhang et al. Journal of Neuroinflammation (2015):
DOI 10.1186/s12974-015-0275-y

SHORT REPORTOpen Access

EP2-PKA signaling is suppressed by triptolide in
lipopolysaccharide-induced microglia activation
Ting Zhang1, Xiaoli Gong2, Guanzheng Hu1 and Xiaomin Wang1*

Abstract
Background: Microglia are key players for the inflammatory responses in the central nervous system. Suppressionof microglial activation and the resulting production of proinflammatory molecules are considered a promisingstrategy 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 foundthat 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.
Keywords: Triptolide, EP2, Microglia, Neuroinflammation, Nitric oxide

Findings
Microglia, the immune-like cells of the brain, play animportant role in inflammatory responses in the central
nervous system (CNS) [1]. 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* Correspondence: xmwang@ccmu.edu.cn
1
Department of Neurobiology, Capital Medical University, Beijing Institute forBrain Disorders and Key Laboratory for Neurodegenerative Disorder, Ministryof Education, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing
100069, China
Full list of author information is available at the end of the article

is thus a very attractive target for researchers and drug
developers [6,7]. However, the deleterious cardiovascularand cerebrovascular side effect of sustained COX-2 inhibition has led to the investigation of its downstream targets[8]. Among these targets, prostaglandin E2 (PGE2) signaling via its EP2 receptor subtype appears to be a major mediator of inflammatory and anaphylactic reactions withinboth the periphery and brain [9]. 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 [10]. 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© 2015 Zhang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

Zhang et al. Journal of Neuroinflammation (2015):

absent in EP2 KO microglia after LPS treatment, indicating that EP2 was necessary for the induction of iNOS and
COX2 after LPS stimulation [10]. Moreover, LPS-activatedmicroglia-mediated neurotoxicity was completely abolished in cultures lacking microglial EP2, indicating thatmicroglial EP2 was critical to LPS-activated microgliamediated neurotoxicity in vitro [10].
Triptolide is the major active component of Tripterygium extracts and possesses potent anti-inflammatory andimmunosuppressive properties [11]. Our group demonstrated for the first time that triptolide possesses potent
neuroprotective properties both in vitro and in vivo [12].
In an LPS-challenged inflammation model of Parkinson’s
disease (PD), intraperitoneal injection with triptolide for24 days significantly improved the behavior of PD rats, decreased DAergic neuron death, and increased DA levels inthe striatum [13]. Further study in primary cultured rat
microglia indicated that triptolide inhibits LPS-induced
microglial activation and suppresses COX-2 expression
and PGE2 release [14]. 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 nitricoxide (NO) production and inducible NO synthase(iNOS) synthesis in primary rat microglial cells, which inturn 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 PengFeiTu (School of Pharmaceutical Sciences, Peking University, Beijing, China). This white crystalline drug has amelting 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), 6Bnz-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 andpenicillin (Life Technologies), 0.2-ml syringe filters, 96and 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 waspurchased from Cell Resource Center, Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences(Beijing, China). MN9D cells were kindly provided byPage 2 of 9

Dr. Bastian Hengerer (Novartis Institute for BioMedical
Research, Basel, Switzerland). The two cell lines weremaintained 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 [14]. Briefly, cerebral
cortices of 0- to 1-day-old SD rats, devoid of meninges andblood 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 themean ± 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 thegroups. The differences were considered to be significant at P < 0.05. The experimental procedures in thisstudy were approved by the Committee on Animal Careand Usage (Capital Medical University), and efforts
were engaged to minimize the number of animal usage
and suffering.
To confirm the anti-inflammatory effect of triptolide,
we first investigated NO and iNOS production in themurine BV2 microglial cell line and in primary rat
microglial cells. BV2 cells and primary rat microglial
cells were cultured in 12-well plates and stimulated withLPS (0.01 μg/mL) for 24 h. At the end of the incubation
period, supernatants were collected. NO measurements
were made using the Griess Reagent System (Promega,
Madison, WI, USA), and iNOS expression was measured
by Western blot. In BV2 cells, LPS significantly induced
NO release to 8 μM compared with 1 μM in naïve cells(Figure 1A). In primary microglia cells, LPS significantly
induced NO release to 12 μM compared with 2 μM innaïve cells (Figure 1B). NO is synthesized by a family of
NOS consisting of three isoforms: endothelial NOS(eNOS), neuronal NOS (nNOS), and inducible NOS(iNOS) [15]. Among them, NO produced from iNOS
[16] promotes the development of neurodegenerative
disorders associated with inflammation [17]. In thepresent study, the increase of NO correlated well with
the induction of iNOS by LPS (Figure 1C, lane 2). BV2cells and primary rat microglia both showed basal release of nitrite, which has been well demonstrated inprevious reports by different laboratories [18-21]. Moreover, Ryuet al. reported that neither iNOS inhibitors nor
nonselective nitric-oxide synthase inhibitors had any effect on the basal release of nitrite [22].

Zhang et al. Journal of Neuroinflammation (2015):

A

C

Page 3 of 9

B

D

Figure 1 Triptolide suppresses NO production and iNOS expression in LPS-activated microglia. (A) Triptolide reduced NO production in
BV2 cells. BV2 cells were stimulated with LPS (0.01 μg/mL) in the presence or absence of different concentrations of triptolide (0, 12.5, 25, 50 nM) for
24 h. At the end of the incubation period, the supernatants were collected for NO measurements. (B) Triptolide reduced NO production in primary rat
microglial cells. Primary rat microglial cells were stimulated with LPS (0.01 μg/mL) in the presence or absence of triptolide (0, 12.5, 25, 50 nM) for 24 h.
At the end of the incubation period, the supernatants were collected for NO measurements. (C) Triptolide reduced iNOS expression in primary rat
microglial cells. Primary rat microglial cells were stimulated with LPS (0.01 μg/mL) in the presence or absence of triptolide (0, 12.5, 25, 50 nM) for 24 h.
The cells were then lysed to detect iNOS expression by Western blot. (D) The protein levels were quantified relative to GAPDH levels and normalized
to the LPS-stimulated group. ***P < 0.001. #P < 0.05, ##P < 0.01, and ###P < 0.001.

To investigate the inhibitory effect of triptolide on LPSinduced 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 adose-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-activatedmacrophages [23].
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 theneuronal toxicity of LPS-stimulated microglia-conditioned
medium. MN9D cell, a fusion of neuroblastoma with miceembryonic ventral mesencephalic cell, was treated withthe conditioned medium for 24 h, and an MTS assay
was used to assess cell viability. The medium frommicroglia treated with only triptolide (50 nM) exhibitedno toxicity. However, a 24 h exposure to medium from
LPS-stimulated microglia caused a significant decreaseof 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.
We confirm the neuroprotective effect of triptolide in
SH-SY5Y cells. The neuroblastoma cell line SH-SY5Y isoften 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

Zhang et al. Journal of Neuroinflammation (2015):

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Figure 2 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 wasassessed 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 (50nM) 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 wasassessed 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.

cells significantly (P < 0.01) increased cell death of SHSY5Y cells. Cell viability decreased to 76% of cells treatedwith medium from untreated microglia. This result is
consistent with that reported by Liu et al. [32], Munch
et al. [33], and Tseng et al. [34]. In contrast, the conditioned media from primary microglia cells pretreatedwith 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 frommicroglia in toxicity of microglia to neuronal cells, we
treated microglial cells with peroxynitrite (ONOO-) decomposition catalysts FeTMPyP [5,10,15,20-tetrakis(nmethyl-4′-pyridyl) porphinato iron (III) chloride]. NO
reacts with superoxide (O2−), producing the highly reactive and toxic peroxynitrite (ONOO-) [35]. In thepresent 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 amountof proinflammatory factors in microglia and protected
neurons from NO stimulation may help establish thepharmacological function of triptolide in neurodegenerative disorders.
Microglial EP2 is critical to the neurotoxicity caused by
the activation of cerebral innate immunity. To investigate
whether EP2 is involved in the anti-inflammatory effect of
triptolide in LPS-stimulated microglia, we first examinedEP2 expression in primary rat microglial cells. We observed the basal expression of EP2 in resting microglia(Figure 3A). As a stimulatory G protein coupled receptor(GPCR), EP2 activation stimulates adenylate cyclase (AC).
This effect results in an elevation of cytoplasmic cAMPlevels, initiating multiple downstream events. To assess thefunctionality of the EP2 receptor in microglia, the cAMPlevel was determined by treating microglia with butaprost(10 μM), a selective EP2 agonist, or with AH6809 (10 μM),
an EP2 antagonist. As shown in Figure 3B, there was atrend of increased intracellular cAMP accumulation in
microglia treated with 10 μM butaprost for 3 h compared
to control. By contrast, the treatment of microglia with
10 μM AH6809 for 3 h decreased the intracellularcAMP accumulation. Butaprost alone increased thebasal level of microglia NO production (Figure 3C).
This may be due to the iNOS expression influenced by

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Figure 3 EP2 signaling pathway regulates NO release in primary rat microglial cells. (A) EP2 expression in primary rat microglial cells.
Immunofluorescent staining of EP2 and CD11b, as well as the nuclear stain DAPI are shown (scale bar, 25 μm). (B) cAMP accumulation in rat
microglia cells left untreated or treated with butaprost (10 μM) or AH6809 (10 μM) for 3 h (n = 3). (C) Butaprost stimulated NO production in
resting microglia. Primary rat microglial cells were stimulated with different concentrations of butaprost (0, 0.1, 1, 10 μM) for 24 h. At the end of
the incubation period, the supernatants were collected for NO measurements. (D) AH6809 suppressed NO production in LPS-activated microglia.
Primary rat microglial cells were unstimulated or stimulated with LPS (0.01 μg/mL) in the presence or absence of AH6809 (0.1, 1, 10 μM) for 24 h.
At the end of the incubation period, the supernatants were collected for NO measurements. ***P < 0.001 and ##P < 0.01.

butaprost; as Quanet al. reported, iNOS mRNA level in
resting rat microglia was increased more than tenfold
after 200 nM and 2 μM butaprost treatment [36]. Another EP2 agonist, ONO-AE1-259 was also reported toincrease iNOS mRNA level in ileal tissue [37]. By contrast, AH6809 inhibited LPS-induced NO synthesis(Figure 3D). Since NO levels in the medium are also affected by proliferation and death of the treated microglia, we observed the cell viability after EP2 agonist andantagonist and the PKA agonist and antagonist treatment in BV2 cells and found no changes (Additional file 1:
Figure S1). Thus, under an innate inflammatory stimulus,
microglia EP2 signaling potentiated a classically activatedproinflammatory response, inducing NO production.
To evaluate the involvement of the EP2 pathway in triptolide’s effect, real-time PCR of EP2 was performed on aStratagene Mx3000P (Agilent, Santa Clara, CA, USA)using the following parameters: 95°C for 10 min, followed
by 40 cycles at 95°C for 15 s, 60°C for 1 min, and 72°C for
1 min using the specific primer sets. The following primer
pairs were employed: 5′ - TTGCTCTTCTGTTCTCTG
CCG - 3′ (upper, 538 to 558) and 5′ - CAGCTGAAGGTATGCGGTCC - 3′ (lower, 642 to 623) for the amplificationof rat EP2 receptor (GenBank accession No. NM_031088)
and 5′ - ATCGCTGACAGGATGCAGAAG - 3′ (upper,
925 to 945) and 5′ - AGAGCCACCAATCCACACAGA 3′ (lower, 1,032 to 1,012) for the amplification of rat βactin. Firstly, we observed the EP2 mRNA level at 1, 3, 6,
12, and 24 h after LPS induction and found that LPS augmented the EP2 expression significantly at 6 h (Figure 4A).

The relative expression level compared with that of resting
microglia increased to nearly 12-fold with LPS, which issimilar as that reported by Noda et al. [38]. Moreover, weobserved a prompt downregulation of EP2 mRNA level
after 12-h LPS treatment. Johansson et al. reported that
stimulation of macrophages with LPS (10 ng/ml) induced arapid increase in EP2 mRNA within 1 h followed by aprompt downregulation by 6 h [39]. The different time ofEP2 mRNA upregulation between macrophages and microglia maybe caused by their local microenvironment, as wellas the differences between periphery and central inflammation, but the similar changes of EP2 expression induced by
LPS indicate that EP2 expression is tightly regulated by inflammatory stimuli.
To investigate the effect of triptolide on LPS-regulated
EP2 expression, microglia were pretreated for 30 min
with triptolide (50 nM) and subsequently stimulatedwith 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 byWestern 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.
To confirm the necessary role of EP2, we treated primary rat microglial cells with butaprost/AH6809 andtriptolide before LPS stimulation. As shown in Figure 5A,
treatment with triptolide or AH6809 (10 μM) alone bothdecreased NO production in LPS-treated microglia toZhang et al. Journal of Neuroinflammation (2015):

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Figure 4 Triptolide regulates EP2 expression in LPS-stimulated microglia. (A) Rat primary microglia cells were stimulated with LPS (0.01 μg/mL)
for 0, 1, 3, 6, 12, and 24 h. The cells were lysed for EP2 expression by real-time PCR analysis. (B) Rat primary microglia cells were stimulated with LPS(0.01 μg/mL) in the presence or absence of triptolide (50 nM) for 6 h. The cells were lysed for EP2 expression by real-time PCR analysis. (C) Primary rat
microglial cells were stimulated with LPS (0.01 μg/mL) in the presence or absence of triptolide (50 nM) for 24 h. The cells were lysed for EP2 expression
by Western blot. (D) The protein levels were quantified relative to GAPDH levels and normalized to the naïve group (untreated cells). **P < 0.01. ***P < 0.001.
##
P < 0.01 and ###P < 0.001.

similar levels as were observed in normal microglia.
Moreover, the combination of triptolide and AH6809
failed to further decrease NO production. Conversely,
the addition of butaprost (0.1 μM) partially reversed
the triptolide-induced inhibition of NO production(Figure 5A). Taken together, these results demonstratethat triptolide inhibits LPS-stimulated NO production
in microglia via a signaling mechanism involving EP2.
These results also indicate that triptolide regulation of
the EP2 pathway is a potential therapeutic mechanism
for the treatment of inflammation-related neurologicaldisorders.
EP2 activation leads to increased levels of cytoplasmiccAMP, which then initiates multiple downstream events via
the PKA or Epac pathways. To differentiate between thesetwo pathways, we added the PKA-specific activator 6-BnzcAMP (6Bnz) or the Epac-specific activator 8-pCPT-2-OMe-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 involvedin 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 downmodulation of LPS-induced TNF-α by PGE2 is dependent
on cAMP-PKA activation. This was concluded given thatthe selective PKA activating cAMP analog 6Bnz but not

the Epac-1 activating analog 8-CPT inhibited TNF-α production [40]. Their recent work also indicates that EP2PKA, rather than Epac-1, is involved in the enhancementof LPS-induced NO [41]. This result suggests that the EP2PKA pathway is very important for the anti-inflammatory
effect of triptolide in microglia. More studies are needed toclarify whether the EP2-PKA signaling pathway is thedominant 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 triptolideLPS 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 triptolideLPS 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 EP2PKA pathway remains unknown. Liu et al. report that
triptolide directly binds to human XPB, a subunit of thetranscription factor TFIIH, leading to the inhibition of
RNA polymerase II-mediated transcription in tumor cells[42]. However, these authors used higher concentrations

Zhang et al. Journal of Neuroinflammation (2015):

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Figure 5 EP2-PKA pathway is important in LPS-stimulated NO production and triptolide inhibition. (A) Primary rat microglial cells weretreated with LPS (0.01 μg/mL), LPS plus AH6809 (10 μM), LPS plus triptolide (50 nM), LPS plus triptolide plus AH6809, and LPS plus triptolide plus
butaprost (0.1 μM) for 24 h. At the end of the incubation period, the supernatants were collected for NO measurements. (B) Primary rat microglial cells
were treated with LPS (0.01 μg/mL), LPS plus triptolide (50 nM), LPS plus triptolide plus butaprost (0.1 μM), LPS plus triptolide plus 8-CPT (10 μM), or
LPS plus triptolide plus 6Bnz (10 μM) for 24 h. At the end of the incubation period, the supernatants were collected for NO measurements. (C) BV2 cells
were treated with LPS (0.01 μg/mL), LPS plus triptolide (50 nM), LPS plus triptolide plus 6Bnz (10 μM), and LPS plus triptolide plus 6Bnz (10 μM) plus
KT5720 (1 μM) for 24 h. At the end of the incubation period, the supernatants were collected for NO measurements. *P < 0.05 and ***P < 0.001.

of triptolide (1 to 100 μM) to inhibit tumor cell proliferation. We used lower concentrations of triptolide (no morethan 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 interferingwith the formation of the TAK1-TAB1 complex, and thatthe binding affinity of triptolide to TAB1 was highly correlated with the inhibitory activity of triptolide against the
MAPK pathway activation in macrophages [23]. Althoughmacrophages 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.

Conclusions
The present study demonstrates that triptolide inhibits
LPS-stimulated NO production in microglia via a signalingmechanism involving EP2 and PKA. The finding that triptolide reduces the proinflammatory factors in microglia andprotects 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 theregulation of innate immunity in the CNS.

Additional file
Additional file 1: Figure S1. Microglia proliferations did not change after
pharmacological treatments. BV2 cells treated with butaprost (0.1 μM),
AH6809 (10 μM), 8-CPT (10 μM), 6Bnz (10 μM), KT5720 (1 μM), and DMSO
(0.1%) for 24 h. Cell viability was assessed by MTS assay.
Abbreviations
LPS: lipopolysaccharide; NO: nitric oxide; iNOS: inducible NO synthase; COX2: cyclooxygenase-2; PGE2: prostaglandin E2; CNS: central nervous system;
PD: Parkinson’s disease; GPCR: G protein coupled receptor; AC: adenylate
cyclase; PKA: protein kinase A; Epac: exchange proteins that are directly
activated by cAMP; 6Bnz: 6-Bnz-cAMP; 8-CPT: 8-pCPT-2-O-Me-cAMP;
MAPK: mitogen-activated protein kinase; DMEM: Dulbecco’s modified Eagle’s
medium; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate
dehydrogenase; PBS: phosphate-buffered saline; PCR: polymerase chain
reaction; SD: standard deviation.

Zhang et al. Journal of Neuroinflammation (2015):

Competing interests
The authors declare that they have no competing interests.

Authors’ contributions
TZ designed the study, performed the experiments, and wrote themanuscript. XMW supervised the design of the study, analyzed the data, andwrote the manuscript. XLG and GZH performed the experiments. All authors
read and approved the final manuscript.

Acknowledgements
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), andthe Beijing Higher Education Young Elite Teacher Project (YETP1667).
Author details
1
Department of Neurobiology, Capital Medical University, Beijing Institute forBrain Disorders and Key Laboratory for Neurodegenerative Disorder, Ministryof Education, No. 10 Xitoutiao, Youanmenwai, Fengtai District, Beijing
100069, China. 2Department of Physiology, Capital Medical University, No. 10
Xitoutiao, Youanmenwai, Fengtai District, Beijing 100069, China.
Received: 29 September 2014 Accepted: 2 March 2015

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