International Journal of Biological Macromolecules 126 (2019) 1158–1166

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Mechanism of the anti-inflammatory activity by a polysaccharide from

Dictyophora indusiata in lipopolysaccharide-stimulated macrophages
Yalin Wang a, Li Lai b, Liping Teng a, Yuhong Li a, Jianqing Cheng a, Jinghua Chen b,⁎, Chao Deng a,⁎
a
Wuxi School of Medicine, Jiangnan University, Wuxi 214122, China
b
School of Pharmaceutics Science, Jiangnan University, Wuxi 214122, China

a r t i c l e i n f o a b s t r a c t

Article history: Dictyophora indusiata polysaccharides (DIP) shows antioxidant, anti-tumor and immunostimulatory activities.
Received 1 October 2018 However, the anti-inflammatory roles of DIP in NLRP3 inflammasome in LPS-stimulated macrophages are still
Received in revised form 2 January 2019 not well defined. In this study, we investigated the mechanism of the anti-inflammatory activity of DIP in LPS-
Accepted 4 January 2019
primed RAW264.7 macrophages. Our data showed that DIP inhibited NF-κB signal pathway via modulating
Available online 6 January 2019
TLR4 expression, phosphorylation of IκBα and nuclear translocation of NF-κB-p65 subunit. Meanwhile, DIP re-
Keywords:
duced inflammasome activation via decreasing NLRP3 expression in cytoplasmic pools, limiting self-assembly
Dictyophora indusiata polysaccharide of NLRP3 inflammasome, as well as the subsequent activation of caspase-1 and the secretion of IL-1β and IL-
Anti-inflammatory activity 18. For the first time, we show that the anti-inflammatory activity of DIP is mediated by inhibiting TLR4/NF-κB
NLRP3 inflammasome signal pathway and NLRP3 inflammasome activation during LPS-induced acute inflammation in RAW264.7
macrophages.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Inflammation is the normal protective reaction that occurs in re-
sponse to infection, tissue injury, trauma or noxious stimuli [1–4]. Lim-
ited and excessive inflammatory response to pathogens can increase
susceptibility to infection and chronic inflammatory condition, respec-
tively [5–7]. Innate immune cells (e.g. macrophages, mast cells, fibro-
blasts, dendritic cells as well as circulating leukocytes) contribute to
the identification of a variety of pathogen- and host-derived danger sig-
nals with surface or intracellular-expressed pattern recognition recep-
tors (PRRs) [8]. These signals include the whole pathogens and
pathogen-associated molecular patterns (PAMPs) (e.g. microbial
nucleic acids, lipoproteins and carbohydrates), and host-derived signals
of cellular damage such as danger-associated molecular patterns
(DAMP) and environmental irritants. The early sensing of dangerous
signals involves PRRs on the cell surface [e.g., membrane-bound Toll-
like receptors (TLRs)] and in the cytoplasm [e.g., NOD-like receptors
(NLRs)] [9].
Innate immune system recognizes Gram-negative bacteria through
TLR4 by identifying lipopolysaccharide (LPS) that is a part of the micro-
bial outer membrane triggering inflammatory response [10]. LPS in-
duced TLR4 activation and signal transduction, which then led to
nuclear factor κappa B (NF-κB) activation and nuclear translocation
⁎ Corresponding authors.
E-mail addresses: jhchenwhut@126.com (J. Chen), dcao@jiangnan.edu.cn (C. Deng).
[11]. Upon activation, NF-κB could bind to the promoter of target
genes encoding inflammatory mediators (e.g. IL-6) [12,13].
NOD-like receptor protein 3 (NLRP3) inflammasomes are cytosol-
localized large multiprotein oligomers consisted mainly of adaptor pro-
tein apoptosis-associated speck-like protein (ASC), NLRP3 and caspase-
1. Caspase-1 is a proteolytic enzyme mediating the cleavage and activa-
tion of secreted IL-18 and IL-1β that are prominent mediators of the in-
flammatory response. Increasing evidence indicates that there might be
a close relationship between NLRP3 inflammasome and inflammatory
diseases including cancer, arthritis, liver diseases and type 2 diabetes
[14–17].
The activation of NLRP3 inflammasomes requires two distinct sig-
nals: the priming signal typically induced by TLR4 that triggers the ex-
pression of pro-IL-1β, pro-IL-18 and the elements of the
inflammasome (NLRP3) through signal transduction pathways (e.g.
NF-κB) [18,19]; the activation of the signals in the second stage induced
assembly of the inflammasome complex consisting of ASC, NLRP3 and
inactive caspase-1, activation of caspase-1 and the cleavage of precursor
cytokines IL-1β and IL-18 [20,21]. Several molecules with similar func-
tion of PAMPs or DAMPs, such as reactive oxygen species (ROS) and ex-
tracellular ATP, may trigger the second signal [22].
Dictyophora indusiata, known as queen of mushrooms, is one of the
most popular edible mushrooms worldwide due to fantastic taste and
nutrition [23]. Also, it is used as the active components for agents with
therapeutic properties. Nowadays, extensive studies have been focused
on the roles of D. indusiata polysaccharide (DIP) with low toxicity and
multiple beneficial potencies, such as antioxidant [24], antitumor

https://doi.org/10.1016/j.ijbiomac.2019.01.022
0141-8130/© 2019 Elsevier B.V. All rights reserved.
 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166 1159

[25,26] and immunostimulatory activities [27]. However, little is known
about the anti-inflammatory roles of DIP in NLRP3 inflammasome in
LPS-stimulated macrophages. In this study, we aim to elucidate whether
DIP can modulate the NF-κB signal transduction pathway and NLRP3
inflammasome-mediated IL-18 and IL-1β secretion in LPS-primed and
ATP-stimulated macrophages.
2. Materials and methods
2.1. Chemicals
LPS extracted from Escherichia coli 0111:B4 was purchased from
Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), penicillin
G, streptomycin and Dulbecco's modified eagle medium (DMEM) were
purchased from Gibco (Carlsbad, CA, USA). Bay11-7082 was purchased
from Beyotime (Shanghai, China).
2.2. DIP preparation
Fruiting bodies of D. indusiata were purchased from Changning,
China. DIP isolation and purification was carried out according to
our previous experiments with slight modification [28]. Briefly,
D. indusiata powder was grinded with ethyl acetate and ethanol to re-
moved lipids. Then the water extract was centrifugated to collect the
polysaccharide-enriched fractions in the supernatant, while the
defatted resultant residue was extracted with distilled water at 90 °C.
Afterwards, the supernatant was precipitated with ethanol and refriger-
ated at 4 °C for 24 h. Upon removal of free proteins via Sevage method,
the yield polysaccharide fraction was further purified through gel
filtration chromatography (Sephadex G-200 chromatogram), followed
by dialysis and lyophilization. The product we obtained was a homoge-
neous component, with a molecular weight and total sugar content
of 650 kDa and 95.67%, respectively. DIP was a β-(1 → 3)-D-glucan
with side branches of β-(1 → 6)-glucosyl units according to gas
chromatography–mass spectrometry and infrared spectrum analysis
[23]. Endotoxin in DIP was very low (b0.01 ng/mL) with a concentration
of 25 μg/mL.
2.3. Cell culture and stimulation
Macrophage-like cell line RAW264.7, purchased from the Cell Bank
of Chinese Academy of Sciences (Shanghai, China), were cultured in
DMEM containing 10% FBS, streptomycin (100 μg/mL) and penicillin
(100 U/mL) at 37 °C in a humidified atmosphere containing 5% CO2.
Cells in the exponential growth phase were used for experiments.
To determine the roles of DIP in modulating LPS-induced NLRP3
inflammasome activation, RAW264.7 cells were divided into the follow-
ing groups: (i) blank control (cells cultured on DMEM medium); (ii) LPS
(1 μg/mL) group; (iii–v) LPS + DIP groups, subject to LPS (1 μg/mL) and
DIP with a concentration of 12.5 μg/mL, 25 μg/mL, or 50 μg/mL for 3 h,

Fig. 1. Inhibitory effects of DIP on LPS-induced expression of TLR4 in RAW264.7 macrophages. (A–C) The expression of TLR4 in the cell surface was detected by flow cytometry. (D) Relative
fluorescence intensity. Different lowercase letters indicated statistical differences (P b 0.05).
1160 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166

respectively. Afterwards, 5 mM ATP was added in LPS group and LPS
+ DIP groups and incubated for 1 h to assess NLRP3 inflammasome
activation.
2.4. Western blot analysis
For inflammasome activation analysis, cellular supernatant was col-
lected, and remaining cells were rinsed twice with ice-cold PBS and
lysed with RIPA lysis buffer containing proteinase inhibitors. Protein ex-
pression was determined by Western blot analysis according to the stan-
dard procedures. Protein content was measured with BCA method.
Denatured proteins were separated in a 10% SDS-PAGE, and then were
transferred to polyvinylidene difluoride membrane and blocked for 1 h
at room temperature. Then they were incubated with the rabbit anti-
pro-caspase-1 (Abcam, UK), anti-NLRP3 (Abcam, UK), anti-phospho-
IκBα (Abcam, UK), anti-IκBα (Abcam, UK), anti-GAPDH (Beyotime Bio-
technology, China) or goat anti-IL-1β (R&D system, China) overnight at
4 °C. Subsequently, the membranes were washed with TBST and incu-
bated with horseradish peroxidase-conjugated goat anti-rabbit or donkey
anti-goat IgG for 1 h. After washing the membrane with TBST thrice for
5 min, proteins were detected using an ECL detection kit (Beyotime,
Shanghai, China) according to the manufacturer's instructions. Bands
were visualized and photographed using Bio-Rad image analysis system
(Bio-Rad, CA, USA). The same membrane was probed with GAPDH for
loading control. Semiquantitative protein quantification was performed
using the ImageJ software (Bio-Rad, CA, USA).

Fig. 2. DIP inhibited LPS-induced NF-κB transcriptional activation. DIP reduced the LPS-induced activation of NF-κB by decreasing phosphorylation of IκB-α, the nuclear translocation of NF-
κB-p65 subunit and the protein secretion regulated by this signaling pathway. (A) Expression of P-IκBα and IκBα protein determined using Western blot analysis. (B, C) Protein semi-
quantification for P-IκBα and IκBα. (D) Nuclear translocation of NF-κB-p65 was observed under a CLSM. (E) Secretion of IL-6 detected by ELISA. Different lowercase letters indicated
statistical differences (P b 0.05).
 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166
2.5. Determination of Caspase-1 activation
Caspase-1 activity was detected using a commercial kit (Beyotime,
Shanghai, China) according to the manufacturer's instructions. In brief,
cellular extract was incubated with Ac-YVAD-pNA in a 96-well microti-
ter plate for 3 h at 37 °C. The absorbance values of pNA were measured
using a microplate reader at 405 nm (Spectra Max M5, Molecular De-
vices, USA).
2.6. Measurement of secretion of IL-6, IL-1β and IL-18
Concentration of mature IL-18, IL-1β and IL-6 in RAW264.7 cells was
determined using a commercial ELISA kit (Invitrogen, USA) according to
the manufacturer's protocol. The tests were performed at least in
triplicate.
2.7. Effect of DIP on NF-κB nuclear translocation
Immunofluorescence method was applied to determine the effects
of DIP on NF-κB nuclear translocation. RAW264.7 cells (1 × 105) were
seeded in a confocal plate and incubated at 37 °C in a humidified atmo-
sphere with 5% CO2 for 24 h. Cells were washed thrice with cold PBS and
fixed with cold paraformaldehyde (4%) solution at room temperature
for 12 min. After washing with cold PBS thrice for 5 min, the cells
were blocked with immunostaining blocking buffer (Beyotime, Shang-
hai, China) at room temperature for 1 h, and then were incubated
with rat monoclonal antibody NF-kB p65 (1:200, Beyotime, Shanghai,
China) overnight at 4 °C. The cells were washed with PBS thrice for
5 min and incubated with Cy3-labeled goat anti-rabbit IgG (Beyotime,
Shanghai, China) for 1 h in the dark. DAPI (Beyotime, Shanghai, China)
was added to each plate and incubated for 5 min. Finally, the cells
were washed with cold PBS thrice and observed under a CLSM.
Fig. 3. Effects of DIP on expression of inflammasome components in priming of inflammasome activation. (A) Western blot analysis of pro-caspase-1 and NLRP3 protein expression. (B–D)
Protein semi-quantifications were shown for pro-caspase-1 and NLRP3. Different lowercase letters indicated statistical differences. (P b 0.05).
1161
2.8. Effects of DIP on the combination of NLRP3 with ASC
The colocalization of NLRP3 with ASC was detected by immunofluo-
rescence staining with a CLSM. Cells were rinsed thrice with cold PBS
and fixed with cold 4% paraformaldehyde solution at room temperature
for 12 min. Next, the cells were blocked with immunostaining blocking
buffer at room temperature for 1 h and then incubated with rat mono-
clonal antibody NLRP3 (1:200, Abcam, UK) and rabbit monoclonal anti-
body ASC (1:200, Abcam, UK) overnight at 4 °C. Cy3-labeled goat anti-
rabbit IgG (Beyotime, Shanghai, China) was added to the cells and incu-
bated for 1 h. DAPI was added to each plate and incubated for 5 min at
room temperature in the dark, followed by observation under a CLSM.
2.9. Detection of ROS generation
ROS generation was determined using commercial kit (Beyotime,
Shanghai, China) according to the manufacturer's protocol. The alive
cells were collected and detected by flow cytometry.
2.10. Effect of TLR4 expression
Flow cytometry was applied to detect the effects of DIP on TLR4 ex-
pression. Briefly, the cells in each group were treated with PE anti-
CD284 (TLR4) antibody (Bio Legend, USA) for 2 h. Finally, cells were
washed with cold PBS thrice and detected by flow cytometry.
2.11. Statistical analysis
All data are presented as the means ± standard deviation (SD). Data
analyses were performed using the SPSS 23.0 software. Significant dif-
ferences were evaluated by one-way ANOVA. P b 0.05 was considered
to be statistically significant.
1162 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166

3. Results 3.2. DIP inhibited the transcriptional activation of NF-κB

3.1. DIP inhibited TLR4 expression
Compared with the LPS-treatment group, DIP (12.5 μg/mL, 25 μg/mL
and 50 μg/mL) significantly reduced fluorescence intensity (P b 0.05,
Fig. 1A–C). This indicated that DIP could inhibit TLR4 expression in
LPS-induced RAW264.7 macrophages. Particularly, DIP (25 μg/mL)
showed the most obvious inhibiting effects on TLR4 expression
(Fig. 1D).
To examine the roles of DIP in LPS-induced TLR4 signal transduction,
we evaluated the activation of the NF-κB pathway. DIP down-regulated
the expression of P-IκBα (Fig. 2A and B) and inhibited NF-κB nuclear
translocation of the p65 subunit (Fig. 2D). Also, it triggered decrease
of IL-6 (Fig. 2E), of which the gene expression was induced by LPS and
regulated through NF-κB in LPS-stimulated RAW264.7 macrophages.
The inhibition effect of NF-κB transcriptional activation by DIP was the
most obvious at a concentration was 25 μg/mL.

Fig. 4. Effects of DIP on the secretion of IL-1β and IL-18 via activating NLRP3 inflammasome in macrophages. (A) Confocal immunofluorescence images of NLRP3 and ASC in macrophages.
(B) Analysis of caspase-1 activity using Ac-YVAD-pNA; the activity of caspase-1 was reflected by the production of pNA. (C) IL-1β protein expression. (D, E) Protein semi-quantification was
shown for supernatant IL-1β and IL-1β of lysate. (F) Secretion of IL-18 was detected by ELISA. Different lowercase letters indicated statistical differences (P b 0.05).
 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166 1163

Fig. 5. IL-1β secretion was inhibited by DIP in LPS-primed RAW264.7 macrophages. (A, B) IL-1β concentrations in culture supernatant were detected by ELISA. Cells were stimulated with
or without 5 mM ATP for 1 h. Different lowercase letters indicated statistical differences (P b 0.05).

3.3. DIP decreased the inflammasome-priming 3.4. DIP inhibited inflammasome activation, IL-18 and IL-1β secretion

Protein expression in supernatant pro-caspase-1 and cytoplasmic
pool of NLRP3 was prominently decreased by the DIP in the LPS-
primed macrophages (Fig. 3A–D). Among these groups, DIP in different
concentrations can suppress the inflammasome-priming, and DIP (25
μg/mL) showed the most evident inhibitory effects.
In blank control, the expression of NLRP3 with ASC was very low,
whereas, colocalization NLRP3 with ASC was obviously increased in
LPS group. Combination of NLRP3 with ASC in experimental groups
was gradually reduced in the 12.5 μg/mL and 25 μg/mL dose groups,
but the inhibitory effects showed attenuation when treating with 50

Fig. 6. Effects of DIP on ROS generation. (A–C) ROS generation in different DIP dose groups (12.5 μg/mL DIP, 25 μg/mL DIP and 50 μg/mL). (D) Relative fluorescence intensity. Different
lowercase letters indicated statistical differences (P b 0.05).
1164 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166

μg/mL DIP (Fig. 4A). DIP significantly inhibited the production of acti-
vated caspase-1, especially at a concentration of 25 μg/mL (Fig. 4B).
DIP significantly inhibited caspase-1-mediated IL-1β (Figs. 4D and
5A) and IL-18 (Fig. 4F) generation, however, the inhibitory effects
showed gradual attenuation with the increase of DIP concentration.
Moreover, DIP also significantly inhibited the secretion of IL-1β when
RAW264.7 macrophages were only primed with LPS in the absence of
any other NLRP3 inflammasome-activating stimuli (Fig. 5B).
3.5. DIP inhibited the ROS production
ROS production involved in NLRP3 inflammasome activation. On
this basis, we investigated the effects of DIP on ROS production by ana-
lyzing the fluorescence intensity of DCF-DA. DIP (12.5 μg/mL, 25 μg/mL
and 50 μg/mL) could significantly inhibit the ROS generation in LPS-
primed and ATP-activated macrophages, and DIP (25 μg/mL) showed
the strongest inhibiting effect on ROS generation (Fig. 6).
3.6. NF-κB was required for the activation of the NLRP3 inflammasome
To elucidate the regulatory relationship of NF-κB in LPS and ATP-
stimulated macrophages, cells were treated with LPS in the presence
or absence of DIP (12.5 μg/mL) or Bay11-7082 (inhibitors with ability
to block NF-κB, 30 μM) for 3 h before stimulating with ATP (5 mM) for
1 h. Bay11-7082 inhibited LPS and ATP induced protein expressions in
NLRP3 inflammasome-priming (P-IkBα, supernatant pro-caspase1 and
NLRP3) and activation phase (supernatant IL-1β, Fig. 7). These results
indicated that NF-κB was critical for the activation of the NLRP3
inflammasome in LPS and ATP-treated cells. DIP (12.5 μg/mL) showed
similar anti-inflammatory effects compared to that of Bay11-7082.
4. Discussion
Inflammasome regulation deficiency involves in numerous chronic
inflammation and autoimmune diseases, such as inflammatory bowel
disease (IBD), systemic sclerosis, systemic lupus erythematosus and
rheumatoid arthritis [29]. NLRP3 inflammasome plays a major role in
the chronic inflammation as it recognizes microbial and cell stress com-
ponents. Additionally, it serves as a platform for caspase-1 activation
and pro-inflammatory cytokine IL-1β and IL-18 during the process of in-
flammatory and autoimmune diseases. Moreover, TLRs are the key me-
diators of innate host defenses in the intestine involving in the mucosal
immune homoeostasis. TLR4 signaling induces the inflammatory re-
sponse through the activation of NF-κB [30]. Thus, TLR4/NF-κB signal
pathway and NLRP3 inflammasome are potential therapeutic targets
for the treatment of inflammatory and autoimmune diseases [31].

Fig. 7. NF-κB was required for the activation of the NLRP3 inflammasome. (A) Western blot analysis findings. (B–H) Semi-quantification analysis of protein in supernatant IL-1β,
supernatant pro-caspase-1, lysis IL-1β, lysis pro-caspase-1, NLRP3, P-IkBα and IkBα. Different lowercase letters indicated statistical differences (P b 0.05).
 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166
Current research on inflammation has focused on the use of natural
products as alternative and effective anti-inflammatory therapies with
low toxicities and minimal adverse effects. These polysaccharides
from bacteria, fungi and mushrooms show high efficacy and lower tox-
icity than other therapeutic agents [32,33]. In a previous study, polysac-
charide of Hericium erinaceus attenuated dextran sodium sulfate (DSS)-
induced colitis in C57BL/6 mice via regulating NF-κB signal pathway and
the secretion of IL-6, IL-1β and TNF-α [34]. Meanwhile, Lycium
barbarum polysaccharides improved hepatic injury through NF-κB and
NLRP3 pathways in a steatohepatitis mouse model subject to a methio-
nine choline deficient diet [35]. In the present study, we found DIP re-
pressed the NF-κB signal transduction pathway and NLRP3
inflammasome-mediated IL-1β and IL-18 secretion in LPS-stimulated
macrophages.
Cell surface receptors, NF-κB signal transduction pathway and the
production of inflammasome components are the priming events es-
sential for inflammasome activation and subsequent IL-1β and IL-18 se-
cretion in the sequential regulation of inflammasome activation. Our
results indicated that DIP could reduce TLR4 expression, NF-κB pathway
activation and signal transduction by inhibiting IκB-α phosphorylation
and nuclear translocation of NF-κB-p65 subunit in LPS-primed macro-
phages. Also, the secretion of IL-6 that was a proinflammatory bio-
marker controlled by NF-κB signaling pathway was inhibited. For
inflammasome components, DIP inhibited the expression of NLRP3
and pro-caspase-1 of the inflammasome components. However, DIP
did not trigger a dose-dependent inhibition for these priming events es-
sential for inflammasome activation, which was possibly related to the
fact that DIP mainly exhibited an effect of inhibiting inflammation at
lower concentrations and an effect of promoting inflammation at a
higher concentration [28].
DIP is reported to modulate inflammasome activation by inhibiting
self-assembly of inflammasome (e.g. the oligomerization of NLRP3
Fig. 8. The anti-inflammatory mechanism of DIP in LPS-primed and ATP-stimulated macrophages.
1165
with ASC), proteolytic cleavage of inactive pro-caspase-1 and the secre-
tion of IL-1β and IL-18 in LPS-primed and ATP-activated macrophages.
We also analyzed IL-1β secretion modulation through DIP in LPS-
primed macrophages in the presence or absence of ATP stimulation.
DIP can inhibit the LPS-induced release of IL-1β independently of
inflammasome-triggered signals. Thus, it inhibited IL-1β secretion in
LPS-primed and ATP-mediated NLRP3 inflammasome activation and
also inhibited IL-1β secretion in LPS-primed and non-ATP-stimulated
inflammasome-activated macrophages. These indicated that LPS was
sufficient to promote IL-1β synthesis in the absence of inflammasome-
activating stimulus independent of the NLRP3 inflammasome sensor in-
duction. However, in LPS-primed macrophages, ATP served as an ago-
nist of the second signal of NLRP3 inflammasome activation triggered
the maturation and secretion of high amounts of IL-1β that was similar
to the previous studies [11,36].
When a LPS-primed macrophage is subjected to an activating stim-
ulus in a permissive cellular context, the NLRP3 inflammasome is acti-
vated by recruiting ASC and caspase-1. However, the exact molecular
events resulting in NLRP3 activation are still not well defined. Several
NLRP3 activation models are mutually interacted, such as pore forma-
tion, ion redistribution, lysosomal disruption, metabolic dysfunction
and mitochondrial dysfunction models. In these models, the common
mechanisms leading to NLRP3 activation were closely related to the
movement of K ions. High levels of extracellular ATP, that may occur
in cases of dying cells adjacent to the inflammasome-containing cells,
also resulted in ion flux by opening the P2X purinoceptor 7 channel.
Once opening of the channel, the NLRP3 inflammasome was activated
by allowing external flow of K ions. In addition, mitochondrial ROS are
also essential for NLRP3 activation (Fig. 8). Many NLRP3 triggers can
cause the increase in mitochondrial ROS production, and NLRP3 is
inhibited by pre-incubating with some antioxidants [8,37]. Therefore,
the inhibitory effects of DIP on IL-1β and IL-18 secretion may be related
1166 Y. Wang et al. / International Journal of Biological Macromolecules 126 (2019) 1158–1166

to the intrinsic antioxidant activity, and the potential inhibition of ROS Acknowledgment
production would be associated with the strong anti-inflammatory ef-
fects of DIP. This work was supported by National Natural Science Foundation of
As DIP could inhibit NF-κB signal pathway via modulating the TLR4 China (No. 31700708) and the Natural Science Foundation of Jiangsu
expression and reduce inflammasome activation via decreasing NLRP3 Province (No. BK20170181).
expression, it could be applied in treating the chronic inflammation
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ROS Reactive oxygen species 1325–1329.
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The authors declare no conflict of interest.