International Immunopharmacology 18 (2014) 1219

Contents lists available at ScienceDirect

International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Extracellular polysaccharide from Bacillus sp. strain LBP32 prevents

LPS-induced inammation in RAW 264.7 macrophages by inhibiting
NF-B and MAPKs activation and ROS production
Ying Diao a,1, Yinqiang Xin a,1, Yi Zhou a, Na Li a, Xiaolong Pan a, Shimei Qi a, Zhilin Qi a, Yimiao Xu a, Lan Luo b,d,
Honggui Wan c,d,, Lei Lan a,d,, Zhimin Yin a,d,
a

Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Science, Nanjing Normal University, Nanjing, Jiangsu, PR China
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, PR China
c
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210-009, PR China
d
Collaborative Innovation Center of Biomedicine for Public Hygiene Emergency and Critical Care, Jiangsu Life Sciences & Technology Innovation Park, Nanjing, Jiangsu, PR China
b

a r t i c l e

i n f o

Article history:
Received 21 June 2013
Received in revised form 15 October 2013
Accepted 17 October 2013
Available online 4 November 2013
Keywords:
EPS
Inammation
NF-B
MAPKs
ASK1
ROS

a b s t r a c t
Extracellular polysaccharides (EPSs) are high-molecular weight sugar-based polymers that are synthesized and
secreted by many microorganisms. Recently, EPSs have attracted particular attention due to their multiple biological functions including anti-inammation. However, studies rarely reported the molecular mechanisms
underlying their functions. We previously puried an EPS from an oligotrophic bacteria (Bacillus sp. LBP32)
found in Lop Nur Desert, which possesses a potent antioxidant activity, while the anti-inammatory effects of
EPS and signaling mechanisms underlying its action have not been claried. In this study, we demonstrated
that EPS signicantly inhibited the LPS-induced release of pro-inammatory mediators, such as nitric oxide
(NO), IL-6 and TNF-, without any signicant cytotoxicity. EPS also downregulated the expression of nitric
oxide synthase (iNOS) induced by LPS. Furthermore, activation of nuclear factor B (NF-B) was abrogated by
EPS through inhibited the phosphorylation of IB kinase (IKK). Activations of Mitogen-activated protein kinases
(MAPKs), including p38 MAPK and c-Jun N-terminal kinase (JNK), were also found to be inhibited by EPS. In addition, the level of intracellular reactive oxygen species (ROS) was also signicantly decreased with the treatment
of EPS. In vivo experiments were conducted and showed that EPS could greatly improve the outcome of mice
with LPS-induced endotoxic shock. Taken together, our data indicate that EPS prevents LPS-induced inammatory response by inhibiting NF-B and MAPKs activation and ROS production.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Inammation is a physiological defense reaction of living tissues
against harmful stimuli, such as pathogens, damaged cells, or irritants.
Normally, this process attempts to remove the injurious stimuli and
promotes wound healing. However, dysregulated activation of inammation and oxidative stress has been recognized as one of the principal
causes of inammatory diseases such as rheumatoid arthritis, diabetes,

Correspondence to: H. Wan, School of Pharmaceutical Science, Nanjing University of

Technology, Nanjing 210009, PR China. Tel.: +86 25 8317 2086; fax: +86 25 8317 2087.
Correspondence to: L. Lan, College of Life Sciences, Nanjing Normal University, No.1
Wenyuan Road, Nanjing 210023, PR China.
Correspondence to: Z. Yin, College of Life Sciences, Nanjing Normal University, No.1
Wenyuan Road, Nanjing 210023, PR China. Tel./fax: +86 2585891305.
E-mail addresses: hg_wan@yahoo.cn (H. Wan), lanlei1978@gmail.com (L. Lan),
yinzhimin@njnu.edu.cn (Z. Yin).
1
These authors contributed equally to this study.
1567-5769/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.intimp.2013.10.021

Alzheimer's disease and even cancers [1,2]. Clinically, the fatal syndrome was originally attributed to lipopolysaccharide (LPS), which is
a major component of the cell walls of Gram-negative bacteria. LPSinduced activation of monocytes and macrophages involves the production of pro-inammatory mediators including tumor necrosis factor
alpha (TNF-), interleukin-1 (IL-1), IL-6, and nitric oxide (NO)
[3,4]. Therefore, regulation of inammatory mediators released from activated macrophages is an important target for the treatment of various
inammatory disorders.
Toll-like receptor 4 (TLR4) is an essential receptor for LPS on immune
cells [5]. The association between LPS and TLR4 triggers two different signal pathways, the myeloid differentiation factor-88 (MyD88)-dependent
and the TIR domain-containing adaptor inducing interferon- (TRIF)-dependent pathways. Activation of MyD88-dependent pathway results
in the rapid phosphorylations of mitogen-activated protein kinase
(MAPKs) and IB kinase (IKK), eventually contributing to the activation
of nuclear factor B (NF-B) signaling [6].
NF-B is a transcription factor that performs a vital function in the
regulation of the expression of genes responsible for a variety of cellular
processes, including inammatory responses, innate and adaptive

Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

immunity, and the pathways involved in cell death and proliferation [7].
In unstimulated cells, NF-B is sequestered in the cytosol in a latent
form bound to inhibitory proteins, IBs. When stimulated with LPS,
IBs will be phosphorylated by IKK and ubiquitinated, thereby releasing
NF-B dimers from the cytoplasmic NF-BIB complex and allowing
activated NF-B to translocate into the nucleus [8].
Among the various signaling pathways upon infection, stressactivated MAPK cascades that converge on c-Jun N-terminal kinases
(JNK) and p38 MAPKs have been characterized as regulators of inammation [9]. It has been shown that LPS stimulation up-regulates the
activities of MAPK family members, JNKs, p38 MAPKs and ERKs, in broblast and macrophage cell lines [10]. The activities of these members are
highly regulated by their upstream kinase MAPK kinases (MKKs) and
MAPK kinase kinases (MAP3Ks). Among a growing number of MAP3Ks,
apoptosis signal-regulating kinase 1 (ASK1) has been considered as a
major factor in regulation of JNK and p38 activation [1113]. Recently,
it has also been shown that ASK1-p38 pathway is involved in mammalian innate immunity [14].
Currently, people paid great attention to natural polysaccharides due
to their applications in the elds of, such as, food, medicine, oil, and of
chemicals. Interest is particularly growing towards extracellular polysaccharides (EPSs), a sort of extracellular polymeric substances secreted by
a broad range of microbial species [15]. The EPSs have many advantages,
including non-toxic and safe, unique physical and chemical properties,
easily separated from bacterial, and can be produced by submerged fermentation in industrial [16]. To date, the biological functions of fungal
EPSs have been documented, including resistance to desiccation, protection against nonspecic and specic host immunity, and adherence
[17,18]. However, we have limited knowledge of immunomodulatory effects of EPS of bacterial origin. Previously, we extracted and puried a
novel bioactive EPS from Bacillus sp. LBP32 in Lop Nur Desert, and we
showed this water-soluble EPS has strong quenching activity on reactive
oxygen species (ROS) and protects cells from UV radiation [19]. During
inammation, ROS can mediate activation of redox-sensitive transcription factors and subsequent expression of inammatory cytokines [20].
Therefore, we went forward to evaluate the anti-inammatory activity
of this kind of EPS to broaden its future applications.
In this study, we used peripheral macrophage RAW264.7 cell line to
examine the potential anti-inammatory properties of EPS upon LPS
stimulation and the underlying molecular mechanisms responsible for
its action. We found that EPS suppressed the LPS-induced release of
pro-inammatory factors and the activations of NF-B and MAPKs
signalings. We also showed that EPS, served as a potent antioxidant, reduced the LPS-induced intracellular ROS accumulation, which may be
correlated to the suppression of NF-B and MAPKs and contribute to
its anti-inammatory action. In addition, the anti-inammatory activity
of EPS was further evaluated in a mouse septic shock model, and EPS obviously enhanced the survival of animals from LPS-induced acute inammation. Our study therefore not only provides more evidence that
EPS possesses anti-inammatory activity, but also provides molecular
insight into mechanisms by which EPS exerts its anti-inammatory
function.
2. Materials and methods
2.1. Reagents and antibodies
EPS was extracted and puried as described previously [19], which is
composed of N-acetylglucosamine, xylose, and mannose in a molar ratio
of 2.36:0.98:1.75 with molecular weight of 9.62 104. Polyclonal antibodies against JNK/SAPK, phospho-JNK/SAPK (Thr183/Tyr185), p38
MAPK, phospho-p38 MAPK (Thr180/Tyr182), p42/p44 MAPK, phosphop42/44 MAPK (Thr202/Tyr204), SEK1/MKK4, phospho-SEK1/MKK4
(Ser257/Thr261), phospho-MKK3/6 (Ser189/207), phosphor-IKK/
(Ser176/177), NF-B p65, LaminB and phospho-ASK1 (Thr845) were
purchased from Cell Signaling Technology (Beverly, MA, USA). Polyclonal

13

antibodies against GAPDH and -Actin were purchased from Bioworld

Technology (Minneapolis, MN, USA). LPS (from Escherichia coli 0111:
B4), N-acetyl-L-cysteine (NAC), and MTT were obtained from SigmaAldrich (St. Louis, MO, USA). CM-H2DCFDA was obtained from Invitrogen
(Carlsbad, CA, USA).
2.2. Cell culture
RAW264.7 macrophage cells, purchased from the CBCAS (Cell Bank
of the Chinese Academic of Sciences, Shanghai, PR China), were cultured
in DMEM (Invitrogen) containing 10% (v/v) fetal bovine serum
(Hyclone) and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin) (Hyclone) at 37 C in a humidied 5% CO2 incubator.
2.3. Cytokine measurement
RAW264.7 cells were plated into 12-well plates 24 h before
pretreated with or without different concentrations of EPS for 2 h,
then were stimulated with LPS (100 ng/ml) for 16 h. After stimulation,
the culture media were collected and centrifuged at 10,000 rpm for
5 min. The amounts of cytokines in the supernatants for IL-6 or TNF, respectively, were determined by using double-antibody sandwich
ELISA (R&D Systems) according to the manufacturer's instructions.
Three replicates were carried out for each of the different treatments.
2.4. Nitrite analysis
NO synthesis was spectrophotometrically determined by assaying
the culture supernatants for nitrite using the Griess reagent (1%
sulfanilic acid, 0.1% N-1-naphthyl-ethylenediamine dihydrochloride,
and 5% phosphoric acid). Absorbance was measured at 550 nm and nitrite concentration was determined using sodium nitrite as a standard.
Three replicates were carried out for each of the different treatments.
2.5. Cell viability assay
Cell viability was determined by MTT assay. Briey, RAW264.7 cells
were seeded into 96-well plates at a density of 2 104 cells per well
24 h before treatment. Cells were treated with various concentrations
of EPS for 24 h followed by incubating with 5 mg/ml of MTT working
solution for 4 h at 37 C. After added 100 l of DMSO to dissolve the
crystals, the absorbance of each well at 570 nm was measured using a
Synergy2 Multi-Mode Microplate Reader (BIO-TEK, INC). Three replicates were carried out for each of the different treatments.
2.6. Western blotting
Cells were rinsed twice with ice-cold phosphate-buffered saline
(PBS) and lysed on ice in a lysis buffer containing 20 mM Tris
(pH 7.5), 2 mM EDTA, 135 mM NaCl, 2 mM DTT, 2 mM sodium pyrophosphate, 25 mM -glycerophosphate, 10% glycerol, 1% Triton X-100,
1 mM Na3VO4, 10 mM NaF, 10 g/ml leupeptin, 10 g/ml aprotinin,
and 1 mM PMSF for 30 min. Lysates were centrifuged (15,000 g) at
4 C for 15 min. Equal amounts of the soluble proteins are denatured
in SDS, electrophoresed on 12% SDS-PAGE, and transferred onto nitrocellulose membranes. Immunoblotting was performed as described
previously [21]. The antibodyantigen complexes were visualized by
the LI-COR Odyssey Infrared Imaging System according to the
manufacturer's instructions using IRDye800 urophore-conjugated antibody (LI-COR Biosciences, Lincoln, NE).
2.7. Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNAs were extracted from cells with Trizol reagent (Gibco) according to the manufacturer's protocol. RT-PCR was carried out by using
two-step RT-PCR System kit (Invitrogen) with following primers (iNOS:

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Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

sense primer 5-cccttccgaagtttctggcagc-3, antisense primer 5-ggctgt

cagagcctcgtggctt-3; GAPDH: sense primer 5-tgaaggtcggtgtgaacggattt
ggc-3, antisense primer 5-tggttcacacccatcacaaacatgg-3). PCR was performed for 30 cycles in a 25 l of reaction mixture and the products
were separated on 1.5% agarose gels and visualized with GoldView.
GAPDH was utilized as a housekeeping gene as indicated.
2.8. Preparation of cytoplasmic and nuclear extracts
Cells were washed with ice-cold PBS and then lysed on ice in a hypotonic buffer (20 mM Hepes (pH 7.9), 10 mM KCl, 1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT, 10% glycerol, 0.1 mM Na3VO4, 1 mM PMSF, and protease inhibitors) with 0.2% NP-40 for 20 min. After centrifugation at
13,000 g for 10 min at 4 C, supernatants were collected as cytoplasmic
fractions, pellets were rinsed twice with the hypotonic buffer and then
resuspended in a high-salt buffer (20 mM Hepes (pH 7.9), 0.42 mM
NaCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.1 mM Na3VO4, 1 mM
PMSF and protease inhibitors) on ice for 15 min. Nuclear extracts
were then collected from supernatants by centrifugation at 13,000 g
for 10 min at 4 C. Protein concentrations of the extracts were determined by Bicinchoninic Acid (BCA) protein assay kit (Pierce).
2.9. Detection of reactive oxygen species
RAW264.7 cells were treated with LPS (100 ng/ml) for 30 min in the
presence or absence of various doses of EPS, and then 10 M of CMH2DCFDA (Invitrogen) was added and incubated for 30 min at 37 C
in the dark. Cells were then washed and resuspended in PBS, and uorescence was measured using Synergy2 Multi-Mode Microplate Reader
after excitation at 485 nm and emission at 535 nm. ROS level was
expressed as percentage over control. Three replicates were carried
out for each of the different treatments.
2.10. Confocal microscopy
The nuclear translocation of NF-B p65 was examined by immunouorescence assay. RAW 264.7 cells were pretreated with or without EPS
(0.48 mg/ml) for 2 h, then stimulated with LPS (100 ng/ml). The cells
were xed with 4% paraformaldehyde in PBS, permeabilized with 0.2%
Triton X-100 in PBS, and were blocked with 3% BSA. Monoclonal antibody against NF-B p65 (1:100 dilution in 1% BSA-PBS) was applied
overnight at 4 C, followed by 1 h of incubation with secondary antibody
conjugated with Texas-Red (Invitrogen). Cells were nuclear-stained with
DAPI and mounted with Vectashield mounting medium. Images were
captured using Nikon A1 microscope (Nikon, Tokyo, Japan). Each sample
was examined under the same uorescence detection parameters for
comparison of accumulation.
2.11. Endotoxin shock model of mice
Laboratory animal handling and experimental procedures were performed in accordance with the requirements of Provisions and General
Recommendation of Chinese Experimental Animals Administration
Legislation and were approved by Science and Technology Department
of Jiangsu Province. Eight-week-old ICR mice (Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China) were used
in this study. Mice were injected i.p. with freshly prepared LPS (7.5 mg/
ml dissolved in saline solution) at the dose of 37.5 mg/kg to induce
endotoxemia. All drugs used for treatment were dissolved in saline solution and given through tail vein injection at 30 min after LPS challenge, saline solution was used as a control treatment. After being
separately treated with saline, EPS (24 or 72 mg/kg) or dexamethasone
(DEX, 1.3 mg/kg, Jinling Pharmaceutical, China), the animals were
returned to their cages and fed ad libitum. The death of the animal
was determined by rigorous physical examinations, including no response to pain, no corneal reex and no spontaneous respirations. The

survival rate was monitored continuously for 72 h and studied in

n = 10 mice in each group.
2.12. Statistical analysis
Data are presented as mean SD. Statistical analysis was performed
by statistics package for social science (SPSS) of 13.0-version. One way
analysis of variance followed by StudentNewmanKeuls multiple comparison tests. Survival of mice was analyzed with KaplanMeier survival
analysis with the log-rank test for between-group comparisons. A value
of p b 0.05 was considered statistically signicant.
3. Results
3.1. EPS inhibited LPS-induced release of inammatory cytokines and
mediators in RAW264.7 macrophages
Pro-inammatory cytokines and mediators play important roles in
inammatory process. To test whether EPS inhibits LPS-induced inammatory response, the productions of the inammatory cytokines (TNF-
and IL-6) and mediators (NO) were assayed. RAW264.7 cells were
pretreated with or without EPS (0.01 mg/ml, 0.1 mg/ml or 0.48 mg/ml,
respectively) for 2 h, and then were stimulated by LPS (100 ng/ml) for
16 h. As shown in Fig. 1AC, it was obvious that LPS stimulation led to
a robust increase in the productions of TNF-, IL-6, and NO, while EPS
signicantly inhibited their productions especially at the highest dose
of 0.48 mg/ml. Next, to exclude a possibility that cytotoxicity of EPS resulted in the inhibitory effects on the LPS-induced production of these
cytokines, we determine the cytotoxicity of EPS by the MTT assay. The
treatment of RAW 264.7 cells with EPS had no effect on cell viability
at concentrations up to 1.92 mg/ml, demonstrating that EPS in noncytotoxic levels suppressed LPS-induced inammatory responses in
macrophages (Fig. 1D).
In addition, since NO was produced by iNOS enzyme, we determined
the effects of EPS on LPS-induced expression of iNOS in RAW264.7 cells.
Cells were pre-incubated with EPS at different concentrations for 2 h
followed by stimulation with 100 ng/ml of LPS. Consistent with the
NO production, LPS induced a dramatic increase of cellular iNOS protein,
however, EPS attenuated such elevation in a dose-dependent manner
(Fig. 1E). Similarly, RAW264.7 cells without the pretreatment of EPS
were expressed with a high level of iNOS mRNA after LPS stimulation,
while such an induced expression of iNOS transcripts was evidently
suppressed by EPS (Fig. 1F). These results indicated that EPS inhibited
the NO production by a mechanism involving the transcriptional regulation of iNOS.
3.2. EPS inhibited LPS-induced NF-B activation
NF-B is an important transcription factor, which plays a critical role
in inammation by regulating the expression of several cytokines involving IL-6 and TNF- [22]. It has also been shown that NF-B can
bind to the 5-promoter region of iNOS gene and regulates its transcription [23]. To investigate a possibility that the inhibitory effects of EPS on
the production of pro-inammatory factors act through suppression of
LPS-induced activation of NF-B signaling, we assessed the effect of
EPS on the nuclear translocation of NF-B p65 subunit. Cells were
pretreated with EPS (0.01, 0.1, 0.48 mg/ml) for 2 h and then stimulated
with LPS (100 ng/ml) for 30 min. As shown in Fig. 3A, comparison with
the constantly expressed nuclear envelope protein Lamin B, LPS stimulation enhanced greatly the protein level of NF-B p65 in the nuclear
fractions, while in the cells pre-incubated with EPS, the level of NF-B
p65 in nucleus was decreased. In addition, we also used Confocal microscopy to verify this observation. In control cells, a majority of NF-B
p65 was distributed in the cytoplasm. After LPS stimulation, most of cytoplasmic p65 was translocated into nucleus as seen as prominent red
staining in the nuclei (Fig. 3B). By contrast, the level of p65 in the nuclei

Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

15

Fig. 1. EPS inhibited the release of pro-inammatory mediators TNF-, IL-6, and NO in RAW264.7 macrophages. (AC) RAW264.7 cells were pretreated with or without EPS (0.01, 0.1, or
0.48 mg/ml) for 2 h and then stimulated with LPS (100 ng/ml) for 16 h. After treatment, the levels of TNF-, IL-6, and NO in the culture supernatants were measured by Griess reagents
and ELISA kits. (D) RAW264.7 cells were treated with indicated concentrations of EPS for 24 h. Cell viability was determined by MTT assay as described in Material and methods. The viability of cells without treatment was taken as 100%. Data were presented as means SD of three independent experiments (*p b 0.05). (E) RAW264.7 cells were pre-incubated with or
without 0.01, 0.1, or 0.48 mg/ml of EPS for 2 h and then stimulated with LPS (100 ng/ml) for 16 h. Cell lysates were prepared and subjected to Western blotting using anti-iNOS antibody.
(F) RAW264.7 cells were incubated with or without 0.48 mg/ml of EPS for 2 h and then stimulated with LPS (100 ng/ml) for 8 h, total RNAs were isolated and the expression of iNOS
mRNA was determined by RT-PCR.

was signicantly decreased by pretreatment with EPS (0.48 mg/ml). All

immunouorescent stainings were performed simultaneously.
To further validate the effect of EPS on the inhibition of NF-B activation, we also detected the phosphorylation of IKK, which is an upstream
kinase for phosphorylation of IB and subsequent IB degradation in
macrophages [24]. In agreement with the suppressive effect of EPS on
the nuclear translocation of NF-B p65, the LPS-induced phosphorylation level of IKK was also attenuated by EPS in a dose-dependent manner (Fig. 3C). Taken together, our results suggested that EPS suppressed
LPS-induced inammatory responses, at least in part, by inhibiting IKK/
NF-B activation in RAW264.7 macrophages.
3.3. Effects of EPS on MAPK pathways
The major intracellular signaling pathways activated by LPS through
binding to its receptor TLR4 on cell membrane are NF-B and MAPK
pathways [25]. Since we showed above that EPS could inhibit the LPSinduced activation of NF-B, we thus went forward to test the effects
of EPS on MAPK pathways. To determine whether inhibition of MAPKs
is involved in the anti-inammatory action of EPS, we examined the
effects of EPS on the LPS-induced phosphorylations of p38, ERK1/2,

and JNK. We found that the phosphorylation levels of p38, ERK1/2,

and JNK were all signicantly increased after the LPS stimulation, and
the activations of p38 and JNK were obviously attenuated by EPS, however, it seems that ERK was not affected by EPS treatment (Fig. 4A).
The above results demonstrated that EPS could inhibit the phosphorylation levels of JNK and p38 MAPK, we then detected whether
EPS affects the upstream kinases necessary for their activation. MKK4
is upstream to JNK, and MKK3/6 is responsive for p38 activation. As
shown in Fig. 4B, the increased phosphorylations of MKK4 and MKK3/
6 induced by LPS were both attenuated by EPS in a dose-dependent
manner. Moreover, we determined the effect of EPS on activation of
ASK1, which is one of a growing number of MAP3Ks identied in the
JNK and p38 pathways [12]. As shown in Fig. 4C, LPS stimulation induced the phosphorylation of ASK1, which was partially prevented by
EPS. All these observations suggested that EPS could suppress, at least
partially, the ASK1-mediated MKK-MAPKs pathways.
3.4. EPS suppressed LPS-induced ROS production
ROS have been proposed as signaling molecules in cellular events
[26]. A recent study showed that LPS-induced intracellular redox state

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Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

Fig. 2. EPS inhibited LPS-induced NF-B activation. RAW264.7 cells were pretreated with or without indicated concentrations of EPS for 2 h, and then were stimulated with LPS
(100 ng/ml) for 30 min. (A) The nuclear fractions were analyzed for detection of NF-B p65 subunit by Western blot analysis. NE: nuclear extracts. (B) RAW264.7 cells were treated
with LPS (100 ng/ml), EPS (0.48 mg/ml), or with both. Microscopic images were captured to show the double staining of p65 (in red) and DAPI (in blue) in cells. (C). Phospho-IKK/
level was determined by Western blot analysis. Lamin B was used as loading control for nuclear fraction, GAPDH was used as loading control for total protein. Blots shown were representative of three independent experiments.

can modulate NF-B activity [27]. Since we previously showed that EPS
could effectively scavenge ROS [28], we thus investigated whether suppressive effects of EPS on the activation of NF-B and MAPKs could be
mediated by ROS. Firstly, we determined the LPS-induced intracellular
accumulation of ROS in the presence or absence of EPS. CM-H2DCFDA,
a H2O2-sensitive probe, was used to monitor the level of ROS. Treatment
of RAW264.7 cells with LPS rapidly increased intracellular ROS level,
which was obviously inhibited by EPS at a concentration of 0.48 mg/ml

(Fig. 5A). Moreover, we used a general accepted antioxidant, NAC, as

a positive control. NAC is known to inhibit the expression of proinammatory genes under inammatory conditions. As expected, NAC
(10 mM) attenuated the LPS-induced production of ROS to the basal
level. Similarly to EPS, NAC inhibited LPS-induced iNOS expression and
LPS-induced phosphorylations of ASK1 and of downstream p38, JNK,
and also the phosphorylation of IKK (Fig. 5B), validating that ROS are essential factors involved in LPS-induced inammatory responses. These

Fig. 3. Effects of EPS on MAPK pathways. RAW264.7 cells were pretreated with or without EPS (0.01, 0.1, or 0.48 mg/ml) for 2 h, then were stimulated with LPS (100 ng/ml) for 30 min.
Cell lysates were prepared and subjected to Western blotting by using antibodies against phospho-p38, phospho-ERK1/2 or phospho-JNK (A), against phospho-MKK4 or phospho-MKK3/6
(B), and by using anti-phospho-ASK1 antibody (C). GAPDH or -Actin was used as loading control. Blots shown were representative of three independent experiments.

Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

17

and saline solution died; only 10% of septic mice with EPS administration (72 mg/kg) died at this time point. By 72 h after LPS challenge,
50% DEX-treated mice died while only 20% EPS-treated mice died.
Therefore, EPS obviously enhanced the survival from LPS-induced
sepsis.
4. Discussion

Fig. 4. EPS suppressed LPS-induced ROS production. (A) RAW264.7 cells were treated with
LPS (100 ng/ml) for 30 min in the presence of EPS (0.48 mg/ml) or NAC (10 mM). Fluorescence of CM-H2DCFDA probe was measured by spectrouorometer after excitation at
485 nm and emission at 535 nm. Results are expressed as the mean SD for each
group from three independent experiments (*p b 0.05). (B) RAW264.7 cells were preincubated with NAC (10 mM) or EPS (0.48 mg/ml) for 2 h followed by stimulation with
LPS (100 ng/ml) for 16 h (for iNOS detection) or 30 min (for phosphorylation level detection). iNOS expression as well as phosphorylations of p38, ERK, JNK and IKK were determined by Western blotting.

results further conrmed that EPS isolated from Bacillus sp. LBP32 serves
as an antioxidant. Therefore, we speculated that the inhibitory effect of
EPS on the LPS-induced activation of MAPKs and NF-B signalings is correlated to the suppression of ROS production.
3.5. EPS enhanced survival rate in a mouse model of sepsis
Finally, to investigate the anti-inammatory role of EPS in vivo, we
established an LPS-induced endotoxic/septic shock mouse model as described in Materials and methods. Following the LPS challenge, mice
were administrated with saline, EPS or dexamethasone (Dex), respectively. As shown in Fig. 6, within 40 h, 80% of the mice receiving LPS

Fig. 5. Effect of EPS on survival of septic mice. Survival of septic mice induced by LPS stimulation (37.5 mg/kg i.p.) was monitored for 72 h and survival for each group (n = 10 in
each group) is expressed by KaplanMeier survival analysis. EPS administration (either
24 mg/kg or 72 mg/kg of body weight) enhanced the survival rate of septic mice. Dex
used as a treatment control (1.3 mg/kg of body weight) did not produce signicant improvement in survival as compared to EPS at the dose of 72 mg/kg.

The EPS we studied was extracted and puried from Bacillus sp.
LBP32 in Lop Nur desert. The Lop Nur desert is located in the southeastern region of the Xinjiang Uygur Autonomous Region. It is extreme
drought and high intensity radiation all the year. Under this condition,
the desert is rich in variety of special oligotrophic bacteria. And the bacteria have adapted to the harmful environment and possess special functions and genes after the long-term natural selection. Our previous study
demonstrated that the EPS from Bacillus sp. LBP32 could effectively scavenge ROS and inhibit lipid peroxidation in vitro [19]. During inammation, ROS can mediate the activation of redox-sensitive transcription
factors, such as NF-B, and the subsequent expression of inammatory
cytokines [20]. Therefore, we speculated that EPS, a potent antioxidant,
could also exhibit anti-inammatory activity. In this study, we found
that EPS inhibited LPS-induced production of pro-inammatory cytokines and determined the cell signaling mechanisms through which
EPS exerts its action by employing macrophage cells, and we also evaluated the efcacy of EPS in endotoxic shock mouse model.
In the inammatory response, macrophages synthesize and release
inammatory cytokines such as IL-6, TNF-, and NO [29]. These cytokines are not only involved in the inammatory response, but also expand the inammation, further deepening the development of tissue
injury and disease. Firstly, we found that EPS could effectively inhibit
the LPS-induced production of IL-6, TNF-, and NO, and could also reduce both the mRNA and protein expression levels of iNOS, which is a
pro-inammatory mediator responsible for NO production (Figs. 1 and
2). Regulation of pro-inammatory mediators has been thought to attenuate inammation responses. It has been demonstrated that NF-B
binds to the 5-promoter region of iNOS gene and regulates its transcription [23]. We therefore determined the effects of EPS on NF-B signaling. Upon LPS stimulation, the expression level of NF-B p65 in the
nuclear fraction and the nuclear translocation of p65 were effectively
suppressed by EPS (Fig. 3A and B). We also showed that EPS inhibited
the LPS-induced activation of NF-B signaling as indicated by the
reduced phosphorylation level of IKK (Fig. 3C). These observations indicate that EPS suppressed the LPS-induced activation of NF-B signaling
probably resulting in the reduced productions of pro-inammatory
cytokines. This nding is consistent with the reported effects of EPS of
other origin on NO production [30].
In addition, the activation of MAPK pathways can regulate gene transcription of inammatory response by activating downstream cytosolic
proteins and nuclear transcription factors such as NF-B. We thus determined the effects of EPS on the activation of MAPKs upon LPS stimulation. The three major MAPKs: ERK1/2, p38, and JNK were determined.
In the study, we found that EPS could suppress the LPS-induced activation of p38 and JNK in a dose-dependent manner, but not of ERK1/2
(Fig. 4A), indicating that inhibitory effects of EPS on production of
pro-inammatory cytokines act through p38 and JNK signalings. This
observation is of particular interesting due to the fact that inhibitors to
p38 MAPK and JNK signaling pathways are more capable of reducing
both the synthesis of pro-inammatory cytokines and their intracellular
signaling [31]. Next, we moved forward to test the activation of MKK4,
MKK3/6, and ASK1, which are upstream kinases of MAPKs, and we
found that EPS could also suppress, at least partially, their activations induced by LPS (Fig. 4B and C). Although it has been reported that MAPKs
are upstream of NF-B [6,32], a controversial report showed that MAPKs
are downstream of NF-B in paclitaxel-induced survival signaling [33].
Therefore, the detailed crosstalk between these pathways is needed to
be elucidated to reveal the precise actions of EPS in macrophage cells.

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Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

Fig. 6. Schematic gure of possible signaling mechanisms of EPS in the inhibition of LPS-induced inammation response.

During inammation, activated macrophages largely increase the

oxygen uptake resulting in massive accumulation of ROS. Although excess of accumulated ROS causes oxidative stress and leads to necrosis or
apoptosis [34], ROS also act as second messengers to activate various
transcription factors and signaling proteins, including NF-B and
MAPKs [35,36]. We previously showed that EPS possesses a strong antioxidant activity and is able to scavenge ROS using in vitro assays. Our
data here further demonstrated that EPS acts as a potent antioxidant
to reduce the level of ROS accumulated in macrophage cells after LPS
stimulation (Fig. 5A). In addition, using NAC as a positive antioxidant
control, we found that NAC also reduced the LPS-induced expression
of iNOS and attenuated the activations of ASK1, p38, JNK, and IKK
(Fig. 5B), suggesting that both EPS and NAC inhibited the activation of
ASK1-p38/JNK pathway probably involving the reducing ROS accumulation. It should be noted that we found that NAC inhibited the LPSinduced activation of p38, JNK, and ERK, while EPS only inhibited the
LPS-induced activation of p38 and JNK. Since EPS, a polysaccharide,
has different chemical structures from NAC and possesses multifunction, we therefore reasoned that EPS reduced the LPS-induced
ROS production and subsequently interfered with the ROS-dependent
activation of p38 and JNK, but by unknown action of EPS, the LPSinduced activation of ERK exhibited unchanged result. It is worth investigating the differential effects of EPS on kinases upstream of ERK, p38,
and of JNK during LPS stimulation. Moreover, NF-B activation is
redox-sensitive, and thus inhibition of NF-B signaling by EPS might
also involve the reduction of ROS level. Therefore, we speculated that
anti-inammatory function of EPS might be attributed, at least partially,
to its antioxidant activity toward ROS in RAW264.7 macrophages. Further work is necessary to investigate the precise correlation between
redox modulation and anti-inammatory action of EPS.
Despite the inhibitory actions of EPS on the LPS-induced activation of
NF-B and MAPKs and accumulation of ROS, we haven't investigated a
possibility that the pre-treatment of EPS induces a tolerance to LPS challenge. Recently, growing evidence showed a protective mechanism
called as endotoxin tolerance, in which immune cells exposed to low
concentrations of endotoxin (e.g. LPS) enter into a transient unresponsive state and are unable to respond to further challenges with endotoxin

[37]. We cannot exclude the possibility of inducing tolerance in

RAW264.7 cells by EPS pre-treatment since macrophages/monocytes
have been shown to be principal cells responsible for the induction of endotoxin tolerance in vivo [38]. Although the molecular mechanism of endotoxin tolerance is unclear, recent progress showed that TLR4-MyD88/
TRIF, plasticity of NF-B signaling, microRNA and gene re-programming
contribute to the development of endotoxin tolerance [37]. We showed
that EPS can inhibit NF-B activation, indicating that it is possible
that EPS could modulate NF-B signaling to induce a transient LPSunresponsivess state, thereby exhibiting an anti-inammatory action in
RAW264.7 cells. In future study, upregulation of anti-inammatory factors such as IL-10, TGF- and IL-1RA and phagocytosis of macrophages,
which are both related to tolerance, should be investigated during the
pre-treatment of EPS.
Finally, we further determined the in vivo value of EPS in a septic
shock mouse model induced by LPS. EPS treatment obviously protected
animals from mortality. Sepsis and septic shock are very harmful disorders with complicated mechanisms. Under the condition of shock, the
imbalance of oxygen demands and oxygen supply will lead to tissue
hypoxia. Although we did not conduct detailed investigation of mechanisms underlying the EPS function in animals, we speculated that the
improved outcome of EPS in septic mouse is correlated to its antioxidant
and anti-inammatory activities. Our future work will be focused on the
mechanisms and therapeutic use of EPS in the treatment of inammatory diseases. Since activities of polysaccharides differ in their chemical
compositions, we also need to conduct a further study to determine
the structure of EPS and its targets in macrophages.
In conclusion, we demonstrated that EPS attenuated the LPS-induced
release of pro-inammatory factors probably via suppressing the activation of NF-B and ASK1-p38/JNK signalings. We also showed that EPS,
served as a potent antioxidant, reduced the LPS-induced intracellular
ROS accumulation which may, at least in part, contribute to the suppression of NF-B and ASK1-p38/JNK and subsequently to the reduced productions of pro-inammatory cytokines in response to LPS. This study
not only provides more evidence that EPS exerts anti-inammatory activity in macrophage cells, but also sheds light on the potential use of
EPS as a therapeutic drug for LPS-mediated sepsis.

Y. Diao et al. / International Immunopharmacology 18 (2014) 1219

Acknowledgments
This work was supported by grants from the National Nature Science
Foundation of China (nos. 81072433, 81172798, and 31071000), the
Jiangsu Provincial Nature Science Foundation (no. BK2012452), and by
the Priority Academic Program Development of Jiangsu Higher Education Institutions (no. 164320H106).

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