The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced
inflammatory response in RAW 264.7 cells
Zili Zhaia,⁎, Sandra E. Gomez-Mejibaa,1, Hua Zhub, Florea Lupub, Dario C. Ramireza,⁎⁎,1
aExperimental Therapeutics Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, United States
bCardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, United States
a b s t r a c t a r t i c l ei n f o
Received 24 July 2011
Accepted 22 December 2011
Aim: Exposure of macrophages to lipopolysaccharide (LPS) induces oxidative and inflammatory stresses, which
cause cell damage. Antioxidant and anti-inflammatory properties have been attributed to the nitrone spin trap
5,5-dimethyl-1-pyrroline N-oxide (DMPO), commonly used in free radical analysis, but these aspects of DMPO
have been little explored. In this study, we sought to establish the anti-inflammatory activity of DMPO, presum-
ably by removing free radicals which otherwise help activate inflammatory response and damage cells.
Main methods: RAW 264.7 macrophages were treated with LPS and/or DMPO for different time points, cell dam-
tion, phosphorylation of MAPKs and Akt, and intracellular reactive oxygen species (ROS) were determined.
Keyfindings:After cells were treated with LPS and/or DMPO for 24 h, DMPO reduced the LPS-induced inflamma-
tory response as indicated by downregulated iNOS expression and production of inflammatory mediators. Ac-
cordingly, DMPO protected cells from LPS-induced cytotoxicity. In order to understand the mechanistic basis of
these DMPO effects, the NF-κB p65 activation and the phosphorylation of MAPKs and Akt were examined. We
found, byassaying cells treatedwith LPS and/or DMPO for 15–60 min, that DMPO inhibitedthe phosphorylation
of MAPKs, Akt, and IκBα, and reduced the NF-κB p65 translocation. Furthermore, we demonstrated that DMPO
inhibited LPS-induced ROS production.
Significance: DMPO showed the anti-inflammatory activity and attenuated LPS-induced cell damage, most likely
by reducing ROS production and thus preventing the subsequent inflammatory activation and damage.
© 2012 Elsevier Inc. All rights reserved.
Nitrone spin traps are a class of synthetic chemicals specifically
designed for trapping free radicals and making them more stable. Two
commonly used nitrone spin traps are 5,5-dimethyl-1-pyrroline N-
free radical trapping capacity, PBN has been studied for its antioxidant
and anti-inflammatory activities (Ahmed et al., 2003). It produces a di-
and injury that include endotoxin shock, ischemia-reperfusion injury,
Given that both DMPO and PBN detect free radicals, e.g., superoxide
and hydroxyl radical, though they may have quite different reaction
rate constants for a specific radical species, DMPO may behave in a sim-
ilar way as PBN in terms of its antioxidant and anti-inflammatory activ-
ities. However, DMPO has been far less studied in this regard. Limited
data demonstrated that preadministration of DMPO reduces the mor-
tality associated with endotoxin shock in the rat (Hamburger and
McCay, 1989) and protects against reperfusion-induced injury or ar-
rhythmias in isolated rat heart models (Tosaki et al., 1992; Zuo et al.,
2009). Recently, when we used DMPO to detect macromolecular free
radicals in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages
(Gomez-Mejibaet al.,2010), we found that DMPO protects against LPS-
induced cytotoxicity and, interestingly, theearlierthetreatmentofcells
with DMPO after LPS induction, the better were the protective effects,
suggesting that DMPO interferes with the LPS-triggered early inflam-
matory signaling. However, this observation needs to be refined.
Exposure of macrophages to LPS induces oxidative stress and an
inflammatory response in which the transcription factor NF-κB
plays a central role (Bhattacharyya et al., 2004). Activation of NF-κB
by LPS depends on toll-like receptor 4-initiated signaling that is
Life Sciences 90 (2012) 432–439
⁎ Correspondence to: Z. Zhai, Department of Medicine, Section of Gastroenterology,
The University of Chicago, Chicago, IL 60637, United States. Tel.: +1 773 834 4705;
fax: +1 773 702 2281.
⁎⁎ Correspondence to: D.C. Ramirez, Laboratory of Experimental and Therapeutic
Medicine-Instituto Multidisciplinario de Investigaciones Biologicas San Luis (IMIBIO-SL)-
CONICET & Department of Molecular Biology-Universidad Nacional de San Luis. Av.
Ejercito de los Andes 950, San Luis, San Luis 5700, Argentina. Tel.: +11 54 9 266
4207483; fax: +11 54 266 422644.
E-mail addresses: firstname.lastname@example.org (Z. Zhai), email@example.com
1Current address: Laboratory of Experimental and Therapeutic Medicine, IMIBIO-SL-
CONICET. San Luis, San Luis 5700, Argentina.
0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/lifescie
modulated by several mitogen-activated protein kinases (MAPKs) in-
cluding ERK1/2, JNK, and p38 (Chan and Riches, 2001; Chung et al.,
2006; Guha and Mackman, 2001). MAPKs are rapidly activated
through phosphorylation in response to LPS and therefore have
been used as a hallmark of LPS-induced signaling (Jang et al., 2006;
Kim et al., 2010). The regulation of NF-κB activity by MAPKs seems
to be dependent on reactive oxygen species (ROS) and free radicals
as messenger molecules (Chung et al., 2006). However, among
them, it is relatively well established that hydrogen peroxide is in-
volved in the cross talk between MAPKs and NF-κB signaling (Gloire
et al., 2006). It is not clear whether and which free radicals involved
in LPS-elicited signaling are targets of DMPO.
The purpose of this study was to test the anti-inflammatory activ-
ity of DMPO in LPS-activated RAW 264.7 macrophages, presumably
by targeting free radicals that act as cell signals in inflammatory re-
sponse. We determined the inhibitory effect of DMPO on LPS-
induced inflammatory response, cell damage, NF-κB translocation
and MAPK activation. Our results showed the experimental effective-
ness of DMPO in controlling LPS-mediated inflammatory conditions,
and suggest that its anti-inflammatory activity might be associated
with the blocking of the upstream inflammatory signaling cascades
including the free radical reactions.
Materials and methods
RAW 264.7 cells were obtained from American Type Culture Col-
lection (TIB-71, Rockville, MD) and grown in DMEM supplemented
with 10% fetal bovine serum at 37 °C in a 5% CO2incubator. Cells be-
tween passages 5 and 20 were used in this study.
LPS and DMPO treatments
LPS (Escherichia coli serotype 055:B5, L2637) was from Sigma
(St. Louis, MO). DMPO was from Alexis Biochemicals (San Diego,
CA). Cells were cultivated on the indicated culture ware and allowed
to attach for at least 2 h, then the medium was removed and replaced
with the indicated medium with DMPO and/or LPS. According to our
earlier studies, LPS at 1 ng/ml was a reasonable concentration that
could induce RAW 264.7 cell activation but caused less cell damage.
DMPO was mostly used at 50 mM in this study. The dose of DMPO
was chosen because it could provide the effective protection against
LPS (1 ng/ml)-induced cytotoxicity but itself showed no detectable
toxicity based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT)-based colorimetric assay.
Cell viability assay
The cytotoxicity of LPS and DMPO was assessed using the MTT
assay. After treatments with LPS and DMPO in 96-well plates, the cul-
ture medium was replaced with 0.5 mg/ml of MTT (Amresco Inc.,
Solon, OH) in phosphate buffered saline (PBS) and cells incubated at
37 °C for 1 h. After aspiration of MTT, the formazan crystals in viable
cells were solubilized in dimethyl sulfoxide and quantified by reading
absorbance at 570 nm with a 630 nm reference using an Infinite 200
microplate reader (Tecan, Research Triangle Park, NC).
Alternatively, the cytotoxicity of LPS and DMPO was determined
by assaying the release of lactate dehydrogenase (LDH) into the
culture medium using a QuantiChrom LDH kit (BioAssay Systems,
To measure the production of proinflammatory cytokines,
RAW 264.7 cells were grown in 6-well plates and treated with LPS
and/or DMPO for 24 h. The culture medium was collected for several
cytokine assays. Tumor necrosis factor-alpha (TNF-α), interleukin
(GM-CSF) were assayed using commercial DuoSet ELISA kits from
R&D Systems (Minneapolis, MN). IL-1β, IL-6, keratinocyte-derived
chemokine (KC), and IL-12p70 were assayed using a MS6000 Mu
ProInflammatory 7-Plex Ultra-Sensitive kit (Meso Scale Discovery,
Gaithersburg, MD) according to the manufacturer’ specifications,
and an MSD Sector Imager 6000 was used to read the plates.
Nitrite accumulation in culture medium was determined as an in-
dicator of nitric oxide (NO) synthesis. RAW 264.7 cells were seeded in
96-well plates and stimulated with LPS in the presence or absence of
DMPO for 24 h. At different time points after LPS treatment, the cul-
ture medium was collected for nitrite measurement using the classi-
cal Griess reaction. In a second set of experiments, DMPO was either
added simultaneously with LPS or up to 24 h after LPS addition. Cul-
ture supernatants were collected for the nitrite assay after cells
were stimulated with LPS for 24 h.
Intracellular ROS production was determined using carboxy-
H2DCFDA, a fluorogenic probe for general ROS such as hydrogen per-
oxide, peroxynitrite, and hydroxyl radical. Cells were incubated in 96-
well plates with 25 μM of carboxy-H2DCFDA (Invitrogen, Carlsbad,
CA) in PBS for 30 min, then washed twice and treated with indicated
concentrations of LPS and DMPO for another 30 min. Fluorescence in-
tensity was measured at Ex/Em=495/529 nm.
For superoxide detection, cells were serum starved overnight in 6-
well plates. Then the medium was changed to PBS containing 10 μM
dihydroethidium (DHE, Invitrogen) with 50 mM DMPO, 1 μM diphe-
nyleneiodonium chloride (DPI, Sigma), or 100 μM apocycin (CalBio-
chem, San Diego, CA) for 20 min at 37 °C. Cells were activated with
the addition of 1 ng/ml LPS plus 1% (v/v) fetal bovine serum in PBS
for 30 min. In the presence of superoxide, cell permeable DHE is oxi-
dized to ethidium, which intercalates with DNA with the emission of
red fluorescence (Ex/Em=488/610 nm). The change in fluorescent
signal indicates the level of intracellular superoxide. Cells were
scraped off and the fluorescence intensity was analyzed by FACS Cali-
bur (BD Biosciences, San Jose, CA) (McAdams et al., 2006). For each
sample, 20,000 events were collected and percent gated cell count
was obtained based on the right shift of red fluorescent signal in the
Preparation of cell lysates
Following treatments with LPS for the indicated times, RAW 264.7
cell activation was stopped by the removal of medium and addition of
ice-cold PBS (Jang et al., 2006). In brief, whole cell lysates were pre-
pared and used to detect proteins of interest. Cells were lysed with
the CelLytic M lysis solution (Sigma) containing 1% (v/v) protease in-
hibitor cocktail (Amresco). Cell debris was removed by centrifugation
at 12,000 g for 15 min at 4 °C, and the resultant supernatants were
stored at −80 °C until use.
To assess NF-κB p65 translocation, subcellular fractions were pre-
pared (Jones et al., 2007; Terra et al., 2007). Cells were lysed in ice-
cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl,
0.25% Nonidet P-40, 0.5 mM dithiothreitol and 1% (v/v) protease in-
hibitors for 10 min. The cytoplasmic fraction was collected by centri-
fugation at 12,000 g for 3 min. The unlysed nuclei were gently
resuspended in ice-cold buffer B (20 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 420 mM NaCl, 25% glycerol and 1% (v/v) protease inhibitors)
and left on ice for 20 min, then a 4-fold volume of buffer C (20 mM
Z. Zhai et al. / Life Sciences 90 (2012) 432–439
HEPES, pH 7.9, 50 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA and
1% (v/v) protease inhibitors) was added, and the nuclear fraction
was collected by centrifugation at 12,000 g for 5 min.
The protein concentrations in cell lysates were determined using a
BCA protein assay kit (Pierce Labs, Rockford, IL) with bovine serum al-
bumin as standard.
Determination of protein nitration
Protein nitration was assessed as a marker for oxidative damage of
proteins in LPS-treated RAW 264.7 cells. Cell lysates, diluted in 0.1 M
bicarbonate buffer, pH 9.6, were incubated in the ELISA plates over-
night at 4 °C. Following washing with 0.05% Tween-20 in PBS and
then blocking with 2.5% cold-water fish skin gelatin (Sigma) for 1 h,
the plates were incubated with rabbit anti-nitrotyrosine (1:1,000 di-
lution, Sigma) at 37 °C for 1 h. The immunocomplexes were detected
using goat anti-rabbit IgG-HRP conjugate and VisiGlo Chemilu HRP
substrate solution (Amresco), and read by a microplate reader.
Western blot analysis
Cell lysates were mixed with 4×SDS NuPAGE sample loading buff-
er (Invitrogen) plus 100 mM 2-mercaptoethanol. After heat denatur-
ation, equal amounts of cellular proteins were separated on 4-12%
reducing NuPAGE Bis-Tris Gels (Invitrogen), followed by electrotrans-
fer onto a nitrocellulose membrane (0.2 μM pore size). After blocking
with 5% non-fat milk in PBS, the immunoblot was performed by incu-
bation with a primary antibody overnight at 4 °C, and then HRP-
conjugated goat anti-rabbit or goat anti-mouse IgG secondary anti-
body for 1 h at room temperature. The immunocomplexes were
visualized using SuperSignal West Pico Chemiluminescent HRP
Substrate (Thermo Fisher Scientific) and recorded with a FluorChem
HD2 imager (Alpha Innotech Corp., San Leandro, CA). The following
primary antibodies were used: anti-NF-κB p65, p44/42 MAPK
(ERK1/2), SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), phospho-
Technology, Inc., Danvers, MA), p53, checkpoint kinase Chk1, histone
H2B, phospho-ERK1/p44 (pT202/Y204), Akt (Epitomics, Burlingame,
CA), iNOS, β-actin (Sigma), phospho-histone H2AX (Ser139) (Active
Motif, Carlsbad, CA), and IκBα (Santa Cruz Biotechnology, Inc., Santa
Real time RT-PCR
RAW 264.7 cells were seeded at 1.6×106cells per well in 6-well
plates with LPS and/or DMPO for 6 h. Total RNA was isolated using a
RNeasy Mini kit (Qiagen, Valencia, CA) with extra on-column DNase
digestion before RNA cleanup using RNase-Free DNase Set (Qiagen).
The integrity, quantity and purity of RNA were examined using Nano-
Drop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE).
For each sample, 0.13 μg of total RNA was reverse transcribed using
the SuperScript III first-strand synthesis system for RT-PCR (Invitro-
gen) with random hexamer primers. Real-time qRT-PCR was used
to determine the relative amount of iNOS mRNA. For a typical reac-
tion, 12.5 μl iTaq SYBR Green 2× supermix, 10 μM primer and 3 μl
of cDNA template were mixed and the final volume was adjusted to
25 μl. ABI Prism 7000 Sequence Detection System (Applied Biosys-
tems) and iTaqTM SYBR Green Supermix (Bio-Rad, CA) were used
for detection and quantitation. The primers for mouse iNOS were 5’-
GGC AGC CTG TGA GAC CTT TG-3’ (forward) and 5’-GCA TTG GAA
GTG AAG CGT TTC-3’ (reverse). The primers for 18S rRNA were 5’-
CGC GGT CCT ATT CCA TTA TTC-3’ (forward) and 5’-CCC GAA GCG
TTT ACT TTG AAA-3’ (reverse). The default PCR conditions were as
follows: initiation: 2 min at 50 °C; hot start of the enzyme: 10 min
at 95 °C; amplification: denaturation at 95 °C for 15 s, followed by
annealing and extension at 60 °C for 1 min; 40 cycles. The specificity
of gene PCR product was evaluated by a melt dissociation curve. Rel-
ative quantification of gene expression was estimated using the ΔΔ Ct
method. The relative expression of iNOS was normalized with 18S
Results are expressed as mean value±SEM. Effects were assessed
using the Student's t test. A difference between treatment groups
with Pb0.05 was considered statistically significant.
DMPO reduces LPS-induced cytotoxicity
To investigate the cytotoxicity of LPS and DMPO, RAW 264.7 cells
were incubated with 1 ng/ml LPS and 50 mM DMPO for 24 h. As
shown in Fig. 1A, LPS decreased cell number as assessed using the
MTT assay. DMPO alone did not affect cell viability, but it suppressed
LPS toxicity. To evaluate LPS-induced protein oxidative damage, we
determined protein nitration and found that DMPO significantly
inhibited LPS-mediated production of protein nitration (Fig. 1B). To
evaluate LPS-induced DNA damage, we determined histone H2AX
phosphorylation and the expression levels of DNA damage sensor
p53 and checkpoint Chk1 in cells exposed to LPS for 24 h. We found
that LPS induced phosphorylation of histone H2AX, but decreased
the expression of p53 and Chk1 (Fig. 1C). However, DMPO inhibited
the LPS effect on the DNA damage parameters. Furthermore, LDH re-
lease into the culture medium was evaluated as an indicator of cell
death. We found that DMPO reduced LPS-induced release of LDH
into the culture medium (Fig. 1D). These data suggest that DMPO
has a protective effect against LPS-induced cell damage.
DMPO reduces LPS-induced inflammatory cytokine production
To investigate the anti-inflammatory effect of DMPO, we deter-
mined the production of several typical inflammatory cytokines,
TNF-α, IL-1β, IL-6, KC, IL-10, IL-12, and GM-CSF, in RAW 264 cells in-
cubated with or without 1 ng/ml LPS and/or 50 mM DMPO for 24 h.
As shown in Fig. 2, LPS stimulated cells to secrete the inflammatory
cytokines. DMPO alone had no effect, but inhibited LPS-induced pro-
duction of these cytokines, suggesting that DMPO inhibits LPS-
induced cell inflammatory response.
DMPO reduces LPS-induced NO production and iNOS expression
NO in combination with inflammatory cytokines is mostly respon-
sible for LPS-mediated cell damage, therefore, we also determined the
effect of DMPO on nitrite accumulation in the culture medium of cells
treated with 1 ng/ml LPS and 50 mM DMPO for different time points
(0–24 h). In agreement with previous reports (Seminara et al.,
2007; Stuehr and Marletta, 1987), there was a lag phase of about
6 h followed by a linear increase in nitrite accumulation. However,
the increasing nitrite production was inhibited when DMPO was pre-
sent in the medium (Fig. 3A).
In LPS-activated macrophages, high levels of NO are produced
through the induction of iNOS. The effect of DMPO on the iNOS induc-
tion was therefore determined. First we used an indirect method
(Zhai et al., 2009). Once macrophages are activated, iNOS gene tran-
scription is initiated at approximately 1 h, whereas its protein expres-
sion occurs some 3–4 h later. If DMPO-mediated decrease in NO
production is through interference with the transcriptional induction
process, earlier intervention with DMPO (i.e. 0.5 h after LPS stimula-
tion) should have a more profound effect on NO production than add-
ing DMPO later. Therefore, in this study, 50 mM DMPO was either
added simultaneously with 1 ng/ml LPS or up to 24 h after addition
Z. Zhai et al. / Life Sciences 90 (2012) 432–439
of LPS. The results showed that after LPS treatment, the earlier addi-
tion of DMPO resulted in more inhibition of NO production
(Fig. 3B), indicating that DMPO inhibits the transcriptional induction
of iNOS. However, there were no remarkable differences in NO pro-
duction for DMPO addition between 0–0.5 h after LPS stimulation.
The inhibitory effect of DMPO on the iNOS expression was further
confirmed. RAW 264.7 cells were treated with LPS and DMPO for 6 h
for iNOS mRNA expression or 24 h for its protein expression. The re-
sults showed that DMPO significantly reduced the expression of
iNOS protein and mRNA (Fig. 3C and D).
DMPO reduces LPS-induced NF-κB p65 translocation and
To determine whether the decreased iNOS expression is due to the
blocking of NF-κB activation by DMPO, we measured the expression
levels of NF-κB p65 in both cytosolic and nuclear fractions of RAW
264.7 cells treated with 1 ng/ml LPS and/or 50 mM DMPO for 1 h. As
shown in Fig. 4A, LPS induced the translocation of NF-κB p65 from
the cytosol to nucleus, but the presence of DMPO inhibited the trans-
location of NF-κB p65. To explain the inhibition of NF-κB p65 nuclear
translocation, we next investigated the effect of DMPO on the IκBα
degradation. As shown in Fig. 4B, IκBα was almost completely de-
graded upon LPS stimulation for 15 min, but DMPO inhibited the deg-
radation of IκBα, suggesting that DMPO inhibits LPS-triggered
upstream signaling of NF-κB activation.
DMPO reduces LPS-induced phosphorylation of MAPKs and Akt
To further explore the molecular mechanism of DMPO, we deter-
mined its effect on the upstream signaling pathways of NF-κB activa-
tion, that is, MAPKs and Akt. Because these kinases are activated
through phosphorylation, their phosphorylation levels were evaluated.
After RAW 264.7 cells were treated with 1 ng/ml LPS and/or 50 mM
DMPO for 15 min, LPS induced the appearance of phosphorylated
ERK1/2, JNK, p38, and Akt, but DMPO showed inhibitory effects on the
phosphorylation of these kinases, especially Akt (Fig. 4C). Either LPS
or DMPO alone had no effect on any total kinase at the time point.
DMPO reduces LPS-induced intracellular ROS production
DMPO has free radical trapping properties that are the basis for its
use in free radical research. Oxygen free radicals are suggested to be
(Gloire et al., 2006). Therefore, we tested the effect of DMPO on
LPS-induced intracellular ROS production. As shown in Fig. 5A,
DMPO inhibited LPS-induced ROS production in a dose-dependent
manner. Of the many ROS, superoxide is important because other ox-
idants can be derived from it. Superoxide was detected in LPS-treated
cells by staining with a fluorescence dye DHE. Our data show that
DMPO blocked any LPS-induced increase in superoxide production
(Fig. 5B). In contrast, the NADPH oxidase inhibitors DPI and apocycin
were only somewhat inhibitory. These data suggest that DMPO in-
hibits LPS-induced intracellular ROS production, and suppressing
ROS production may weaken the downstream NF-κB activity and ac-
cordingly reduce various biochemical events that otherwise cause ex-
cessive oxidative and inflammatory damage.
Inflammation mediators are important for the host defense. How-
ever, improper upregulation of the inflammatory mediators is re-
sponsible for the origin and progression of many pathological
conditions. For example, induced TNF-α and NO are known to be
strong inducers of cell damage and apoptosis (Comalada et al.,
2003; Gotoh et al., 2002; Xaus et al., 2000). In the present study, we
first evaluated the protective effect of DMPO against LPS-induced
cell damage using several methods, i.e., MTT-based mitochondrial
Fig. 1. DMPO reduces LPS-induced oxidative damage and cell death. RAW 264.7 cells were treated with LPS (1 ng/ml) and DMPO (50 mM) for 24 h. (A) Cell viability was assessed by
the MTT assay. (B) Protein nitration as a marker of protein oxidative modification was determined by ELISA. (C) Equal amounts of whole cell lysates were prepared and analyzed by
Western blot with antibodies against p53, Chk1, and phospho-histone H2AX. The detection of these proteins was estimated with histone H2B as a loading control. (D) As a marker
of cell death, LDH release into culture medium was measured using a colorimetric assay. The data are representative or expressed as the mean±SEM of two independent exper-
iments run in triplicate. *Pb0.05 vs the baseline control; **Pb0.001 vs the baseline control; #Pb0.05 vs the LPS treatment group.
Z. Zhai et al. / Life Sciences 90 (2012) 432–439
enzyme activity, LDH release, protein nitration, and histone H2AX
phosphorylation (Fig. 1).
Chk1 is an important checkpoint in the cell cycle and DNA damage
response. In response to damaged DNA induced by genotoxic stress
such as ROS, Chk1 is phosphorylated and activated, and peaks at
about 90 min following LPS stimulation (Sharma et al., 2010). It has
been reported that the Chk1 gene was down-regulated 3 h after LPS
treatment in RAW 264.7 macrophages (Nemeth et al., 2003). Our ob-
served decrease in Chk1 protein expression 24 h after LPS treatment
may be due to the collective result of the downregulated gene expres-
sion and the degraded phosphorylated protein. In our study, LPS-
induced DNA damage was also indicated by phosphorylation of the
variant histone H2AX (Fig. 1C), one of the earliest biochemical events
at the sites of DNA strand breaks (Celeste et al., 2003). Histone H2AX
phosphorylation is an upstream signaling of Chk1 activation
(Reinhardt and Yaffe, 2009). The data of Chk1 protein expression
and histone H2AX phosphorylation in combination with other cell
damage assays confirmed the protective effect of DMPO against LPS-
induced cell damage.
The protective properties of DMPO in LPS-induced cell damage
could be largely attributed to its inhibition of the overproduction of
toxic inflammatory mediators including TNF-α, IL-1β and NO (Fig. 2
and 3). However, the inhibitory effects of DMPO on all inflammatory
mediators observed might just be secondary to the perturbation
of common signaling pathways triggered by LPS. To explore the
mechanistic basis of DMPO for its anti-inflammatory activity, we
first determined NO production in RAW 264.7 cells by adding DMPO
at different time points after LPS treatment (Fig. 3B). The results
showed that DMPO added at the onset of LPS-induced iNOS expres-
sion (b 3 h) resulted in a stronger inhibitory effect on NO production,
suggesting that DMPO blocks iNOS gene induction. This reasoning
was supported by the analysis of iNOS protein and mRNA expression
(Fig. 3). The inhibition of iNOS expression by DMPO could be further
extended to its ability to inhibit the upstream signaling as evidenced
by the decreased NF-κB p65 translocation and IκBα degradation
NF-κB plays a critical role in inflammatory conditions and, there-
fore, inhibition of NF-κB activation has become an important anti-
inflammatory pharmacological manipulation (Gupta et al., 2010;
Uwe, 2008). NF-κB activation is multifactorial in nature, involving a
coordinated activation of many kinases (Jones et al., 2007), including
MAPKs and Akt. Therefore, the inhibition of NF-κB translocation by
Fig. 2. DMPO reduces LPS-induced production of inflammatory cytokines. RAW 264.7 cells were incubated with LPS (1 ng/ml) and/or DMPO (50 mM) for 24 h, then culture super-
natants were collected for determination of TNF-α (A), IL-1β (B), IL-6 (C), KC (D), IL-10 (E), IL-12p70 (F), and GM-CSF (G) by commercial assay kits. The data are expressed as the
mean±SEM of four independent experiments run in duplicate. *Pb0.05 vs the baseline control; **Pb0.001 vs the baseline control; #Pb0.05 vs the LPS treatment group.
Z. Zhai et al. / Life Sciences 90 (2012) 432–439
DMPO may not be a single-event consequence, but a cumulative re-
sult of inhibition of MAPKs, Akt, and some other signaling molecules.
MAPKs and Akt are phosphorylated as early as 10 min following LPS
stimulation and thus comprise an important part of the early signal-
ing events of LPS-initiated macrophage activation (Sharma et al.,
2010). The phosphorylated MAPKs and Akt regulate and activate the
NF-κB signaling pathway via phosphorylation of many possibly over-
lapping signaling molecules including IKKα/β and IκBα (Madrid et al.,
2001; Yang et al., 2000). Therefore, we investigated the effect of
DMPO on the phosphorylation of MAPKs and Akt in RAW 264.7 cells
Fig. 3. DMPO reduces LPS-induced NO production and iNOS protein and mRNA expression. (A) Time course of NO production. RAW 264.7 cells were treated with LPS (1 ng/ml) in
the presence or absence of DMPO (50 mM) for different time points, and the nitrite accumulation in culture medium was determined by the Griess reaction. (B) Time course of
inhibition of NO production by DMPO. Cells were incubated in the presence or absence of LPS (1 ng/ml) for 24 h. DMPO (50 mM) was either added simultaneously with LPS
(0 h) or up to 24 h after addition of LPS. The nitrite accumulation in the culture medium was determined 24 h after LPS stimulation. (C) Cells were treated with LPS (1 ng/ml)
and DMPO (50 mM) for 24 h, then whole cell lysates were assayed by Western blot for iNOS expression. (D) Cells were treated with LPS (1 ng/ml) and DMPO (50 mM) for 6 h.
Total RNA was extracted and assayed for iNOS mRNA expression by real time RT-PCR analysis. The data are representative or expressed as the mean±SEM of two or three inde-
pendent experiments run in triplicate. *Pb0.05 vs the baseline control; #Pb0.05 vs the LPS treatment group.
Fig. 4. DMPO reduces LPS-induced nuclear translocation of NF-κB p65, degradation of IκBα, and phosphorylation of ERK1/2, JNK, p38, and Akt. (A) RAW 264.7 cells were incubated
with LPS (1 ng/ml) and/or DMPO for 1 h, then cytosolic and nuclear fractions were isolated and assayed for NF-κB p65 by Western blot. (B) RAW 264.7 cells were treated with LPS
(1 ng/ml) and/or DMPO (50 mM) for 15 min, then equal amounts of whole cell lysates were assayed for IκBα degradation by Western blot. (C) RAW 264.7 cells were incubated with
LPS (1 ng/ml) and/or DMPO for 15 min, then whole cell lysates were prepared and subjected to Western blot analysis for MAPKs and Akt. Western blot detection of respective total
kinase was considered to be a loading control. The data are representative of at least two experiments.
Z. Zhai et al. / Life Sciences 90 (2012) 432–439
stimulated with LPS for 15 min, and found that the phosphorylation
and activation of these kinases were to some extent inhibited by
DMPO (Fig. 4C). Although DMPO's inhibition of the signaling mole-
cules of MAPKs and Akt seems to be unselective, it is likely involved
in a common mechanistic pattern. Since free radicals are involved in
the upstream signaling of these kinases (Asehnoune et al., 2004), it
is proposed that DMPO functions through trapping free radicals that
activate these kinases. We have confirmed the inhibitory effect of
DMPO on intracellular ROS (Fig. 5A) and in particular superoxide pro-
duction (Fig. 5B). It should be noted that DHE staining for superoxide
was performed after serum withdrawal overnight in order to reduce
the background levels of intracellular superoxide and to expectedly
increase superoxide production upon addition of serum and LPS
(Gurjar et al., 2001; Lim and Clement, 2007). Inhibited ROS produc-
tion by DMPO will undoubtedly result in weakened downstream sig-
naling activities, lower production of inflammatory mediators, and
less cell damage.
NF-κB signaling is redox-regulated (Chung et al., 2006; Gloire et
al., 2006; Yao et al., 2007). In LPS-stimulated macrophages, superox-
ide may be the first ROS induced. Superoxide can be produced by
NAPDH oxidase whose activation is dependent on the phosphoryla-
tion of the cytosolic subunit p47phox. It has been suggested that Akt
is responsible for p47phoxphosphorylation (Hoyal et al., 2003). From
superoxide, many other reactive oxygen and nitrogen species, e.g.,
hydrogen peroxide, peroxynitrite, hydroxyl radical, and hypochlorous
acid, can be directly or indirectly derived. These oxidants are thought
to be important mediators in LPS-triggered NF-κB activation (Gloire
et al., 2006). Although superoxide is produced early in response to
LPS and inhibition of superoxide production by, for example, DPI,
shows potential anti-inflammatory activity (Miesel et al., 1995; Qian
et al., 2007), DMPO is a poor trap of superoxide with a rate constant
of 10 M-1s-1(Finkelstein et al., 1979). In contrast, the reaction of hy-
droxyl radicals with DMPO is very fast, k=2×109M-1s-1(Finkelstein
et al., 1979; Makino et al., 1991). As for NO, though its large produc-
tion requires the induction of iNOS, macrophages contain constitutive
eNOS to maintain low levels of NO (Buras et al., 2000; Connelly et al.,
2003); however, it is known that DMPO is ineffective in trapping NO
(Pou et al., 1994) and it cannot alter eNOS expression (data not
shown). In addition to these small molecule reactive species, macro-
molecular free radicals may have a role in NF-κB activation. It is pos-
sible that some kinases or kinase-interacting molecules may produce
protein-centered radicals when phosphorylated and activated. Hy-
droxyl radical, peroxynitrite, and hypochlorous acid are known to ox-
idize proteins with the formation of protein-centered radicals as
intermediates (Davies et al., 1991; Hawkins and Davies, 1998; Lopes
de Menezes and Augusto, 2001; Gomez-Mejiba et al., 2009 and refer-
ences therein; Ramirez et al. 2005). The precise mechanism of DMPO
in blocking free radicals as cell signals warrants further studies.
Our previous data showed that 50 mM DMPO provided the most ef-
fective protection against LPS-induced cell damage but itself did not
show detectable cell toxicity based on the MTT assay. Increased DMPO
concentrations may produce cell damage and lose the protective effect
against LPS action. For example, 100 mM DMPO generated a protective
effect only commensurate with 12.5 mM DMPO. However, DMPO at
50 mM could not inhibit LPS-mediated macrophage activation
completely as indicated by production of proinflammatory cytokines
and NO, suggesting that there may be both ROS-dependent and ROS-
independent LPS-induced signaling pathways. Chandel et al. had
reported that LPS activates NF-κB via a ROS-independent mechanism
in the J774.1 macrophage cell line model (Chandel et al., 2000). Consid-
eringthat DMPO is a spin trap, that is, DMPO has a potential to trap free
radicals, its effect on ROS-independent signaling pathways is expected
to be rather limited or ineffective, but could not be excluded.
inflammatory response and cell damage in a macrophage cell line.
Based on our data we propose that the anti-inflammatory effect of
DMPO is likely through trapping or inhibiting ROS that are upstream
signaling molecules, thus preventing later inflammatory response and
damage. Further studies will be needed to detect the free radicals
trapped by DMPO in the early signaling events, to explore the possible
uate its anti-inflammatory effectiveness in animal models. DMPO has
been used as a spin trap for over 40 years (Taniguchi and Madden,
2000). The present study will help look at this “old” spin trap in new
ways, including its anti-inflammatory use and mechanism of action.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
The project was supported by Award Number 5R00ES015415-04
to DCR from the National Institute of Environmental Health Sciences.
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institute of En-
vironmental Health Sciences or the National Institutes of Health. The
authors would like to thank Dr. Ann Motten of Duke University for
editing the manuscript.
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