International Journal of Biological Macromolecules 132 (2019) 393–405

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Polysaccharide from Scutellaria baicalensis Georgi ameliorates colitis via

suppressing NF-κB signaling and NLRP3 inflammasome activation
Li Cui a, Wei Wang a, Yi Luo a, Qing Ning a, Zhi Xia a, Juan Chen a, Liang Feng b, Hua Wang a, Jie Song a,
Xiaobin Tan a, Wei Tan a, Chengcheng Wang a, Xiaobin Jia a,⁎
a
Affliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, Jiangsu, PR China
b
School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, Jiangsu, PR China

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

Article history: The purpose of the study was to extract, separate, purify and character Scutellaria baicalensis Georgi polysaccha-
Received 16 November 2018 rides, and to further explore the potential mechanism on colitis. Scutellaria baicalensis Georgi was extracted with
Received in revised form 14 March 2019 water and precipitated with alcohol. The obtained crude polysaccharide was fractionated sequentially by DEAE-
Accepted 29 March 2019
52 ion exchange column and Sephadex G-100 gel colum. Then, we obtain the purified fraction, named SP1-1. SP1-
Available online 30 March 2019
1 (4.56 × 105 Da) was mainly composed of mannose, ribose, glucuronic acid, glucose, xylose and arabinose with
Keywords:
molar ratios of 2.14:3.61:1:2.86:5.98:36.39. FI-TR indicated SP1-1 had the characteristic absorption of polysac-
Polysaccharide charides with a pyranoid ring. SEM displayed honeycomb structure. Oral administration of SP1-1 significantly de-
Colitis creased disease activity index, ameliorated dextran sulfate sodium-induced colonic pathological damage, and
NF-κB reduced myeloperoxidase activity. Additionally, SP1-1 markedly suppressed the proinflammatory cytokines
NLRP3 inflammasome level, including IL-1β, IL-18 and TNF-α, in the serum and colon of DSS-induced mice and THP-1-derived macro-
phages. Furthermore, a decreased CD11b+ macrophage infiltration in colons and inactivation of caspase-1 in
peritoneal macrophages were detected in SP1-1-treated mice. The mechanisms responsible for these effects of
SP1-1 were attributed to its inhibition on NF-κB signaling and NLRP3 inflammasome activation. These findings
support SP1-1 as a novel drug candidate in the treatment of colitis.
© 2019 Published by Elsevier B.V.

1. Introduction Nuclear factor-kappa B (NF-κB) and inflammasome are key compo-

Ulcerative colitis (UC) is one of the common types of inflammatory
bowel diseases (IBD), which has an effect on the intestinal mucosa (rec-
tum, colon and caecum) with the typical features of repeated outbreak
and protracted course of abdominal pain and diarrhea [1,2]. It happens
more generally in western countries, resulting in enormous economic
loss, meanwhile seriously impairing life quality and sometimes
lifethreatening [3,4]. It has been reported that UC contributed to risk
of cancerization of colitis, and demonstrated that 40% UC patients with
N25 years finally developed colorectal cancer [5]. Although the mecha-
nism of colitis is not fully understood, plenty of evidence indicates
that the induction and progression of this disease might result from a
dysregulated immune response to intestinal microbiota [6]. Patients
with UC have abundant macrophages in their mucosa, secreting excess
proinflammatory mediators cytokines, including interleukin (IL)-1β, IL-
6, IL-18 and tumor necrosis factor (TNF)-α [7]. Macrophages will trigger
and even aggravate the severity of colitis.
⁎ Corresponding author.
E-mail address: xiaobinjia_nj@126.com (X. Jia).
nents of inflammatory process and their dysregulation contributes to
UC for the ability to induce the overproduction of proinflammatory
cytokines. NF-κB is a critical transcription factor associated with the
synthesis and release of inflammatory cytokines and chemokines [8].
Inhibition of NF-κB activation is an effective strategy for preventing
experimental models of IBD and blocking inflammatory cytokine
production in IBD patients [9]. Notably, NLRP3 inflammasome was
extensively studied and played a critical role in intestinal homeostasis
and inflammation. The NLRP3 inflammasome was a multiprotein
platform comprising the NLRP3 protein, the adaptor protein ASC and
caspase-1, could further activate IL-1β and IL-18 in different tissue
environments [10–12]. In dextran sulfate sodium (DSS)-induced acute
colitis model, NLRP3 gene knockout or medical inhibition of the
NLRP3 inflammasome activation both exerted protective effects on
mice, demonstrating NLRP3 as a key mediator in mouse colon inflam-
mation [13–15].
Currently, polysaccharides have gained widespread attention owing
to its noteworthy pharmacological activities with fewer side effects.
Currently, polysaccharides exhibited remarkable curative effect in the
treatment of colitis [16,17], suggesting its potential application as natu-
ral alternative medicine in colonic inflammation treatment. Scutellaria

https://doi.org/10.1016/j.ijbiomac.2019.03.230
0141-8130/© 2019 Published by Elsevier B.V.
394 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405
baicalensis Georgi is the monarch drug of Huangqin Tang, a traditional
Chinese formula, which has been used in China for a wide range of dis-
orders, especially in gastrointestinal inflammation [18]. Flavone and
polysaccharide are regarded as the important effective components of
Scutellaria baicalensis Georgi. The flavone contains baicalin, baicalein,
wogonoside, wogonin, oroxylin, and so on. Cui et al. reported that
baicalin attenuated experimental colitis through inhibiting TLR4/NF-
κB pathway activation [19]. Zhang et al. suggested that baicalin may al-
leviate inflammatory infiltration in dextran sodium sulfate-induced
chronic ulcerative colitis via inhibiting IL-33 expression [20]. Baicalein
and wogonoside could ameliorate colitis [21–23]. However, high-dose
wogonin exacerbated DSS-induced colitis by up-regulating effector T
cell function and inhibiting Treg cell [24]. The effect of polysaccharide
on colitis has never been explored. Based on the protective effect of
other polysaccharides against colitis, we speculated that polysaccha-
rides from Scutellaria baicalensis Georgi may exhibit a prophylactic and
therapeutic role in colitis. To demonstrate this hypothesis, we explored
the protective effects and potential mechanism of polysaccharides from
Scutellaria baicalensis Georgi on an acute colitis mice model induced by
DSS and LPS-stimulated THP-1-derived macrophages.
2. Materials and methods
2.1. Materials and reagents
Antibodies against inhibitor of NF-κB (IκBα), p-IκBα, inhibitor of NF-
κB kinase α (IKKα), p-IKKα, IKKβ, p-IKKβ, p-NF-κB p65, NF-κB p65,
ASC, NLRP3, caspase-1, cleaved caspase-1, pro-caspase-1, cleaved IL-
1β, cleaved IL-18, β-actin, lamin B were purchased from Santa Cruz Bio-
technology (Santa Cruz, CA). Adenosine triphosphate (ATP), lipopoly-
saccharide (LPS), phorbol myristate acetate (PMA) and 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)
were purchased from Sigma Chemical Co. (St. Louis, MO). Dextran sul-
fate sodium (DSS, 36–50 kDa) was purchased from MP Biomeicals (Au-
rora, OH). Immunohistochemistry kit was from KeyGEN Biotech Inc.
(Nanjing, China). Myeloperoxidase (MPO) activity assay kit was from
Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits
for IL-1β, IL-18 and TNF-α were purchased from Wuhan Boster Biolog-
ical Engineering Co., Ltd. (Wuhan, China). FITC-anti-CD11b was ob-
tained from Abcam (Cambridge, UK). Monosaccharide references
(mannose, ribose, glucuronic acid, glucose, xylose and arabinose) and
glucan reference were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). Ultrapure water was purified by the Milli-Q water purifica-
tion system (Millipore, Bedford, Ma, USA). All the other reagents and
chemicals were from standard commercial sources.
2.2. Animals and cell lines
C57BL/6 mice (8–10 weeks, 20 ± 2 g, male and female) were
purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai,
China). They had access to food and water ad libitum, were kept under
a 12-h light/dark cycle, and were housed at 25 °C with a relative humid-
ity of 45%. Animal welfare and experimental procedures were
conducted strictly according to protocols approved by the Animal Ethics
Committee of Jiangsu Provincial Academy of Chinese Medicine.
Human THP-1 cells were obtained from the Cell Bank of the Shang-
hai Institute of Cell Biology (Shanghai, China). The cells were main-
tained in RPMI 1640 medium, supplemented with 10% fetal calf serum
(FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS at 37
°C with 5% CO2 in a humidified atmosphere. THP-1 cells were stimulated
by phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) overnight to dif-
ferentiate into macrophages [25]. Culture was exchanged with fresh
culture medium every 3 days. Peritoneal macrophages were obtained
from the peritoneal cavity as reported before [26]. Cells were washed
twice in PBS and suspended in RPMI-1640 medium containing
10,000 U/ml penicillin, 10 mg/ml streptomycin and 10% FCS. The
macrophages were cultured in 24-well microplates for 40 min at 37 °C
in a moist atmosphere of 5% CO2. Under the conditions, adherent macro-
phages were obtained for further experiments.
2.3. Extraction and purification SP1-1
Water extraction with alcohol precipitation was used to refine poly-
saccharides [27]. Briefly, Scutellaria baicalensis Georgi (400 g) were
ground to a fine power, immersed in petroleum ether (60 °C–90 °C) at
room temperature overnight. The solid residue was collected by filtra-
tion and the procedure was repeated twice to defat. The pretreated sam-
ple was extracted by water, and the water extraction solution was
precipitated by the addition of ethanol to a final concentration of 80%
(v/v) and kept at 4 °C for 24 h. Then, the supernatant was removed by
filtration, the obtained crude Scutellaria baicalensis Georgi polysaccha-
ride was redissolved in water, and deproteinized five times using the
Sevage method [28]. The supernatant was collected and desiccation in
vacuo. The crude polysaccharide was redissolved in deionized water
and applied to DEAE-52 column (2.6 cm × 50 cm), eluting at a folw
rate of 0.8 ml/min with deionized water and 0.1, 0.3, 0.5 and 1.0 mol/L
NaCl. The resulting fractions were combined according to the carbohy-
drate content quantified by the phenol‑sulfuric acid method. Then, the
deionized water fraction was further fractionated with size-exclusion
chromatography on a Sephadex G-100 gel column (1.6 cm × 60 cm),
and eluted at a flow rate of 0.2 ml/min with deionized water. The frac-
tion was collected, concentrated, dialyzed and lyophilized to obtain a
purified polysaccharide (SP1-1). The SP1 was then stored in a bottle
desiccator at room temperature for further study.
2.4. Preliminary characterization of SP1-1
2.4.1. Total polysaccharide and total protein determination
The phenol–sulfuric acid method was performed for the determina-
tion of total sugars using glucose as standard [29]. Protein was analyzed
using Bradford method, using bovine serum albumin (BSA) as standard
[30].
2.4.2. Molecular weight
The homogeneity and molecular weight of SP1-1 was determined by
HPLC, which was carried out by HPLC, which was equipped with a LC-
10AT chromatograph (Shimadzu, Tokyo, Japan), refractive index
detection (RID-10A), TSK guard column PWH (7.5 mm × 75 mm;
Tosoh Corporation, Tokyo, Japan) and TSK-GEL G4000PWXL column
(7.8 mm × 300 mm; Tosoh Corporation, Tokyo, Japan). The column
oven was controlled by the Shimadzu Class-VP 5.0 chromatography
workstation, and the column temperature was maintained at 30 °C.
The flow rate of the mobile phase (0.05 mol/L NaCl) was 0.8 ml/min.
The injection volume was 10 μl. The calibration curve was made based
on a series dextran of known molecular weight as standard.
2.4.3. Monosaccharide compositional analysis
SP-1-1 (10 mg) was hydrolyzed with 1 ml 4 M trifluoroacetic acid
(TFA) at 110 °C for 8 h. Then, methanol was employed to remove the ex-
cess TFA, followed by evaporation to dryness. The hydrolysate was dis-
solved in aquadistillate (100 μl), neutralization with a stoichiometric
amount of 3 M sodium hydroxide. After reaction, the mixture was cen-
trifuged and the supernatant was collected. Take the supernatant 100 μl,
and successively add 0.5 M methanol solution of PMP 100 μl and 0.3 M
sodium hydroxide 100 μl, react in water bath at 70 °C for 60 min. After
being cooled to room temperature, 150 μl of 0.2 M HCl was used to neu-
tralize the resultant solution and evaporated to dryness. The residue
was redissolved in deionized water, and the excess PMP was removed
by the addition of chloroform [30]. The aqueous layer was filtered
through a 0.45 μm membrane and analyzed by a HPLC system, equipped
with a Zorbax Extend-C18 (4.6 mm × 250 nm, 5 μm), detected at
250 nm with column temperature at 25 °C. The mobile phase was a
 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405
mixture of acetonitrile (A) and phosphate buffered saline (KH2PO4,
0.05 M, pH 6.8; B) with a gradient elution: 0–5 min, 83%–82% B;
5–10 min, 82%–81% B; 10–30 min, 81%–80% B; 30–35 min, 80%–83% B.
The injection volume was 10 μL. The standard monosaccharides
derivatizated with PMP and measured by the same procedure.
2.4.4. Infrared spectrum and UV spectrum analysis
The IR spectra of the polysaccharides were carried out using a Fou-
rier transform IR spectrophotometer (FT-IR) (Nicolet Avatar-370,
USA). SP1-1 was ground with KBr powder and then pressed into pellets
for FT-IR measurement in the frequency range of 400–4000 cm−1. SP1-1
was dissolved to 1 mg/ml solution and scanned from 200 to 400 nm
with an UV spectrophotometer (Model SP-752, China).
2.4.5. SEM analysis
The microstructure of the refined polysaccharide was evaluated by
scanning electron microscope. The sample particles were fixed on the
silicon wafer. The shape and surface characteristics were observed and
recorded using a SEM (S-3400N, Hitachi, Japan). SEM images were
taken at an accelerating voltage of 5 kV, with magnifications of ×300,
×1000, ×3000 and ×10,000.
2.5. Acute toxicity test
To identify the maximum tolerated dose of SP1-1 in mice, we per-
formed the acute toxicity test. Ten female C57BL/6 mice and ten male
C57BL/6 mice were given the maximum dose of SP1-1 by intragastric
administration (maximum concentration 500 mg/ml, maximum gavage
volume 0.4 ml/10 g), respectively. Observation for a week, to see
whether the animal's activity, fur, diet and feces had any abnormal
changes, and whether there were poisoning symptoms and death. At
the end of experiment, the mice were euthanized. Major organs includ-
ing heart, liver, spleen, lung and kidney were isolated and stained with
hematoxylin and eosin (HE).
2.6. Induction of colitis and design of drug treatment
Colitis was induced in mice with 3% DSS (w/v), which was dissolved
in drinking water, continuously for 7 days. Normal mice were given
water. The mice were divided into six group (n = 10/group). The
mice in low-dose, medium-dose and high-dose SP1-1 group received
50, 100, 200 mg/kg SP1-1 per day by gastric lavage. The mesalazine
group received 100 mg/kg mesalazine per day by gastric lavage [31].
Water was given orally to mice in the normal control and model groups.
The animals in each group were treated from day 1 to day 10. On the
11th day, after 24 h fasting, blood samples obtained via the orbital
sinus were centrifuged at 3000 ×g at 4 °C for 10 min, and the superna-
tants were stored at −70 °C. Then, the mice were sacrificed by cervical
dislocation. Distal colonic specimens were frozen in liquid nitrogen or
fixed immediately in a 10% (w/v) formalin solution for further analyses.
2.7. Clinical scoring and histological analysis
Mice were observed for body weight, stool consistency and the pres-
ence of gross blood in feces and at the anus every day. The disease activ-
ity index (DAI) was determined by assigning well-established and
validated scores [32]. Briefly, the following parameters were used for
calculation: i) diarrhea (0 point = normal, 2 points = loose stools, 4
points = watery diarrhea); ii) hematochezia (0 point = no bleeding,
2 points = slight bleeding, 4 points = gross bleeding). Histological
score was carried out based on the following five parameters. For lesion
range, 0 point was given for 0%, 1 point for 1%–25%, 2 points for 26%–
50%, 3 points for 51%–75%, and 4 points for 76%–100%. Lesion depth
was scored 0 for no lesion, 1 point for mucosal epithelium, 2 points for
mucoderm, 3 points for submucosal layer, and 4 points for muscular
layer and serous layer. Crypt damage was scored 0 for intact crypts, 1
395
point for loss of the basal one third, 2 points for loss of the basal two
thirds, 3 points for loss of entire crypt and intact epithelial surface, 4
points for the both loss of change of crypt. For severity of inflammation,
0 point for no signs of inflammation, 1 point light leukocyte infiltration,
2 points for low leukocyte infiltration, 3 points for moderate leukocyte
infiltration, 4 points for high leukocyte infiltration. Fibrous tissue prolif-
eration was scored 0 point for no fibrosis, 1 point for light fibrosis, 2
points for low fibrosis, 3 points for moderate fibrosis, 4 points for exten-
sive fibrosis.
2.8. Assay of colonic myeloperoxidase activity
Neutrophil infiltration into inflamed colonic mucosa was quantified
by myeloperoxidase (MPO) activity assessment. On the 11th day, mice
were sacrificed and then the colonic tissue segments were homoge-
nized in a solution containing 5% EDTA/NaCl buffer (pH 4.7) followed
by a centrifugation at 10,000 ×g for 10 min at 4 °C. The pellet was
then resuspended in 0.5% hexadecyl trimethyl ammonium bromide
(TMB) buffer (pH 5.4), and then centrifuged for 30 min at 20,000 ×g
and 4 °C. An aliquot (50 μL) of the supernatant was added to a reaction
mixture of 1.6 mM tetramethylbenzidine and 0.1 mM H2O2 and incu-
bated at 37 °C. The absorbance was measured spectrophotometrically
at 690 nm, and the result was expressed in unit per mg tissue.
2.9. Cytokine analysis by ELISA
Inflammatory cytokines, including IL-1β, IL-18 and TNF-α, in colon
tissues and serum, and THP-1-derived macrophages were quantified
by their respective ELISA kits according to the manufacturer's instruc-
tions (KeyGEN Biotech. Co., Ltd., Nanjing, PR China). For the ELISA of
IL-1β, IL-18 and TNF-α, colons from mice in each group were homoge-
nized in 1 ml of ice-cold RIPA lysis buffer containing 1% protease inhib-
itor cocktail and 1% phosphatase inhibitor cocktail. The homogenate
was centrifuged (15,000 ×g, 4 °C) for 15 min, and the supernatant was
transferred to 96-well plates. The amount of IL-1β, IL-18 and TNF-
αwere quantified according to the standard curve.
2.10. Western blot analysis
The protein lysates were separated by 10% SDS-PAGE gel and then
transferred onto a PVDF membrane blocking with 5% skim milk. Subse-
quently, the blocked membrane was incubated with the primary anti-
bodies. Membranes were rinsed with TBST for four times (10 min/
time) and then incubated with the horseradish peroxidase-bound sec-
ondary antibody (1:5000) in a shaker for 1 h at room temperature.
Chemo luminescence reagents were added for the visualization of the
protein bands. The quantification of proteins was analyzed by Image-
Pro Plus (IPP) software.
2.11. Immunohistochemistry analysis
Immunohistochemistry was performed as previously described [33].
Colon tissues were preserved by perfusion fixation with a solution of 4%
paraformaldehyde and blocked by paraffin. All sections were cut to 5 μm
thick, and were heated in 10 mM sodium citrate buffer (pH 6.0) to re-
trieve antigens. The endogenous peroxidase activity was blocked by in-
cubation with hydrogen peroxide in methanol. Sections were incubated
with citrate buffer solution at 100 °C for 10 min. Then, sections were
blocked with 10% horse serum. Primary antibodies anti-NLRP3
(1:200), anti-IL-1β (1:200), anti-IL-18 (1:200), anti-p65 NF-κB
(1:200), were diluted for application at the incubation of tissues,
followed by the secondary antibody. All sections were visualized and
photographed under a light microscope (Olympus IX71, Japan). The
positive expression was determined by IPP software.
396 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405

2.12. Immunofluorescence of colon tissues
The immunofluorescence was carried out on the paraffin-embedded
colonic tissue sections (5 μm). In brief, the sections were deparaffinized,
rehydrated and washed in 1% PBS–Tween buffer. Sequentially, they
were treated with 3% hydrogen peroxide, blocked with 10% goat
serum and incubated with CD11b primary antibody in PBS containing
1% BSA for 1 h at room temperature. The slides were counter-stained
with DAPI. The reaction was terminated by thorough washing in
water for 20 min. Images were obtained by confocal laser-scanning mi-
croscope (Olympus FV1000, Lake Success, NY). The parameters of image
acquisition were identical for tissues in different groups.
2.13. Real-time RT-PCR
Real-time PCR was performed as described previously [34]. Briefly,
RNA samples were reverse transcribed to cDNA and subjected to quan-
titative PCR, which was performed with the LightCycler® 96 Real-Time
PCR System (Roche, Basel, Swiss) using AceQ qPCR SYBR Green Master
Mix (Vazyme, Nanjing, China). Conditions for amplification were
1 cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60 °C
for 30 s, and 95 °C for 10 s. In this study, the samples were analyzed in
triplicate. The primer sequences used in this study were as follows: IL-
1β forward, 5′-CTT CAG GCA GGC AGT ATC ACT C-3′; IL-1β reverse, 5′-
TGC AGT TGT CTA ATG GGA ACG T-3′; IL-18 forward, 5′-GCC TCA AAC
CTT CCA AAT CA-3′; IL-18 reverse, 5′-TGG ATC CAT TTC CTC AAA GG-
3′; TNF-α forward, 5′-CGA GTG ACA AGC CTG TAG CCC-3′; TNF-α re-
verse, 5′-GTC TTT GAG ATC CAT GCC GTT G-3′; NLRP3 forward, 5′-GAT
CTT CGC TGC GAT CAA CAG-3′, NLRP3 reverse, 5′-CGT GCA TTA TCT
GAA CCC CAC-3′; pro-caspase-1 forward, 5′-TTT CCG CAA GGT TCG
ATT TTC A-3′, pro-caspase-1reverse, 5′-GGC ATC TGC GCT CTA CCA
TC-3′; β-actin forward, 5′-TGC TGT CCC TGT ATG CCT CT-3′; β-actin re-
verse, 5′-TTT GAT GTC ACG CAC GAT TT-3′.
followed by Tukey's test using SPSS version 13.0 software. Statistical sig-
nificance was indicated by the p value b0.05.
3. Results and discussion
3.1. Physical characteristics of SP1-1
The carbohydrate content of SP1-1 was 72.64%, and there was no ob-
vious protein content in Bradford method. Fig. 1A shows the chromato-
gram of SP1-1. A single, symmetric peak indicates a high purity of the
polysaccharide. The average molecular weight was calculated to be
4.56 × 105 Da according to the calibration curve. The calibration curve
was obtained from a series dextran: lgMw = −0.6552 tR + 10.273, R2
= 0.9903. As shown in Fig. 1B, SP1-1 was mainly composed of mannose
(Man), ribose (Rib), glucuronic acid (GluA), glucose (Glc), xylose (Xyl)
and arabinose (Ara) with molar ratios of 2.14:3.61:1:2.86:5.98:36.39.
The infrared spectroscopy (IR) of SP1-1 was shown in Fig. 2A. The
polysaccharide displayed a broad and intense peak at 3405 cm−1 for
O\\H stretching vibration as well as a weak absorption peak at
2934 cm−1 for C\\H stretching vibrations [36]. The signals at
1731 cm−1 and 1639 cm−1 were attributed to asymmetric stretching
of the carboxylate anion group (C = O) [37]. The peak at 1417 cm−1
was caused by C\\O stretching vibrations. The bands at 1384 cm−1 cor-
respond to symmetrical deformations of the CH2 groups. The signals at
1314 cm−1 and 1248 cm−1 were assigned to symmetric stretching of

2.14. Nuclear and cytoplasmic extraction

After the treatment with indicated stimuli, THP-1 cells were har-
vested by centrifugation and washed with PBS for three times. Nuclear
and cytosol fractions were prepared using the Nuclear/Cytosol Fraction-
ation Kit (BioVision, Mountain View, CA) according to the protocol of
the manufacturer. A Bio-Rad protein assay dye, using the Bradford
method, was applied to measure the protein concentration in the nu-
clear and cytoplasmic fractions. The obtained extractions were stored
at −70 °C for further investigation.

2.15. Immunoprecipitation (IP)

IP was carried out as previously described [35]. Whole-cell lysates

were incubated with anti-NLPR3 antibody (1:100), anti-ASC antibody
(1:100) and anti-caspase-1 antibody (1:100). Samples were stored at
−20 °C for further experiment.

2.16. Assay of caspase-1 activity

The activity of caspase-1 was measured using a caspase-1 activity

assay kit according to the manufacturer's instructions. This activity de-
tection kit is based on the fact that caspase-1 can catalyze the produc-
tion of yellow pNA (p-nitroaniline) from ac-yvad-pna (acetyl-tyr-val-
ala-asp p-nitroanilide). Then, the activity of caspase-1 can be detected
by measuring absorbance at 405 nm.

2.17. Statistical analysis

Fig. 1. Chromatographic spectrum of SP1-1 from Scutellaria baicalensis Georgi and its
All data in this study are taken from three independent experiments monosaccharide composition. (A) Chromatographic spectrum. (B) Monosaccharide
and then expressed as means ± standard deviation (S.D.). Statistical composition. a, PMP derivative of monosaccharide reference substances; b, PMP
analysis was carried out by one-way analysis of variance (ANOVA) derivative of SP1 sample (1-Man, 2-PMP, 3-Rib, 4-GluA, 5-Glc, 6-Xyl, 7-Ara).
 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405 397

Fig. 2. Spectrum analysis and surface morphology characterization of SP1-1 from Scutellaria baicalensis Georgi. (A) FT-IR spectra of SP1-1. (B) UV spectra. (C) SEM figures (a, ×300
magnification; b, ×300 magnification; c, ×1000 magnification; d, ×10,000 magnification).

C = O. Each particular polysaccharide has a specific band in the
1000–1200 cm−1 region. This region is dominated by ring vibrations
overlapped with stretching vibrations of (C–OH) side groups and the
(C–O–C) glycosidic band vibration. The absorption at 1040 cm−1 indi-
cated a pyranose form of sugar [29,36,37]. Additionally, as shown in
Fig. 2B, there was no absorption between 260 and 280 nm in the UV
spectrum, suggesting that there was no protein and nucleotide. In Brad-
ford method, no obvious protein was found, further confirming that
protein has been removed.
Analysis of surface morphology by SEM is a qualitative tool to char-
acterize polysaccharides. As shown in Fig. 2C, an obvious honeycomb
structure was observed under light microscope with 10,000× magnifi-
cation. The morphological characteristics were possibly caused by the
changes of physicochemical properties or the extraction and separation
process.
3.2. Acute toxicity test
During the experiment, the animal's activity, fur, diet and feces
had no changes. After a week of observation, no mice died. Then,
the toxicity of SP1-1 to the mice was evaluated by examination of
the HE-staining sections of major organs. As shown in Fig. 3, no obvi-
ous damage could be observed in the organ sections, including heart,
liver, spleen, lung and kidney, indicating that SP1-1 has no toxicity to
the mice.

Fig. 3. Toxicity of SP1-1 to mice was evaluated by HE staining of tissue section.

398 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405

3.3. SP1-1 attenuated DSS-induced experimental colitis
Throughout the experiment, there was two mice dead in model
group, one mouse dead in low-dose SP1-1. A 3% DSS in drinking water
for 7 days should not result in mortality. The dosage in low-dose SP1-
1 group was 50 mg/kg, far lower than the dosage in toxicity test. The
probable cause was accidental death rather than drug toxicity. The ob-
servation of signs and symptoms is an important part of disease
diagnosis. Colitis model presented characteristics of remarkable appear-
ance of diarrhea/loose feces and obvious fecal blood, leading to impor-
tant DAI enhancement [38]. Comparatively, it was ameliorated by SP1-
1 treatment (Fig. 4A). Histological analysis showed lesion range, lesion
depth, crypt damage, severity of inflammation and fibrous tissue prolif-
eration in the colon specimens of colitis mice, however, SP1-1 improved
pathological changes in a dose-dependent manner (Fig. 4B,C). The MPO
activity of colonic tissues was enhanced in DSS group compared with

Fig. 4. SP1-1 treatment ameliorated DSS-induced colon damage in mice. (A) DAI was determined. (B) Serial sections of colon tissues were stained with HE. a, control group; b. DSS group; c.
mesalazine group; d. low-dose SP1-1 + DSS group; e. medium-dose SP1-1 + DSS group; f. high-dose SP1-1+ DSS group. a, control blank; b, model group (DSS); c, mesalazine group; d,
low-dose SP1-1; e, medium-dose SP1-1 and f, high-dose SP1-1. (C) Histological scores of each group were determined. (D) MPO activity in the colonic tissues. Data are expressed as means
±SD (n = 8). ⁎⁎p b 0.01, compared with control group; &&p b 0.01, &&&p b 0.001, compared with model group; #p b 0.05, ##p b 0.01, compared with model group.
 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405 399

the control group, whereas, it was decreased by SP1-1 oral administra-
tion (Fig. 4D, #p b 0.05, ##p b 0.01). These data indicated that SP1-1 had
the protective effect on DSS-induced colitis in mice.
3.4. Cytotoxicity of SP1-1 to THP-1 derived macrophages
We investigated the protective mechanisms of SP1-1 against UC on
THP-1 derived macrophages in vitro. The viability of THP-1 derived mac-
rophages under different concentrations (20, 40, 80, 160, 320, 640, 1280
μg/ml) of SP1-1were estimated. The result showed that there were no
significant changes in cell viability (%) with increasing concentration of
SP1-1, indicating no obvious cytotoxicity of SP1-1 on THP-1 derived mac-
rophages. Even so, lower concentrations (20, 40 and 80 μg/ml) of SP1-1
were picked for further study on its protective mechanisms against UC.
3.5. SP1-1 regulated inflammatory cytokines production
IL-1β, IL-18 and TNF-α were important inflammasome-related fac-
tors that were known to be upregulated in DSS-induced inflammation
[39]. We prepared serum and colon homogenates, then analyzed the cy-
tokine production by ELISA. As shown in Fig. 5A,B, the levels of IL-1β, IL-
18 and TNF-α in serum and colon were both increased markedly after
DSS treatment. However, mesalazine significantly decreased the ex-
pression levels of these inflammatory cytokines. This increase could
also be decreased after SP1-1 treatment. Subsequently, we further con-
firmed the anti-inflammatory effect of SP1-1 in vitro. The result revealed
that SP1-1 inhibited the production of IL-1β, IL-18 and TNF-α induced
by LPS in a dose-dependent manner (Fig. 5C). Additionally, mRNA levels
of IL-1β, IL-18 and TNF-α were also significantly inhibited by SP1-1

Fig. 5. SP1-1 inhibited inflammatory cytokine production. (A,B) Colitis was induced by DSS. The expression levels of the inflammatory cytokines, including IL-1β, IL-18 and TNF-α, both in
the serum and colon tissue were determined by ELISA. (C,D) SP1-1 inhibited inflammatory cytokines production in THP-1-derived macrophages. The cells were treated with LPS (1 μg/ml)
alone or with indicated concentrations of SP1-1 for 6 h followed by 45 min incubation of 5 mM ATP. The expression levels of inflammatory cytokines were analyzed by ELISA and real-time
RT-PCR. Data are expressed as means±SD (n = 3). ⁎⁎p b 0.01, compared with control group; &&p b 0.01, compared with DSS group, #p b 0.05, ##p b 0.01, compared with DSS group.
400 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405

(Fig. 5D). The above results indicated that SP1-1 suppressed the expres-
sion of IL-1β, IL-18 and TNF-α.
3.6. SP1-1 attenuated macrophage infiltration and reduced cleaved
caspase-1 expression of peritoneal macrophages in DSS-induced colitis
mice
To further investigate the mechanism through which SP1-1 regu-
lated intestinal inflammation and recovery, we evaluated the effect of
SP1-1 on inflammatory cells infiltration in the colon tissues. CD11b is
expressed on the surface of many leukocytes including monocytes,
neutrophils, natural killer cells, granulocytes and macrophages [40].
Therefore, we examined macrophage infiltration in colon tissues from
each experimental group. Compared with control group, more
CD11b+ macrophages in the mucosa of the lesion site were detected
in DSS-treated mice. However, the infiltrating macrophages were
down-regulated by mesalazine and SP1-1 (Fig. 6A). Macrophages incu-
bated with DSS secreted high levels of IL-1β in a caspase-1-dependent
manner [14]. We evaluated the modulation of caspase-1 activation
in vivo from model and SP1-1 groups. The result indicated that SP1-1

Fig. 6. SP1-1 attenuated macrophage infiltration and suppressed caspase-1 activation in DSS-treated mice. (A) Serial sections of colonic tissue were immunostained with DAPI (blue) and
anti-CD11b-FITC (green) and observed by confocal laser-scanning microscope, 400×. (B) Peritoneal macrophages were isolated from control, mesalazine-treated and SP1-1-treated colitis
mice at day 11. After 5 mM ATP stimulation for 45 min, proteins were collected for western blot. Data are expressed as means±SD (n = 3). ⁎⁎p b 0.01, compared with control group; &&p b
0.01, compared with DSS group, #p b 0.05, ##p b 0.01, compared with DSS group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405 401

Fig. 7. SP1-1 suppressed the activation of NF-κB signaling pathway. (A) Protein expressions of NF-kB p65, IL-1β and IL-18 in colonic tissues were detected by immunohistochemistry.
(B) Western blot assay for the protein levels of p-IKKα, IKKα, p-IKKβ, IKKβ, p-IκBα and IκBα. THP-1-derived macrophages were treated with various concentrations of SP1-1 in the
absence or presence of LPS (1 μg/ml) for 6 h. (C) The protein levels of total and phosphorylated NF-kB p65 were determined and quantitatively analyzed both in cytoplasmic and
nuclear in THP-1-derived macrophages. (D) Cells were treated with various concentrations of SP1-1 in the absence or presence of LPS for 6 h, then immunostained with DAPI (blue)
and anti-p-NF-kB p65 (green) and observed using a fluorescence microscope. a- control, b-LPS, c-LPS + 20 μg/ml SP1-1, d-LPS + 40 μg/ml SP1-1, e-LPS + 80 μg/ml SP1-1. The original
amplification was 200×. Data were expressed as means±SD (n = 3). ⁎⁎p b 0.01, compared with control group; #p b 0.05, ##p b 0.01, compared with LPS group. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)
402 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405

remarkably suppressed caspase-1 activation in vivo compared with con-
trol group (Fig. 6B).
3.7. SP1-1 inhibited the activation of NF-kB signaling
NF-κB is a transcription factor that promotes transcription of genes
encoding pro-inflammatory cytokines and has been implicated in the
pathogenesis of various inflammation-related diseases [41,42]. NF-κB
signaling is considered as a critical switch in the regulation of immune
and inflammatory responses by controlling the expression of inflamma-
tory cytokines [43]. Therefore, the suppression of NF-κB activation could
be a very effective strategy to prevent experimental models of UC and
block inflammatory cytokine production in UC patients [9,44]. To
investigate the possible molecular mechanism of SP1-1, the activation
of NF-κB signaling was estimated. Strong nuclear NF-κB p65 staining
was observed in colonic tissues of DSS-induced mice compared with

Fig. 8. SP1-1 inhibited the activation of NLRP3 inflammasome. (A) Down-regulation of SP1-1 on DSS-induced NLRP3, IL-1β and IL-18 protein expression in colon by immunhistochemistry.
(B) The mRNA expression of NLRP3 in colonic tissue was determined. (C) The protein expressions of NLRP3, caspase-1, cleaved caspase-1, IL-1β, pro-IL-1β, IL-18 and pro-IL-18 were
determined by western blot. Data are expressed as means±SD (n = 3). ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001, compared with control group; &&p b 0.01, compared with DSS group, #p b 0.05, ##p b
0.01, compared with DSS group.
 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405 403

the control group. Notably, the number of NF-κB p65 in nucleus was re-
duced by SP1-1 in a concentration-dependent manner (Fig. 7A). Multi-
ple evidences indicated that the phosphorylation of IKKα and IKKβ as
well as the phosphorylation and degradation of IκBα were crucial for
the activation of the NF-κB signaling pathway [45]. Therefore, we eval-
uated the regulatory effect of SP1-1 on IKKα, IKKβ and IκBα, which
were important upstream regulators of the inflammation-related NF-
κB signaling pathway. In THP-1-derived macrophages, LPS up-
regulated the p-IκBα level and down-regulated total IκBα expression.
But SP1-1 significantly reversed the effect of LPS by suppression of p-
IκBα and increase of total IκBα expression. Additionally, an obvious in-
crease of p-IKKα and p-IKKβ and a decrease of total IKKα and IKKβ were
observed in LPS group. Interestingly, these processes were reversed by
SP1-1 (Fig. 7B). These results suggested that LPS-induced IKKα/β-IκBα
signaling pathway was effectively inhibited by SP1-1, resulting in NF-
κB inactivation. Furthermore, the expression level of p-NF-kB p65 in nu-
cleus was increased while its value was decreased in cytoplasm by LPS
stimuli in THP-1-derived macrophages. However, the nuclear
localization of p65 subunit of NF-κB was inhibited in SP1-1-treated
groups (Fig. 7C,D). As shown in Fig. 6A, SP1-1 treatment dramatically
suppressed inflammatory cytokines production, including IL-1β and
IL-18 in DSS-induced mice colon, in line with the blockade of the inflam-
matory cytokines production observed in LPS stimulated THP-1 derived
macrophages (Fig. 5). These results indicated that SP1-1 significantly
blocked the NF-κB signaling pathway in experimental colitis.
3.8. SP1-1 inhibited the activation of NLRP3 inflammasome
The NLRP3 inflammasome is multi-protein complex (contain NLRP3,
ASC and pro-caspase-1) which recognizes microbial and danger compo-
nents and serves as a platform for caspase-1 activation and pro-
inflammatory cytokine IL-1β maturation [46]. More and more attention
has been paid to NLRP3 inflammasome because of its clinical impor-
tance in autoimmune, infectious and metabolic diseases. It was reported
that NLRP3 inflammasome played a crucial role in DSS-induced colitis
[47]. We have demonstrated that SP1-1 decreased IL-1β and IL-18

Fig. 9. SP1-1 suppressed the activation of NLRP3 inflammasome in THP-1-derived macrophages. (A) THP-1-derived macrophages were treated with LPS (1 μg/ml) alone or with indicated
concentrations of SP1-1 for 6 h. The mRNA levels of NLRP3 and pro-caspase-1 were determined by real-time RT-PCR. Data are expressed as means±SD (n = 3). ⁎⁎p b 0.01, compared with
control group; #p b 0.05, ##p b 0.01, compared with LPS group. (B,C) THP-1-derived macrophages were treated with LPS (1 μg/ml) alone or with indicated concentrations of SP1-1 for 6 h
followed by 45 min incubation of 5 mM ATP. The expression levels of cleaved caspase-1, NLRP3 and ASC protein were determined by western blot. Data are expressed as means±SD (n =
3). ⁎⁎p b 0.01, compared with control group; #p b 0.05, ##p b 0.01, compared with LPS + ATP group. (D) The caspase-1 activity was determined. Data are expressed as means±SD (n = 3).
⁎⁎p b 0.01, compared with control group; #p b 0.05, ##p b 0.01, compared with LPS + ATP group. (E) Proteins were isolated and immunoprecipitated with an antibody against ASC. Data are
expressed as means±SD (n = 3). ⁎p b 0.05, ⁎⁎p b 0.01, compared with control group; #p b 0.05, ##p b 0.01, compared with LPS + ATP group.
404 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405
production, two NLRP3 inflammasome-dependent inflammatory cyto-
kines, both in DSS-induced mice and LPS-induced THP-1-derived mac-
rophages (Fig. 5). Subsequently, we investigated whether SP1-1 could
inhibit NLRP3 inflammasome activation in DSS-induced colitis.
Caspase-1 cleavage is a critical step for the NLRP3 inflammasome activa-
tion [48]. After DSS exposure, the expression levels of NLRP3 and ASC
proteins were markedly increased in comparison with those of control
group. Whereas, treatment with SP1-1 led to significant attenuation of
protein expressions of NLRP3 and ASC in a dose-dependent manner
(Fig. 8). Additionally, cleaved caspase-1, cleaved IL-1β, and cleaved IL-
18 were remarkably reduced after SP1-1 treatment (Fig. 8C,D). Our
in vivo study proved that the colonic caspase-1 activity was significantly
suppressed by SP1-1 as well (Fig. 6B). As shown in Fig. 9A, SP1-1 sup-
pressed the elevated mRNA levels of NLRP3 and pro-caspase-1 in LPS-
induced THP-1-derived macrophages. Additionally, we evaluated the
expressions of related proteins when NLRP3 inflammasome was acti-
vated by ATP. The results suggested that SP1-1 markedly inhibited the
related protein expression of activated NLRP3 inflammasome and
caspase-1 activity in THP-1-derived macrophages (Fig. 9B–D). Further-
more, we explored the mechanism how SP1-1 suppressed the activa-
tion of inflammasome. As depicted in Fig. 9E, immunoprecipitation
analysis demonstrated that SP1-1 blocked the association of ASC with
NLRP3 or pro-caspase-1.
Some polysaccharides have been reported to possess the activity of
relieving colitis. But the underlying mechanisms were different. Jing
et al. found that CPN polysaccharides have the good prebiotic properties
and shown good application prospects in the prevention and treatment
of acute colitis [49]. Miao et al. found that pectic polysaccharides ex-
tracted from Rauvolfia verticillata (Lour.) Baill. var. hainanensis Tsiang
ameliorated ulcerative colitis via regulating the MAPKs and NF-κB path-
ways in dendritic cells [50]. Tao et al. reported that polysaccharides from
Chrysanthemum morifolium Ramat ameliorate colitis rats via regulation
of the metabolic profiling and NF-κB/TLR4 and IL-6/JAK2/STAT3 signal-
ing pathways [51]. Cai et al. demonstrated that compound polysaccha-
rides ameliorated the experimental colitis of rats induced by TNBS by
modulating the gut microbiota composition and function profiles,
which makes it possible to be used as prebiotic agents to treat gut
dysbiosis in colitis individuals [52].
4. Conclusions
Polysaccharides are one major active ingredient of Scutellaria
baicalensis Georgi. However, its research on structural characteristics
and pharmacological activity is limited. Scutellaria baicalensis Georgi is
often used to treat gastrointestinal diseases. Therefore, we carried out
this research to obtain pure polysaccharide and explore the possible
mechanism for its activity to treat colitis. After purifying using DEAE-
52 and Sephadex G-100 gel chromatography, a novel fraction polysac-
charide SP1-1 was successfully obtained from Scutellaria baicalensis
Georgi, with 72.64% carbohydrate and no protein. The molecular weight
was 4.56 × 105 Da, mainly made of Man, Rib, GluA, Glc, Xyl and Ara. The
characteristic absorptive bands of purified SP1-1 were evaluated by in-
frared spectrum and UV spectrum. There was an obvious honeycomb
structure from SEM. Our work demonstrated that SP1-1 significantly at-
tenuated DSS-induced experimental colitis in mice. The mechanism of
SP1-1 action involved inhibition of NF-kB signaling and NLRP3
inflammasome activation, and provided some evidence for its potential
use in the treatment of colitis.
Acknowledgments
This work was supported by the Natural Science Foundation of China
(Nos. 81403121, 81803756), Young Medical Talents Project of Jiangsu
Province (QNRC2016636), “Double First-Class” University Project of
China Pharmaceutical University (CPU2018GF07, CPU2018GY11 and
CPU2018PZQ19), Key Special Items of Research on Modernization of
Traditional Chinese Medicine (2018YFC1706900), China Postdoctoral
Science Foundation Grant (No. 2016M601864), Jiangsu Province Post-
doctoral Science Foundation Grant (1601023C), and the National Sci-
ence and Technology Major Projects for the Major New Drugs
Innovation and Development (2017ZX09301051).
References
[1] S. Luo, R. Wen, Q. Wang, Z. Zhao, F. Nong, Y. Fu, S. Huang, J. Chen, L. Zhou, X. Luo,
Rhubarb Peony Decoction ameliorates ulcerative colitis in mice by regulating gut
microbiota to restoring Th17/Treg balance, J. Ethnopharmacol. 231 (2019) 39–49.
[2] J. Zhou, T. Wang, Y. Dou, Y. Huang, C. Qu, J. Gao, Z. Huang, Y. Xie, P. Huang, Z. Lin, Z.
Su, Brusatol ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced experimental
colitis in rats: involvement of NF-kappaB pathway and NLRP3 inflammasome, Int.
Immunopharmacol. 64 (2018) 264–274.
[3] R. Marchioni Beery, S. Kane, Current approaches to the management of new-onset
ulcerative colitis, Clin. Exp. Gastroenterol. 7 (2014) 111–132.
[4] K.A. Matkowskyj, Z.E. Chen, M.S. Rao, G.Y. Yang, Dysplastic lesions in inflammatory
bowel disease: molecular pathogenesis to morphology, Arch. Pathol. Lab. Med. 137
(3) (2013) 338–350.
[5] R. Shivashankar, W.J. Tremaine, W.S. Harmsen, E.V. Loftus Jr., Incidence and preva-
lence of Crohn's disease and ulcerative colitis in Olmsted County, Minnesota from
1970 through 2010, Clin. Gastroenterol. Hepatol. 15 (6) (2017) 857–863.
[6] A.D. Kostic, R.J. Xavier, D. Gevers, The microbiome in inflammatory bowel disease:
current status and the future ahead, Gastroenterology 146 (6) (2014) 1489–1499.
[7] Y.R. Mahida, V.E. Rolfe, Host-bacterial interactions in inflammatory bowel disease,
Clin. Sci. 107 (4) (2004) 331–341.
[8] C. Palmela, J. Torres, M. Cravo, New trends in inflammatory bowel disease, GE Port. J.
Gastroenterol. 22 (3) (2015) 103–111.
[9] Z. Li, D.K. Zhang, W.Q. Yi, Q. Ouyang, Y.Q. Chen, H.T. Gan, NF-kappaB p65 antisense
oligonucleotides may serve as a novel molecular approach for the treatment of pa-
tients with ulcerative colitis, Arch. Med. Res. 39 (8) (2008) 729–734.
[10] P.J. Shaw, M.F. McDermott, T.D. Kanneganti, Inflammasomes and autoimmunity,
Trends Mol. Med. 17 (2) (2011) 57–64.
[11] S.L. Masters, A. Simon, I. Aksentijevich, D.L. Kastner, Horror autoinflammaticus: the
molecular pathophysiology of autoinflammatory disease, Annu. Rev. Immunol. 27
(2009) 621–668.
[12] A.J. van Beelen, Z. Zelinkova, E.W. Taanman-Kueter, F.J. Muller, D.W. Hommes, S.A.
Zaat, M.L. Kapsenberg, E.C. de Jong, Stimulation of the intracellular bacterial sensor
NOD2 programs dendritic cells to promote interleukin-17 production in human
memory T cells, Immunity 27 (4) (2007) 660–669.
[13] T.J. Lin, S.Y. Yin, P.W. Hsiao, N.S. Yang, I.J. Wang, Transcriptomic analysis reveals a
controlling mechanism for NLRP3 and IL-17A in dextran sulfate sodium (DSS)-in-
duced colitis, Sci. Rep. 8 (1) (2018), 14927.
[14] C. Bauer, P. Duewell, C. Mayer, H.A. Lehr, K.A. Fitzgerald, M. Dauer, J. Tschopp, S.
Endres, E. Latz, M. Schnurr, Colitis induced in mice with dextran sulfate sodium
(DSS) is mediated by the NLRP3 inflammasome, Gut 59 (9) (2010) 1192–1199.
[15] V. Neudecker, M. Haneklaus, O. Jensen, L. Khailova, J.C. Masterson, H. Tye, K. Biette, P.
Jedlicka, K.S. Brodsky, M.E. Gerich, M. Mack, A.A.B. Robertson, M.A. Cooper, G.T.
Furuta, C.A. Dinarello, L.A. O'Neill, H.K. Eltzschig, S.L. Masters, E.N. McNamee,
Myeloid-derived miR-223 regulates intestinal inflammation via repression of the
NLRP3 inflammasome, J. Exp. Med. 214 (6) (2017) 1737–1752.
[16] R. Kaur, M. Gulati, S.K. Singh, Role of synbiotics in polysaccharide assisted colon
targeted microspheres of mesalamine for the treatment of ulcerative colitis, Int. J.
Biol. Macromol. 95 (2017) 438–450.
[17] J. Lv, Y. Zhang, Z. Tian, F. Liu, Y. Shi, Y. Liu, P. Xia, Astragalus polysaccharides protect
against dextran sulfate sodium-induced colitis by inhibiting NF-kappacapital VE, Cy-
rillic activation, Int. J. Biol. Macromol. 98 (2017) 723–729.
[18] P. Chen, X. Zhou, L. Zhang, M. Shan, B. Bao, Y. Cao, A. Kang, A. Ding, Anti-
inflammatory effects of Huangqin tang extract in mice on ulcerative colitis, J.
Ethnopharmacol. 162 (2015) 207–214.
[19] L. Cui, L. Feng, Z.H. Zhang, X.B. Jia, The anti-inflammation effect of baicalin on exper-
imental colitis through inhibiting TLR4/NF-kappaB pathway activation, Int.
Immunopharmacol. 23 (1) (2014) 294–303.
[20] C.L. Zhang, S. Zhang, W.X. He, J.L. Lu, Y.J. Xu, J.Y. Yang, D. Liu, Baicalin may alleviate
inflammatory infiltration in dextran sodium sulfate-induced chronic ulcerative coli-
tis via inhibiting IL-33 expression, Life Sci. 186 (2017) 125–132.
[21] X. Luo, Z. Yu, C. Deng, J. Zhang, G. Ren, A. Sun, S. Mani, Z. Wang, W. Dou, Baicalein
ameliorates TNBS-induced colitis by suppressing TLR4/MyD88 signaling cascade
and NLRP3 inflammasome activation in mice, Sci. Rep. 7 (1) (2017), 16374.
[22] T. Hong, G.B. Jin, S. Cho, J.C. Cyong, Evaluation of the anti-inflammatory effect of
baicalein on dextran sulfate sodium-induced colitis in mice, Planta Med. 68 (3)
(2002) 268–271.
[23] Y. Sun, Y. Zhao, J. Yao, L. Zhao, Z. Wu, Y. Wang, D. Pan, H. Miao, Q. Guo, N. Lu,
Wogonoside protects against dextran sulfate sodium-induced experimental colitis
in mice by inhibiting NF-kappaB and NLRP3 inflammasome activation, Biochem.
Pharmacol. 94 (2) (2015) 142–154.
[24] W. Xiao, M. Yin, K. Wu, G. Lu, B. Deng, Y. Zhang, L. Qian, X. Jia, Y. Ding, W. Gong,
High-dose wogonin exacerbates DSS-induced colitis by up-regulating effector T
cell function and inhibiting Treg cell, J. Cell. Mol. Med. 21 (2) (2017) 286–298.
[25] Y. Wang, H. Wang, C. Qian, J. Tang, W. Zhou, X. Liu, Q. You, R. Hu, 3-(2-Oxo-2-
phenylethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(1 1bH)-one
(compound 1), a novel potent Nrf2/ARE inducer, protects against DSS-induced coli-
tis via inhibiting NLRP3 inflammasome, Biochem. Pharmacol. 101 (2016) 71–86.
 L. Cui et al. / International Journal of Biological Macromolecules 132 (2019) 393–405
[26] Z. Yu, M. Kong, P. Zhang, Q. Sun, K. Chen, Immune-enhancing activity of extracellular
polysaccharides isolated from Rhizopus nigricans, Carbohydr. Polym. 148 (2016)
318–325.
[27] T. Yang, S. Zhang, R. Wang, D. Li, Y. Hu, J. Nie, X. Zhao, Q. Wang, Y. Chen, Y. Zheng, P.
Chen, Polysaccharides from Rhizoma Panacis Majoris and its anti-oxidant activity,
Int. J. Biol. Macromol. 86 (2016) 756–763.
[28] J. Su, L. Jiang, J. Wu, Z. Liu, Y. Wu, Anti-tumor and anti-virus activity of polysaccha-
rides extracted from Sipunculus nudus(SNP) on Hepg2.2.15, Int. J. Biol. Macromol.
87 (2016) 597–602.
[29] Y. Fan, X. Wu, M. Zhang, T. Zhao, Y. Zhou, L. Han, L. Yang, Physical characteristics and
antioxidant effect of polysaccharides extracted by boiling water and enzymolysis
from Grifola frondosa, Int. J. Biol. Macromol. 48 (5) (2011) 798–803.
[30] C. Wang, L. Feng, J. Su, L. Cui, L. Dan, J. Yan, C. Ding, X. Tan, X. Jia, Polysaccharides
from Epimedium koreanum Nakai with immunomodulatory activity and inhibitory
effect on tumor growth in LLC-bearing mice, J. Ethnopharmacol. 207 (2017) 8–18.
[31] T. Boeing, P. de Souza, T.J. Bonomini, L.N.B. Mariano, L.B. Somensi, R.M. Lucinda, A.
Malheiros, L.M. da Silva, S.F. Andrade, Antioxidant and anti-inflammatory effect of
plumieride in dextran sulfate sodium-induced colitis in mice, Biomed.
Pharmacother. 99 (2018) 697–703.
[32] F. Tao, C. Qian, W. Guo, Q. Luo, Q. Xu, Y. Sun, Inhibition of Th1/Th17 responses via
suppression of STAT1 and STAT3 activation contributes to the amelioration of mu-
rine experimental colitis by a natural flavonoid glucoside icariin, Biochem.
Pharmacol. 85 (6) (2013) 798–807.
[33] S. Nakamura, H. Li, A. Adijiang, M. Pischetsrieder, T. Niwa, Pyridoxal phosphate pre-
vents progression of diabetic nephropathy, Nephrol. Dial. Transplant. 22 (8) (2007)
2165–2174.
[34] X. Liu, W. Zhou, X. Zhang, P. Lu, Q. Du, L. Tao, Y. Ding, Y. Wang, R. Hu, Dimethyl fu-
marate ameliorates dextran sulfate sodium-induced murine experimental colitis by
activating Nrf2 and suppressing NLRP3 inflammasome activation, Biochem.
Pharmacol. 112 (2016) 37–49.
[35] H. Gu, X. Wang, S. Rao, J. Wang, J. Zhao, F.L. Ren, R. Mu, Y. Yang, Q. Qi, W. Liu, N. Lu, H.
Ling, Q. You, Q. Guo, Gambogic acid mediates apoptosis as a p53 inducer through
down-regulation of mdm2 in wild-type p53-expressing cancer cells, Mol. Cancer
Ther. 7 (10) (2008) 3298–3305.
[36] Y. Zhu, Q. Li, G. Mao, Y. Zou, W. Feng, D. Zheng, W. Wang, L. Zhou, T. Zhang, J. Yang, L.
Yang, X. Wu, Optimization of enzyme-assisted extraction and characterization of
polysaccharides from Hericium erinaceus, Carbohydr. Polym. 101 (2014) 606–613.
[37] L. Yang, T. Zhao, H. Wei, M. Zhang, Y. Zou, G. Mao, X. Wu, Carboxymethylation of
polysaccharides from Auricularia auricula and their antioxidant activities in vitro,
Int. J. Biol. Macromol. 49 (5) (2011) 1124–1130.
[38] T. Sha, K. Igaki, M. Yamasaki, T. Watanabe, N. Tsuchimori, Establishment and valida-
tion of a new semi-chronic dextran sulfate sodium-induced model of colitis in mice,
Int. Immunopharmacol. 15 (1) (2013) 23–29.
[39] W. Liu, W. Guo, J. Wu, Q. Luo, F. Tao, Y. Gu, Y. Shen, J. Li, R. Tan, Q. Xu, Y. Sun, A novel
benzo[d]imidazole derivate prevents the development of dextran sulfate sodium-
induced murine experimental colitis via inhibition of NLRP3 inflammasome,
Biochem. Pharmacol. 85 (10) (2013) 1504–1512.
405
[40] A.M. Serra, J. Waddell, A. Manivannan, H. Xu, M. Cotter, J.V. Forrester, CD11b+
bone marrow-derived monocytes are the major leukocyte subset responsible
for retinal capillary leukostasis in experimental diabetes in mouse and
express high levels of CCR5 in the circulation, Am. J. Pathol. 181 (2) (2012)
719–727.
[41] T. Lawrence, M. Bebien, G.Y. Liu, V. Nizet, M. Karin, IKKalpha limits macrophage NF-
kappaB activation and contributes to the resolution of inflammation, Nature 434
(7037) (2005) 1138–1143.
[42] M. Bewtra, C.M. Brensinger, V.T. Tomov, T.B. Hoang, C.E. Sokach, C.A. Siegel, J.D.
Lewis, An optimized patient-reported ulcerative colitis disease activity measure de-
rived from the Mayo score and the simple clinical colitis activity index, Inflamm.
Bowel Dis. 20 (6) (2014) 1070–1078.
[43] I. Siddique, I. Khan, Mechanism of regulation of Na-H exchanger in inflammatory
bowel disease: role of TLR-4 signaling mechanism, Dig. Dis. Sci. 56 (6) (2011)
1656–1662.
[44] I. Atreya, R. Atreya, M.F. Neurath, NF-kappaB in inflammatory bowel disease, J. In-
tern. Med. 263 (6) (2008) 591–596.
[45] A.B. Lentsch, P.A. Ward, The NFkappaBb/IkappaB system in acute inflammation,
Arch. Immunol. Ther. Exp. 48 (2) (2000) 59–63.
[46] A. Stutz, D.T. Golenbock, E. Latz, Inflammasomes: too big to miss, J. Clin. Invest. 119
(12) (2009) 3502–3511.
[47] X. He, Z. Wei, J. Wang, J. Kou, W. Liu, Y. Fu, Z. Yang, Alpinetin attenuates inflamma-
tory responses by suppressing TLR4 and NLRP3 signaling pathways in DSS-induced
acute colitis, Sci. Rep. 6 (2016), 28370.
[48] E. Latz, T.S. Xiao, A. Stutz, Activation and regulation of the inflammasomes, Nat. Rev.
Immunol. 13 (6) (2013) 397–411.
[49] Y. Jing, A. Li, Z. Liu, P. Yang, J. Wei, X. Chen, T. Zhao, Y. Bai, L. Zha, C. Zhang, Absorp-
tion of Codonopsis pilosula Saponins by coexisting polysaccharides alleviates gut
microbial dysbiosis with dextran sulfate sodium-induced colitis in model mice,
Biomed. Res. Int. 2018 (2018), 1781036.
[50] X.P. Miao, X.N. Sun, Q.S. Li, L.J. Cui, X.Y. Wang, G.F. Zhuang, T.Z. Deng, Pectic
polysaccharides extracted from Rauvolfia verticillata (Lour.) Baill. var.
hainanensis Tsiang ameliorate ulcerative colitis via regulating the MAPKs and
NF-kappaB pathways in dendritic cells, Clin. Exp. Pharmacol. Physiol. 46 (1)
(2019) 48–55.
[51] J.H. Tao, J.A. Duan, W. Zhang, S. Jiang, J.M. Guo, D.D. Wei, Polysaccharides from Chry-
santhemum morifolium Ramat ameliorate colitis rats via regulation of the metabolic
profiling and NF-kappa B/TLR4 and IL-6/JAK2/STAT3 signaling pathways, Front.
Pharmacol. 9 (2018) 746.
[52] Y. Cai, W. Liu, Y. Lin, S. Zhang, B. Zou, D. Xiao, L. Lin, Y. Zhong, H. Zheng, Q. Liao, Z. Xie,
Compound polysaccharides ameliorate experimental colitis by modulating gut
microbiota composition and function, J. Gastroenterol. Hepatol. (2018), https://doi.
org/10.1111/jgh.14583.