Carbohydrate Polymers 278 (2022) 118898

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effects of laminarin zwitterionic carboxylate and sulfonate on the intestinal

barrier function and gut microbiota
Yun-Feng Li a, Veerabagu Udayakumar b, Malairaj Sathuvan a, Yang Liu a, Xiaojuan Liu a,
Yi-Qing Zhang a, Wan-Ying Ma a, Wancong Zhang c, d, *, Shijie Tang c, d, *, Kit-Leong Cheong a, **
a
Guangdong Provincial Key Laboratory of Marine Biotechnology, Department of Biology, College of Science, Shantou University, Shantou 515063, Guangdong, China
b
Laboratorio de Nanocelulosa y Biomateriales, Departamento de Ingeniería Química, Biotecnología y Materiales, Facultad de Ciencias Físicas y Matemáticas, Universidad
de Chile, Santiago 8370456, Chile
c
Department of Plastic Surgery and Burn Center, Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong, China
d
Plastic Surgery Institute of Shantou University Medical College, Shantou, Guangdong, China

A R T I C L E I N F O A B S T R A C T

Keywords: Ulcerative colitis (UC) has become a global chronic disease that keeps increasing. This study was to explore the
Laminarin treatment effectiveness of two functional zwitterionic laminarins, zwitterionic sulfonate (LZS) and zwitterionic
Zwitterionic sulfonate carboxylate (LZC), in dextran sulfate sodium (DSS) induced mouse model. FT-IR and NMR techniques were used
Zwitterionic carboxylate
to characterize the aforementioned functional zwitterion. Compared to UC mice, the composition and diversity of
Colitis
IBD
gut microbiota were significantly increased in the treated mice. Specifically, the composition of Bacteroidetes
Gut microbiota increased and the level of Firmicutes decreased. Moreover, we demonstrated the alleviation of colitis by LZS and
Colon tissue LZC reflected by the improved integrity of intestinal mucosa, which includes increased number of goblet cells,
mucin protein production, maintenance of collagens, as well as the lower extent of intestinal fibrosis. These
findings indicated the potentials of LZC and LZS as promising agents to prevent colitis via adjusting gut
microbiota and maintaining intestinal barrier integrity.

1. Introduction accelerating incidence in newly industrialized countries in Asia and

Inflammatory bowel disease (IBD), which comprises Crohn's disease
and ulcerative colitis, is a chronic idiopathy caused by an imbalanced
immune system and an inflamed gastrointestinal tract. Even now, the
IBD remains as a challenge to public health worldwide. IBD is a lifelong
disease and characterized by episodes of abdominal pain, profuse diar­
rhea, weight loss, bloody stools, inflammation, and ulceration in the
rectum (Ananthakrishnan, 2015). Traditionally, IBD is a Western dis­
ease, with the highest prevalence values reported in the western world,
such as North America (ulcerative colitis, 286 per 100,000 individuals in
the USA; Crohn's disease, 319 per 100,000 individuals in Canada) and
Europe (ulcerative colitis, 505 per 100,000 individuals in Norway;
Crohn's disease, 322 per 100,000 individuals in Germany). The preva­
lence of IBD exceeded 0.3% in North America, Oceania, and many
countries in Europe (Ng et al., 2017). Nowadays, IBD has been consid­
ered as one of the most prevalent gastrointestinal diseases with
Africa. Although the pathogenesis of IBD is not well understood, the
currently accepted hypothesis lies in the disruption of the intestinal
barrier and the dysregulation of commensal gut microbiota (Kudelka
et al., 2020).
The gastrointestinal tract is a specialized organ with an enormous
surface area that is optimized to efficiently absorb water, nutrients, and
minerals from food. In the meantime, it constitutes an essential barrier.
The intestinal epithelium is lined with epithelial cells representing
intercellular junctions that function as a barrier between the inner and
outer environments. In addition, the intestinal epithelial system is home
to abundant innate and adaptive immune cells, which function tightly
with the intestinal epithelial cells to maintain intestinal homeostasis
(Peterson & Artis, 2014). The main function of the intestinal barrier is to
allow the transport of essential nutrients while restricting pathogen
substances (Camilleri et al., 2012). Increasing pieces of evidence showed
that the loss of barrier integrity may cause various diseases, including

* Corresponding authors at: Department of Plastic Surgery and Burn Center, Second Affiliated Hospital, Shantou University Medical College, Shantou, Guangdong,
China.
** Corresponding author.
E-mail addresses: 02wczhang@stu.edu.cn (W. Zhang), sjtang3@stu.edu.cn (S. Tang), klcheong@stu.edu.cn (K.-L. Cheong).

https://doi.org/10.1016/j.carbpol.2021.118898
Received 31 August 2021; Received in revised form 11 November 2021; Accepted 12 November 2021
Available online 17 November 2021
0144-8617/© 2021 Elsevier Ltd. All rights reserved.
Y.-F. Li et al.
IBD, diabetes, obesity, multiple sclerosis, and exacerbate disease pro­
gression (Chelakkot et al., 2018). Intestinal barrier permeability may
therefore be a prognostic marker for disease pathophysiology. More­
over, the human gut contains a vast number of microorganisms, which
function in tandem with the host's defenses and the immune system to
protect the host from pathogen colonization and invasion (Fong et al.,
2020). Dysbiosis of the gut microbiota is associated with the patho­
genesis of both intestinal and extra-intestinal diseases, such as IBD,
metabolic syndrome, allergic asthma, and cardiovascular disease
(Pickard et al., 2017). The gut microbiota may play an important role in
the prevention and treatment of IBD, which normally involve increasing
protective bacterial species and reducing harmful bacteria. Therefore,
the beneficial bacteria not only produce functional metabolites but also
form homeostatic colonization to maintain an intestinal mucosal barrier.
Consequently, targeting the intestinal barrier integrity and balancing
gut microbiota hold promises for the therapy and prevention of IBD.
Laminarin is considered one of the most abundant polysaccharides
found in the marine ecosystem (Xu et al., 2017). Laminarin presents in
the cell wall of brown algal mainly found in Laminaria/Saccharina spe­
cies. Laminarin is a β-glucan type polysaccharide that consists of a
backbone of β-1,3-linked-glucose residues with branching β-1,6-glucose
residues, while the proportion ratio of β-1,6 to β-1,3 is roughly 1:3 (Cui
et al., 2021). The molecular weight of Laminarin is about 5 kDa, and the
polymerisation range between 20 and 25 glucose moieties (Kadam et al.,
2015). Laminarin has been found to exert potent pharmacological
properties, including immuno-modulatory (Zhu et al., 2019), antioxi­
dant (Chen et al., 2021), anticoagulant (Kadam et al., 2015), and anti­
cancer activities (Zargarzadeh et al., 2020). Meanwhile, laminarin has
been reported to have a therapeutic effect on DSS-induced colitis in pigs
(Rattigan et al., 2020). It was recently reported that the chemical de­
rivatives of laminarins are more effective than the native laminarins.
The sulfated laminarin derived from Fucus evanescens effectively pre­
vented the migration of breast adenocarcinoma cells with higher anti­
cancer activity in vitro (Malyarenko et al., 2017). Moreover, the sulfated
laminarin derived from Dictyota dichotoma exhibited significant anti­
cancer activity against melanoma cells by inhibiting cell proliferation,
colony formation, and cancer cell migration (Usoltseva et al., 2018).
Zwitterion is a type of electrically neutral molecule that contains
both cations and anions in its molecular structure. Recently, zwitterions
with immunomodulation, antifouling, non-thrombogenic, and cell-
compatible properties have attracted wide scientific interest, espe­
cially in biomedical applications (Chen & Yang, 2020; Li et al., 2020). It
has been recently reported that zwitterionic Chitooligosaccharides has
immune activating ability to tumor-associated macrophages (Zhang
et al., 2019). To the best of our knowledge, there are few papers about
zwitterionic laminarins and their effects on gut microbiota and intestinal
barrier. Combining these aspects above, we hypothesized that the
zwitterionic laminarin carboxylate and sulfonate possesses a protective
effect against IBD via regulating inflammatory reactions and microbial
community composition. The main objective of this study was to explore
laminarins functionalized with zwitterionic carboxylate and zwitter­
ionic sulfonate. The zwitterionic laminarin was characterized via
instrumental analysis methods including FT-IR and NMR. Then, we
explored the therapeutic effects of zwitterionic laminarin on IBD, as well
as its possible mechanisms of action at tissue level using dextran sulfate
sodium (DSS)-induced colitis mouse model. We also investigated the
effects of zwitterionic laminarin on intestinal microflora community
structure.
2. Materials and methods
2.1. Materials
Laminarin was purchased from Tokyo Chemical industry (Tokyo,
Japan). DSS was purchased from Aladdin, Beijing. Methyl isonicottinate,
1,3-propanesultone was purchased from Macklin, Shanghai. DNeasy
2
Carbohydrate Polymers 278 (2022) 118898
PowerSoil kit was obtained from QIAGEN. All other chemical reagents
were of reagent grade.
2.2. Synthesis of laminarin zwitterion carboxylate and laminarin
zwitterion sulfonate
The functionalization of laminarin was prepared according to the
literature report with minor modification (Song et al., 2019). Dissolve
0.15 g of Laminarin (LAM) and 0.01 g of methyl isonicotinate in 3 mL of
toluene at the same time. And the mixtures were refluxed at 120 ◦ C
under N2 atmosphere with vigorous stirring for 48 h. The solvent from
the reaction mixture was removed with a rotavapor at 65 ◦ C, to get the
dry solid and labeled as LPY.
In order to synthesize the laminarin zwitterion carboxylate (LZC),
the dried LPY (0.1 g) was dissolved in methanol (1 mL) with stirring at
room temperature. The γ-butyrolactone was dissolved in 0.5 mL of
DMSO and added dropwise into the solution. The reaction mixture was
stirred and heated at 80 ◦ C for 48 h. The reacted product was precipi­
tated in cold the mother liquor was decanted to give the brown solid.
In order to construct the laminarin zwitterion sulfonate (LZS), the
dried LPY (0.1 g) was dissolved in 1 mL of DMSO and stirring at room
temperature. Then 1,3-propanesultone was dissolved in 0.5 mL of DMSO
and added dropwise into the solution. The reaction mixture was stirred
and heated at 80 ◦ C for 48 h, subsequently cooled to room temperature
and poured into ice-cold acetone to get the brown viscous liquid.
2.3. FT-IR spectrum
FTIR spectra were recorded with an FTIR spectrometer (Nicolet iS50,
USA) in the frequency range 4000–400 cm− 1 at a resolution of 4 cm− 1.
2.4. NMR
The dried sample was weighed to 80 mg, dissolved in D2O, lyophi­
lized, and repeated three times to completely replace the hydrogen in
the sample with deuterium‑hydrogen; the sample was then dissolved in
D2O with traces of tetramethylsilane standard reference. Then trans­
ferred to an NMR tube for detection on a Bruker AVANCE 500 spec­
trometer (Bruker BioSpin Corporation, Billerica, MA, USA), and 1H NMR
and 13C NMR data were recorded at room temperature.
2.5. Animals and treatment
Female C57BL/6 mice 7–9 weeks old, sourced from the Guangdong
Experimental Animal Centre. The living conditions of the experimental
animals were controlled at a temperature of 23 ◦ C ± 2 ◦ C, humidity of
50 ± 10%, 12-hour light/dark cycle, drinking water ad libitum, and food
of commercial grade animal chow. Animal experiments were conducted
according to the regulations of Shantou University Laboratory Animal
Centre and permission was obtained from the Shantou University
Medical Animal Care and Welfare Committee (SUMC 2020-330). The
experimental mice were randomly divided into 4 major groups, control
group (NC, n = 5), colitis group (DSS, n = 5), laminarin treatment group
(LAM, n = 15) group, zwitterionic carboxylate treatment group (LZC, n
= 15) and zwitterionic sulfonate treatment group (LZS, n = 15). Acute
colitis was induced with 3% DSS (w/v) aqueous solution in all groups
except the NC group, which was treated with water only. After 8 d of
colitis induction, each treatment group was randomly divided into three
different groups according to the oral dose (75, 150, and 300 mg/kg)
and labeled (L, M, and H, respectively) in front of the group name to
represent the three doses (low, medium and high), with 5 mice in each
dose group. The body weight of the mice was recorded daily. After 14
days of administration, the animals were sacrificed and the faeces of
each group were collected and immediately frozen to − 80 ◦ C for sub­
sequent experimental testing. The colon was separated from the prox­
imal rectum, washed and stored at − 80 ◦ C in the refrigerator.
Y.-F. Li et al. Carbohydrate Polymers 278 (2022) 118898

2.6. 16S ribosomal RNA isolation and quantitative real-time PCR 3382 2893 1635 1460 1373 1075 892

A
LAM
The total DNA was prepared according to a previous method (Xu
et al., 2020). In brief, the extraction of total DNA from mouse faecal
samples with the DNeasy PowerSoil Kit (QIAGEN). The extracted LZC

genomic DNA was used as a template to select the V4 region of 16S rRNA
on a PCR system (Biometra, Göttingen, Germany) with PCR amplifica­
LZS

tion was performed with front-end primer 343F (5′ -TACG­

GRAGGCAGCAG-3′ ), back-end primer 798R (5′ -AGGGTATCTAATCCT-
3′ ), magnetic bead purification, agarose gel electrophoresis for band
3500
detection and Qubit for concentration detection. 4000 3000 2500 2000 1500 1000

Wavenumbers(cm-1)

2.7. Bioinformatic analysis

B H2,H3,H4,H5,H6
i
The PCR amplification products were sequenced in high throughput
using the Illumina sequencing system, the original double-ended se­ H1
quences were de-hybridised using Trimmomatic software, chimeric se­
quences were detected and removed from the sequences using UCHIME, ii
sequence similarity greater than or equal to 97% was assigned to an OTU
unit using Vsearch software, and all representative sequences were an­ NCH2
notated against the database Silva (version 123) using the QIIME soft­
ware package. iii B
A

2.8. Histopathological examination NCH2

The colon tissue was thoroughly washed in PBS buffer, then soaked
in 10% formalin for 24 h, paraffin-embedded and sectioned (5 μm).
Sections were treated with hematoxylin-eosin (H&E) staining, alcian- C i
blue/periodic acid Schiff (AB-PAS) staining (AB-PAS) and Masson C2,C3,C4,C5,C6

staining respectively, and then observed using light microscopy. C1

2.9. Immunohistochemistry

Paraffin sections were dewaxed to water, antigen repaired, endoge­ ii

C2,C3,C4,C5,C6
nous peroxidase blocked, serum blocked, primary and secondary anti­
bodies added respectively, DAB stained, hematoxylin re-stained nuclei C=O C1
and sealed after dehydration. The hematoxylin-stained nuclei are blue
iii
and the positive expression of DAB is brownish-yellow. A
C2,C3,C4,C5, C6 NCH2 B

2.10. Statistical analysis C=O

C1

Data are expressed as mean ± SD. Statistical differences between the

two data sets were analysed using one-way analysis of variance
(ANOVA). Data were analysed by data processing software and differ­
ences were considered statistically significant at p < 0.05.
3. Results and discussion
3.1. Spectroscopic characteristic of LZC and LZS
The structures of the derivate compounds forming the laminarin
zwitterion carboxylate complex and laminarin zwitterion sulfonate
complex are denoted as LZC and LZS, respectively. Fourier transform
infrared (FT-IR) spectrometry was used to monitor the structural
changes in laminarin (Fig. 1A). As the hydroxyl groups formed inter­
molecular and intramolecular hydrogen bonds, the OH stretching vi­
bration, which was a broad intense band at 3400–3300 cm− 1 in the FT-
IR spectrum. Vibrations between 1200 cm− 1–950 cm− 1 (sugar region)
and 950 cm− 1–750 cm− 1 (anomeric region) are important structural
regions for structural characterization of laminarin (Rajauria et al.,
2021). IR spectrum of the LZC displayed additional peaks, which are
characteristic peaks of the derivate carboxylate group signal at 1460
cm− 1and 1373 cm− 1. The absorption peaks at these locations indicate to
the carboxylate C– – O vibration and the carboxylate C–O stretching
vibration (Lin et al., 2017). On the other hand, the LZS shows an increase
in the strength of the S– – O stretching vibration at 1373 cm− 1, 1182
Fig. 1. (A) FT-IR spectra, (B) 1H NMR, (C) 13C NMR of native laminarin,
carboxylate laminarin, and sulfonated laminarin.
cm− 1, 1075 cm− 1 and 1036 cm− 1, which verifies the success of the
modification (Khan et al., 2020; Sagbas et al., 2015). Absorption peaks
at 1000 cm− 1–800 cm− 1 primarily are owed to the variable angle vi­
bration and bending vibration of hydroxyl groups (Qiu et al., 2020). The
absorption peaks at 892 cm− 1 and 1075 cm− 1 are indicative of
β-configuration and β-1,3 linkage, respectively (Liu et al., 2021). Bands
in the 956 cm− 1 region correspond to the C–O–S symmetrical
stretching vibration, associated with the C–O–SO3 group (Wang et al.,
2015), indicating that the native polysaccharide was sulfonylated.
The FT-IR absorbent peaks for the CH3 symmetrical stretching vi­
bration, CH3 deformation vibration, CH2 asymmetric stretching vibra­
tion, CH2 bending vibration, CH stretching vibration, and C– –O
stretching vibration, which demonstrated the basic structure of lami­
narin. In the NMR spectra (Fig. 1B–C), the signals of the laminarin unit
in the region (δH 3.25–4.50, δC 55–103) were observed (Fig. 1B (i) and C
(i)) as an intense peak but after functionalized with zwitterion carbox­
ylate and zwitterion sulfonate the signals are weak (Fig. 1B (ii & iii) and
C (ii & iii)). These peaks were attributed to C6/H6, C5/H5, C4/H4, C3/

3
Y.-F. Li et al.
H3, C2/H2 and C1/H1 in the laminarin unit. More importantly, the
≡N–CH2 peaks at δH 3.76 and δC 49.4 in Fig. 1B (ii & iii) and C (ii & iii)
confirm the successful functionalization of zwitterion carboxylate and
zwitterion sulfonate with laminarin unit. The four doublet peaks around
δH (7.76 & 7.78) and δH (8.66 & 8.70) be assigned to pyridinium (a)
–CH and –NCH hydrogens (Fig. 1B (ii & iii)), respectively. It is further
confirming from δC (128.2 & 128.4) and δC (144.0 & 144.5) in Fig. 1C (ii
& iii). The region around δH 2.60–3.30 (Fig. 1B (ii)), δC 20.0–36.0
(Fig. 1C (iii)) and δH 2.00–3.00 (Fig. 1B (iii)), δC 20.0–36.0 (Fig. 1C (ii))
corresponds to alkyl groups (b) of the carboxylate and sulfate anions,
respectively.
Based on these results, it can be concluded that the zwitterion
carboxylate and zwitterion sulfonate have been successfully synthe­
sized, respectively.
3.2. LZC and LZS improve IBD-induced gut dysbiosis in mice
Mice without intestinal bacteria have been reported to be much less
resistant to dextran sodium sulfate and thus colitis (Maslowski et al.,
2009). The intestinal microbiota is closely linked to the morphology,
structure and intestinal cell renewal of the gut. The morphology of the
gut in mice without enterobacteria was abnormal, with a significantly
reduced total intestinal surface area, shorter ileal villi, and smaller in­
testinal crypts (Natividad & Verdu, 2013).
Accumulating evidence demonstrated that the gut microbiota plays a
significant role in the development of IBD and its associated complica­
tions; therefore, we analysed the effects of LZC and LZS on the gut
microbiota composition by multiplex sequencing covering the V4 re­
gions of 16S rRNA.
Among the classifiable sequences, 15 phyla were identified, while
Fig. 2. LZS and LZC regulate gut microbiota diversity and composition. (A) Gut microbiota composition at phylum level, (B) Firmicutes/Bacteroidetes (F/B) ratio,
**p < 0.01 compared with the DSS group; ##p < 0.01 compared with the CN group. (C) Heatmap analysis of top 30 genus.
4
Carbohydrate Polymers 278 (2022) 118898
Bacteroidetes, Firmicutes, and Proteobacteria were representing the most
dominant lineages in the samples. Compared to the mice in the control
group, significantly increased abundance of the phylum Firmicutes and
reduced abundance of phylum Bacteroidetes were observed in the DSS-
induced mice. Interestingly, LAM, LZC, and LZS (Fig. 2A) restored the
composition of Bacteroidetes and decreased the level of Firmicutes in
mouse gut microbiota in the NC group. Firmicutes and Bacteroidetes are
two main kinds of dominant phyla in the gut, and recently it has been
widely accepted that the Firmicutes/Bacteroidetes (F/B) ratio is important
in maintaining normal intestinal homeostasis (Magne et al., 2020; Xie &
Cheong, 2021). The alteration of the F/B ratio is regarded as a critical
marker of the gut microbiota and associated with the pathogenesis of
both intestinal and extra-intestinal disorders such as obesity and IBD.
Our results showed that LAM, LZC, and LZS restored the intestinal mi­
crobial balance by affecting the F/B ratio (Figure 2B). While the thera­
peutic effect of LAM decreased with increasing concentration, the
therapeutic effects of LZC at all three concentrations were significantly
increased compared to LAM. The therapeutic effect of LZS did not vary
among all concentrations. LAM, LZC, and LZS all showed therapeutic
effects in regulating intestinal microbiota, approximately recovered the
level of the DSS-induced symptoms to the level of the normal group.
Indeed, microbial utilization of complex polysaccharides is a major
driving force in shaping the composition of the intestinal microbiota
(Zheng et al., 2020). In the gut, the Bacteroidetes phylum is considered
the primary degrader of polysaccharides (Lapébie et al., 2019).
Polysaccharide-degrading enzymes produced by Bacteroides, the so-
called carbohydrate-active enzymes partially degraded the poly­
saccharide into small oligosaccharides that are imported into the peri­
plasm (Lapébie et al., 2019).
The distinct changes of gut microbiota at the genus level and several
Y.-F. Li et al. Carbohydrate Polymers 278 (2022) 118898

predominant taxa in each group are presented in Fig. 2C. Specifically,
the taxonomic composition with the high relative abundance at the
genus level mainly included Helicobacter, Lachnospir­
aceae_NK4A136_group, Odoribacter, Alistipes, Prevotellaceae_UCG-001,
Ruminococcaceae_UCG-014, and Bacteroides. Compared with the NC
group, the relative abundances of Lachnospiraceae and GCA-900066575
were significantly increased, while that of the Ruminococcaceae was
decreased in the DSS-induced group. Mice treated with LZC and LZS
exhibited a significant decrease in the relative abundance of GCA-
900066575. At the same time, it was observed that the relative abun­
dance of Alloprevotella showed a dose-dependent relationship as the
administration concentrations of LZC and LZS increased, indicating that
the administration of high concentrations of LZC and LZS could
contribute to the increase of Alloprevotella in the gut microbiota of mice.
Meanwhile, the change of the relative abundance of Lachnospir­
aceae_NK4A136_group suggested that the administration concentrations
of HLZC could reduce the proportion of genus in the gut microbiota of
mice compared with the DSS group, thus contributing to the data more
in favor of the NC group, indicating that HLZC affected the proportion of
gut microbiota in mice and showed a trend closer to the NC group. An
intestinal bacterium, Lactobacillus rhamnosus GG, was found to repair the
intestinal mucosal state of DSS-induced colitis mice by promoting the
renewal of epithelial cells (Swanson et al., 2011). The composition of the
gut microbiome even affects the immune system by regulating immune
mediators, and then affects the intestinal barrier (Qiu et al., 2021).
These suggest that the intestinal microbiota is closely related to the in­
testinal barrier and that dysbiosis of the intestinal flora may facilitate
disruption of the intestinal barrier.
3.3. Effects of LZC and LZS on the tissue layer
The mucosal injury and colonic inflammation were investigated by
pathological examination of the colon tissue using H&E staining. As
shown in Fig. 3A, the H&E-stained colon sections of the control group
revealed the normal arrangement of the mucosa, submucosa, and mus­
culosa layers. In addition, the crypt structures were relatively well
preserved and a number of goblet cells appeared with basal nuclei. The
H&E staining indicated that severe injuries were triggered in the DSS-
induced group (Fig. 3B) when compared to the control group. Specif­
ically, the colon sections presented damaged crypt structure, loss of
goblet cells, massive infiltration of inflammatory cells, muscularis
thinning, and disruption of the epithelial layer, which are typical

A B

C D E

F G H

I J K

Fig. 3. Effect of LZC and LZS on the histopathological injury and gut barrier integrity in colon tissues of DSS-induced colitis mice. Representative images of H&E
staining. (A) control group; (B) DSS-induced colitis mice group; (Observably colonic tissue injury were mucosal erosions and ulcerations (indicated by arrows),
neutrophil infiltrations (black arrow), and the basal cells of mucosal injury (red arrow)); (C) LLAM group; (D) MLAM group; (E) HLAM group; (F) LLZC group; (G)
MLZC group; (H) HLZC group; (I) LLZS group; (J) MLZS group; (K) HLZS group. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

5
Y.-F. Li et al.
symptoms of UC development. Intriguingly, LZC and LZS treatments
significantly reversed the DSS-induced tissue damage in colon tissue,
with prominent muscularis mucosa, normal submucosa, and musculosa
observed. Low concentrations of LZC improved the structural integrity
of the columnar epithelium and the lamina propria more significantly
compared to low concentrations of LAM. Meanwhile, low concentrations
of LZS were more effective in reducing inflammatory cell infiltration and
maintaining a more intact lamina propria, while there was some thick­
ening of the submucosa after treatment with medium and high con­
centrations of LZS. Despite that all three groups showed significant
therapeutic effects, the LZS and LZC were more effective in treating the
structural integrity of the mucus layer than the LAM. In particular, the
LZS was more effective in reducing inflammatory cell infiltration and the
LZC was more effective in maintaining the normalized submucosa.
3.4. LZC and LZS increase the number of goblet cells
The mucus layers are at the first line of defense as an important part
of the intestinal barrier (Hu et al., 2018). Mucus is formed and secreted
from goblet cells at surfaces. These mucus layers serve to prevent
pathogens and toxins from direct contact with intestinal epithelium
(Capaldo et al., 2017). Mucins are the main component of the mucus
Fig. 4. Representative Alcian blue periodic acid Schiff stained mouse colonic tissue sections with a magnification of 100 fold. (A) control group; (B) DSS-induced
colitis mice group; (C) LLAM group; (D) MLAM group; (E) HLAM group; (F) LLZC group; (G) MLZC group; (H) HLZC group; (I) LLZS group; (J) MLZS group; (K)
HLZS group.
6
Carbohydrate Polymers 278 (2022) 118898
layer, and they are classified into neutral and acidic subtypes depending
on the sugar type in the chains. The neutral mucins are rich in glycogen,
while the acidic mucins containing sulphomucins or sialomucins in­
crease mucus viscosity and resistance against bacterial enzymes (Truter
et al., 2017). The distribution of this histologically identifiable mucin
can indirectly indicate the quality of the biofilm in a particular colon
segment. Given the importance of the mucus layers in intestinal barrier
integrity, we therefore further evaluated the effects of LZS and LZC on
intestinal mucosa by the mucin area in the Alcian blue and Alcian blue
periodic acid Schiff staining (AB-PAS) colon sections, respectively.
Neutral mucins were stained in red, acidic mucins were stained in blue,
and mixed mucins (contained neutral and acidic mucins) were stained in
purple.
As shown in Fig. 4A, a number of goblet cells covered by mucus were
observed in the control group, and the mucus layers showed uniform
distribution with a complete layer. In contrast, in DSS-induced mice, the
goblet cells were severely destroyed, the mucus was greatly reduced and
mucus layer distribution was destructed. As shown in Fig. 4, the mucin
areas of colitis mice were significantly increased in the order of LZS <
LAM < LZC. Significant restorative effects were observed after oral
administration of all three polysaccharides, with LZC being particularly
impressive. These results demonstrated that LZC and LZS could generate
Y.-F. Li et al.
recovery in UC or inhibit colitis associated deterioration through
reducing mucus layer and goblet cell destruction. This observation was
in line with previous studies that reported the destruction of the intes­
tinal barrier integrity is the primary pathological characteristic of IBD.
3.5. Expression of MUC2 in the treatment of LZC and LZS
To confirm the increased area of mucus layer and the number of
goblet cells in the colonic tissue of LZC and LZS treated mice, we further
performed immunohistochemical analysis to determine the expression
profile of MUC2. As a predominant intestinal gel-forming mucin, MUC2
is a large, net-like insoluble molecule secreted from goblet cells and
makes up the skeleton of the mucus layer (Johansson & Hansson, 2016).
In the control groups (Fig. 5A), MUC2 expression by goblet cells was
observed in the proximal and distal colon from the crypt base to the
surface epithelium. The DSS-induced group showed (Fig. 5B) decreased
MUC2 levels when compared to the control group. However, the accu­
mulation of MUC2 in the surface epithelium became more pronounced
in the LZC and LZS-treated groups.
A previous study noted that the mucin-rich mucus layer acts as an
essential intestinal barrier, which not only primarily plays protective
Fig. 5. Immune histochemical-stained sections for MUC2 (100×); (A) Control group showing reactions in many cells; (B) DSS group showing group showing re­
actions in few cells; (C) LLAM group; (D) MLAM group; (E) HLAM group; (F) LLZC group; (G) MLZC group; (H) HLZC group; (I) LLZS group; (J) MLZS group; (K)
HLZS group.
7
Carbohydrate Polymers 278 (2022) 118898
and lubricative roles but also impacts the localization of gut microbiota,
while providing glycans to the bacterial residents of the gut, especially
Bacteroides (Zafar & Saier, 2021). Impressively, another report
demonstrated that oral administration of Dendrobium huoshanense
polysaccharides could strengthen the biochemical barrier function in
female C57BL/6 mice by elevating the expression and secretion of MUC2
mucin in diverse regions of the gut (Xie et al., 2019). Consistently, our in
vivo results of MUC2 expression suggested that these two zwitterionic
laminarins were able to restore the expression of mucins in epithelial
cells during UC.
3.6. LZS and LZC attenuate colonic fibrosis
To investigate the effects of LZC and LZS on intestinal fibrosis in
chronic DSS-induced colitis, the colonic sections were stained with
Masson's trichrome method.
The abnormal accumulation and deposition of collagen are the basis
of fibrosis, and the collagen deposition can be shown by the blue color
staining area in the mucosa and submucosa. As shown in Fig. 6, the
expression of collagen in the DSS group was significantly increased
compared with the control group, which might be related to the long-
Y.-F. Li et al. Carbohydrate Polymers 278 (2022) 118898

Fig. 6. Effects of LZS and LZC on collagen expression in colonic mucosa of DSS-induced mice. Representative image of Masson's trichrome staining of the colonic
tissues of (A) control group; (B) DSS-induced colitis mice group; (C) LLAM group; (D) MLAM group; (E) HLAM group; (F) LLZC group; (G) MLZC group; (H) HLZC
group; (I) LLZS group; (J) MLZS group; (K) HLZS group. Extracellular matrix is blue in color and the original magnification ×100. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)

term irritation caused by acute inflammation in the intestinal mucosal. 4. Conclusion

Interestingly, after LAM, LZC, and LZS treatments, the collagen depo­
sition was significantly decreased compared with the DSS group. Among
them, the LZC group had the least collagen fibers, followed by the fewer
collagen fibers in the LAM group and the LZS group. Although the
treatment effect of LZS was slightly inferior to that of LZC, the collagen
deposition was significantly improved than that of the DSS group. These
results suggested that LZC and LZS treatments could reduce the risk of
intestinal fibrosis in UC.
The development of fibrosis caused by chronic intestinal inflamma­
tion is an important phenomenon involved in the pathogenesis of IBD
(Shih et al., 2014). Colonic fibrosis results in a stiff colon that leads to the
loss of tissue function to carry out peristalsis or to resorb fluids. Our
results demonstrated that the treatments of LZC and LZS promoted the
maintenance of collagens, prevented intestinal fibrosis, and alleviated
the development obstruction of the colon. Consistent results showed that
rhamnogalacturonan isolated from Acmella oleracea (L.) R.K., as a
complex pectic polysaccharide containing uronic acid groups, can
reduce the severity of DSS-induced colitis through conserving collagen
homeostasis and increasing cell proliferation (Maria-Ferreira et al.,
2018).
The conventional therapy for UC, such as the use of glucocorticoids,
is focused on maintaining remission. Unfortunately, adverse effects such
as headache and nausea remain a major concern for the long-term use of
glucocorticoids. In this sense, treatments with LZS and LZC not only
improved the sign and symptoms of acute UC but also modulated gut
microbiota. Furthermore, LZS and LZC were able to increase the
numbers of mucus-secreting goblet cells, promote the area of the mucus
layer, and increase the expression of MUC2. Therefore, it was concluded
that the LZC and LZS had significant influences on both the gut micro­
biota and intestinal barrier integrity. LZC and LZS hold promise as
preventive or protective agents for attenuating UC and may provide new
clues for the development and utilization of zwitterionic poly­
saccharides, while the detailed mechanisms need more investigations in
future studies.
CRediT authorship contribution statement
Yun-Feng Li: Investigation, Data curation, Writing - original draft.
Veerabagu Udayakumar, Malairaj Sathuvan, Yang Liu, Xiaojuan Liu, Yi-
Qing Zhang, Wan-Ying Ma: Investigation, Data curation, Writing -

8
Y.-F. Li et al. Carbohydrate Polymers 278 (2022) 118898

review & editing. Shijie Tang: Investigation, Conceptualization, Data laminarans from far eastern brown seaweeds and their sulfated derivatives. Journal
of Applied Phycology, 29(1), 543–553.
curation, Writing - review & editing. Wancong Zhang: Conceptualiza­
Maria-Ferreira, D., Nascimento, A. M., Cipriani, T. R., Santana-Filho, A. P.,
tion, Methodology, Supervision. Kit-Leong Cheong: Conceptualization, Watanabe, P. D. S., Sant'Ana, D. D. M. G., & Baggio, C. H. (2018).
Methodology, Supervision. All authors reviewed and approved the final Rhamnogalacturonan, a chemically-defined polysaccharide, improves intestinal
manuscript. barrier function in DSS-induced colitis in mice and human Caco-2 cells. Scientific
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