Preventive Effect of Lactobacillus plantarum Isolated from Altay
Traditional Fermented Yogurt on DSS-induced Colitis in Mice
Xin Zhou
949th Hospital of Army, TuanJie Road No. 219, Altay City, Altay Prefecture, Xinjiang Uygur Autonomous Region, China
Keywords: Lactobacillus plantarum, Colitis, DSS.
Abstract: In this study, Preventive effect of Lactobacillus plantarum isolated from Altay traditional fermented yogurt
on DSS-induced colitis in mice has been measured. The BALB/c mice were randomly divided into five
groups: NC group (normal controls), DSS group (14-day orally administration of saline + 7-day 4% DSS
exposure from day 8), low-dose LP-A group (LP-A-L, 1×108 CFU/kg, 7-day orally administration of LP-A-
L + 7-day 4% DSS exposure from day 8), high-dose LP-A group (LP-A-H, 1×109 CFU/kg, 7-day orally
administration of LP-A-H + 7-day 4% DSS exposure from day 8), positive control group (sulfasalazine,
500mg/kg, 14-day orally administration of sulfasalazine + 7-day 4% DSS exposure from day 8 ). According
to the results, the LP-A group, especially the LP-A-H group has significantly increased the length of mice
colon, reduced the mice serum levels of pro-inflammatory cytokines, and increased seurm levels of anti-
inflammatory cytokines. In the histopathological observation of mice colon tissues, congestion and cell
infiltration were alleviated, and the goblet cells were increased by the LP-A treatment. Based on the results
presented above, this study will lay a certain theoretical foundation for the development of probiotic strain
with the effect of preventing colitis.
1 INTRODUCTION
In many areas of China, fermented dairy products are
an indispensable part of the daily diet. In addition,
many ethnic minority areas have greatly reduced the
incidence of digestive system diseases due to their
special dietary habits (Basso, Camara, Sales-Campos
2019).
The Kazakh people in the Altay region of
Xinjiang are very good at using a variety of milk
sources to ferment dairy products, such as fermented
cow milk, fermented camel milk, fermented horse
milk.
Traditional fermented dairy products mostly use
natural inoculation, and the fermentation process is a
relatively open system. The microorganisms are very
diverse and the dynamic changes of microorganisms
are also very complicated. In the long-term
fermentation and domestication process, a relatively
stable flora and ecological environment have
gradually formed, at the same time, abundant and
excellent microbial resources have been screened and
retained. The benefits of lactic acid bacteria in
fermented foods to human health have been
extensively studied (He, Zeng, Lv, Mao, Wang
2015).
Inflammatory bowel disease (IBD) is a chronic
inflammation of the gastrointestinal tract. In the past
ten years, with the increase in the incidence, IBD has
become a public health problem of world. IBD is
mainly composed of ulcerative colitis (UC), Crohn's
disease (CD) and undetermined IBD (Almana,
Mohammed, 2019). In recent years, the incidence and
prevalence of UC in China have increased year by
year. 9% to 11% of UC patients eventually die of
colon cancer and rectal cancer. Therefore, UC is
listed as a modern refractory disease by the World
Health Organization (Feuerstein, Cheifetz 2014).
The pathogenesis of colitis is complicated, and the
specific mechanism has not been studied clearly.
However, existing studies have shown that the
occurrence of colitis are related to immune
abnormalities, family inheritance, eating habits and
other factors. At the same time, the balance of pro-
inflammatory cytokines and anti-inflammatory
cytokines, oxidative stress, and impaired intestinal
mucosal barrier function are closely related to the
pathogenesis of colitis (Patil, Moss, Odze 2016).
968
Zhou, X.
Preventive Effect of Lactobacillus Plantarum Isolated from Altay Traditional Fermented Yogurt on DSS-induced Colitis in Mice.
DOI: 10.5220/0011374800003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 968-972
ISBN: 978-989-758-595-1
Copyright
c
2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
At present, the most commonly used treatment
methods are aminosalicylate anti-inflammatory drugs
(sulfasalazine, mesalazine and derivatives, etc.),
corticosteroids (prednisone, prednisone, budesonide,
etc.), immunosuppressive drugs (thiopurine,
methotrexate), and TNF antagonists (infliximab
adalimumab) (Nyambe, Koekemoer, Van de Venter
2019). The newest developments include integrin
antagonists that inhibit T cell adhesion and
antagonists of pro-inflammatory factors. At the same
time, probiotics have been used to improve the
clinical symptoms of UC patients (Shen, Zhu, Quan
2018). The use of probiotics to intervene in colitis is
a research hotspot in the field of digestion. Therefore,
it is very important to develop probiotics with the
activity of preventing UC (Grover, Rashmi,
Srivastava, Batish. 2012).
In this study, we will research the preventive
effect of Lactobacillus plantarum isolated from Altay
traditional fermented yogurt on DSS-induced colitis
in mice. Lactobacillus plantarum is very
representative of probiotics. In addition, food-derived
probiotics are safer and more available. Our study
will lay a certain theoretical foundation for the
development of probiotic strain with the effect of
preventing UC.
2 MATERIALS AND METHODS
2.1 Experimental Strain
The Lactobacillus plantarum strain was isolated from
traditional fermented yogurt in the Altay region of
Xinjiang, China. We named it LP-A.
2.2 Animal Experiments
Fifty male BALB/c mice (6 weeks old) were obtained
from Chongqing Medical University, Chongqing,
China (SCXK (YU) 2017-0001). After 7 days of
adaptive feeding, the mice were randomly divided into
five groups: NC group (normal controls), DSS group
(14-day orally administration of saline + 7-day 4%
DSS exposure from day 8), low-dose LP-A group
(LP-A-L, 1×10
8
CFU/kg, 7-day orally administration
of LP-A-L + 7-day 4% DSS exposure from day 8),
high-dose LP-A group (LP-A-H, 1×10
9
CFU/kg, 7-
day orally administration of LP-A-H + 7-day 4% DSS
exposure from day 8), positive control group
(sulfasalazine, 500mg/kg, 14-day orally
administration of sulfasalazine + 7-day 4% DSS
exposure from day 8 ). On the last day of the
experiment, the mice were fasted for 12 h. All mice
were sacrificed and the whole blood was taken from
the inferior vena cava, centrifuged at 4000 rpm for 10
min, and collected the serum. The mice colon tissues
were obtained.
2.3 Determination of IL-1Β, IL-6, IL-4,
IL-10, TNF-Α Level in Mice Serum
The serum levels of interleukin-1β (IL-1β), IL-6, IL-
4, IL-10, and tumor necrosis factor-α (TNF-α) were
quantified by commercial kits which were purchased
from BioLegend Inc., USA.
2.4 Histopathological Observation
Colon tissues of mice were put into a pre-prepared
fixative (10% formalin) to denature and coagulate the
protein of tissues and cells. The water of tissue were
gradually removed by alcohol from low-
concentration to high-concentration. Then put the
tissue block in transparent agent xylene to be
transparent, and replace the medium alcohol in the
tissue block with xylene. The transparent tissue block
were put in the melted paraffin. After the paraffin was
completely immersed in the tissue block and
embedded, then cut into thin slices. After
hematoxylin-eosin (H&E) staining, gum were added
to the slices and covered with a cover glass for
mounting. The sections were observed under the
microscope to analyze the morphological changes of
mice colon tissues.
2.5 Data Statistical Analysis
The SPSS 19.0 statistical software (SPSS Institute
Inc., Chicago, IL, USA) was used to analyze the data
of each group using the one-way ANOVA method to
detect the significant differences between the groups
(p < 0.05).
3 TEST RESULTS AND
DISCUSSIONS
3.1 Colon Length of Mice
When UC occurs, colon wall thickening, edema, colon
congestion and hypertrophy, and ulcers all shorten the
length of the colon. Therefore, the reduced length of
the colon can indirectly reflect intestinal
inflammation. As shown in Table 1, the length of the
colon in the DSS group was significantly shortened.
In the LP-A groups, the length of the colon
Preventive Effect of Lactobacillus Plantarum Isolated from Altay Traditional Fermented Yogurt on DSS-induced Colitis in Mice
969
significantly increased compared with the DSS group
(p < 0.05).
Table 1: Colon length of mice.
Groups Colon length (cm)
NC 8.75 ± 1.07
a
DSS 6.97 ± 0.22
e
LP-A-L 7.18 ± 0.62
d
LP-A-H 7.55 ± 0.49
c
Sulfasalazine 7.80 ± 0.67
b
Significant differences were found between
groups with different letter representations (p < 0.05).
3.2 Serum Levels of Pro-inflammatory
Cytokines
The imbalance between pro-inflammatory cytokines
and anti-inflammatory cytokines is regarded as an
important pathogenesis of UC (Macdonald,
Mlonteleone, Pender 2000). IL-1β can increase the
cytokines produced by macrophages, such as IL-6,
TNF-a and IL-8, so that neutrophils can accumulate
to the inflammation site and enter the intestinal
lesions, thereby causing a series of intestinal lesions,
such as colonic epithelium injury, crypt abscess,
which eventually cause the onset of UC (Lee, Lee,
Choi, Lee, Kim, Ye 2012, Rafa, Benkhelifa,
AitYounes, Saoula, Belhadef, Belkhelfa, Boukercha,
Toumi, Soufli, Moralès, de Launoit, Mahfouf,
Nakmouche, TouilBoukoffa 2017). In Table 2, the
high levels of pro-inflammatory cytokines were
shown in the DSS group. After orally administration
of LP-A, all the cytokines were significantly lower
than the DSS group (p < 0.05), especially the LP-A-
H group were similar with the sulfasalazine group.
Table 2: Serum levels of pro-inflammatory cytokines.
Groups
IL-1β
(ng/L)
IL-6
(pg/mL)
TNF-α
(ng/L)
NC 31.47 ± 4.07
e
21.32 ± 3.21
e
11.22 ± 2.04
e
DSS 75.24 ± 5.12
a
60.48 ± 6.02
a
44.01 ± 3.13
a
LP-A-L 62.57 ± 5.25
b
39.88 ± 5.11
b
30.19 ± 3.08
b
LP-A-H 56.64 ± 4.19c 30.42 ± 2.79
c
27.35 ± 2.11
c
Sulfasalazine 49.86 ± 3.23
d
30.02 ± 2.84
d
22.27 ± 2.32
d
Significant differences were found between groups with different letter representations (p < 0.05).
3.3 Serum Levels of
Anti-inflammatory Cytokines
IL-4 can inhibit the production of IL-1, TNF-a by
human monocytes and the formation of superoxide
anions derived from monocytes. IL-10 is mainly
produced by mononuclear macrophages, which can
inhibit NK cell, Th1 cell response and macrophage
cytokine synthesis (Lopetuso, Chowdhry, Pizarro
2013). Studies have shown that IL-10 synthesis is
reduced in UC patients, and IL-10 knockout mice can
develop spontaneous colitis (Keubler, Buettner,
Häger, Bleich 2015). As shown in Table 3, the LP-A
groups, especially the LP-A-H group has
significantly increased serum levels of anti-
inflammatory cytokines compared to the DSS group
(p < 0.05).
Table 3: Serum levels of anti-inflammatory cytokines.
Groups
IL-4
(pg/mL)
IL-10
(ng/L)
NC 63.38 ± 8.43
a
181.10 ± 33.62
a
DSS 34.42 ± 4.19
e
94.55 ± 25.15
e
LP-A-L 43.91 ± 4.08
d
112.61 ± 30.28
d
LP-A-H 54.16 ± 3.98
c
149.49 ± 29.13
c
Sulfasalazine 56.22 ± 4.15
b
166.67 ± 32.27
b
Significant differences were found between groups with different letter representations (p < 0.05).
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
970
3.4 Histopathological Observation of
Mice Colon Tissues
According to Figure.1, the histopathological section
of the mice colon tissues showed that the epithelial
cells of the colon tissue in the normal group were
intact, the mucosal layer was tightly connected, and
there was no obvious inflammatory cell infiltration.
At the same time, the goblet cells and crypt structures
were clearly visible. While in the DSS group, a large
number of inflammatory cells infiltrate were
obeserved, the glands were arranged disorderly, and
goblet cells were greatly reduced. After treatment
with LP-A, congestion, edema, and cell infiltration
were alleviated, and the goblet cells were increased
compared with the DSS group. In the LP-A groups,
LP-A-H had the more obvious effect on improving
colon tissue.
Figure 1: Histopathological observation of mice colon tissues (H&E staining, 500×).
4 CONCLUSION
This study confirmed the preventive effect of
Lactobacillus plantarum isolated from Altay
traditional fermented yogurt on DSS-induced colitis
in mice. Lactobacillus plantarum is very
representative of probiotics. In addition, food-derived
probiotics are safer and more available. According to
the results, the LP-A group, especially the LP-A-H
group has significantly increased the length of mice
colon, reduced the mice serum levels of pro-
inflammatory cytokines (IL-1β, IL-6, and TNF-a),
and increased seurm levels of anti-inflammatory
cytokines (IL-4, IL-10). In the histopathological
observation of mice colon tissues, DSS induced
congestion and cell infiltration were alleviated, and
the goblet cells were increased by the LP-A
treatment. Based on the results presented above, this
study will lay a certain theoretical foundation for the
development of probiotic strain with the effect of
preventing colitis.
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