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Bacillus coagulans MZY531
alleviates intestinal mucosal
injury in immunosuppressive mice
via modulating intestinal barrier,
inammatory response, and gut
microbiota
Zhongwei Zhao
1,2, Manqing Sun
1, Xinmu Cui
1, Jiaxin Chen
1, Chunhong Liu
2 &
Xuewu Zhang
1*
Bacillus coagulans has a potential role in improving intestinal injury. However, the specic mechanism
is still unclear. In this study, the protective eect of B. coagulans MZY531 on intestinal mucosa injury
in cyclophosphamide (CYP)-induced immunosuppressed mice were investigated. The results indicated
that the immune organ (thymus and spleen) indices of B. coagulans MZY531 treatment groups were
signicantly increased compared to the CYP group. B. coagulans MZY531 administration promotes
the expression of immune proteins (IgA, IgE, IgG, and IgM). B. coagulans MZY531 could upregulate
the ileum levels of IFN-γ, IL-2, IL-4, and IL-10 in immunosuppressed mice. Moreover, B. coagulans
MZY531 restores the villus height and crypt depth of the jejunum and alleviates injury of intestinal
endothelial cells caused by CYP. Furthermore, the western blotting results showed that B. coagulans
MZY531 ameliorated CYP-induced intestinal mucosal injury and inammatory via up-regulates
the ZO-1 pathway and down-regulates the expression of the TLR4/MyD88/NF-κB pathway. After
treatment with B. coagulans MZY531, the relative abundance of Firmicutes phylum was dramatically
increased, as well as the genera of Prevotella and Bidobacterium, and reducing harmful bacteria.
These ndings suggested that B. coagulans MZY531 has a potential immunomodulatory activity on
chemotherapy-induced immunosuppression.
Normal gastrointestinal function is essential for the absorption of nutrients, while gastrointestinal changes can
lead to serious defects in the intestinal barrier and gastrointestinal diseases1,2. Tight junction proteins, includ-
ing ZO-1, occludin, and claudin-1, constitute intestinal epithelial cells as a physical barrier, and their complex
interactions maintain the integrity of the intestinal barrier and reduce intestinal permeability3. Intestinal leakage
can activate immune cells to secrete inammatory cytokines, which in turn increases intestinal permeability and
causes systemic inammation4. In addition, microorganisms in the gastrointestinal tract can also damage the
intestinal barrier function by promoting mucosal reaction, reducing the expression of tight junction proteins.
Aer reaching the blood, bacteria can trigger inammation by binding TLR4 receptors expressed on the liver
through the portal vein5,6.
Cyclophosphamide (CYP) was an alkylated anticancer agent which was widely used in the treatment of vari-
ous cancers. However, long-term treatment of CYP may could result various side eects such as acute cytotoxicity,
immunosuppression, and gastrointestinal mucosal barrier damage. To improve this situation, some protective
agents are increasingly being used to alleviate adverse side eects in chemotherapy patients. Certain anti-para-
sitics have also been reported to have an immunomodulatory activity such as levamisole. Levamisole is a drug
widely used to enhance the immunity of various human diseases, including leprosy, rheumatoid arthritis, and
in adjuvanted therapy of colorectal cancer. In recent years, more and more attention has been paid to alleviating
CYP induced immunosuppression based on gut microbiota regulation. In 2013, Viaud etal. demonstrated that
OPEN
1Medical College, Yanbian University, Yanji 133002, Jilin, People’s Republic of China. 2College of Special Education,
Changchun University, Changchun 130022, People’s Republic of China. *email: zhangxuewu@ybu.edu.cn
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CYP alters microbiota composition in the small intestine and induces the translocation of selected species of
Gram-positive bacteria into secondary lymphoid organs7. A few years later, Xie etal. discovered that Lactobacil-
lus plantarum intervention could protect the intestinal mucosal injury and intestinal barrier function in mice
induced by CYP by regulating intestinal ora imbalance8. erefore, regulating intestinal ora, inammation,
and intestinal barrier may be a novel potential therapeutic strategy for the treatment of the intestinal injury.
Probiotics are considered a novel choice to reduce the side eects of chemotherapy on patients. Probiotics
can maintain the balance of intestinal microorganisms and protect the intestinal integrity of epithelial cells by
increasing the mucus layer and the expression of TJ9. Bacillus coagulans, as a probiotic, can stay in the gut for a
longer time; it also has a strong adhesive ability and signicant immunomodulatory eect10. It has been reported
that some active components in the fermentation supernatant of B. coagulans can form a biological protective
barrier in the human intestinal tract, promote the immune response of the digestive tract mucosa, and thus
improve intestinal immunity11. In addition, B. coagulans 13002 can stimulate the growth of Bidobacterium and
Lactobacillus and reduce the intestinal side eects caused by cyclophosphamide12. Furthermore, B. coagulans
TL3 can protect rats from inammation caused by endotoxin and inhibit the reproduction of harmful bacteria by
blocking the expression of the TLR4 pathway so as to enhance intestinal immunity13. Moreover, previous stud-
ies have shown that B. coagulans 13002 and fructooligosaccharides signicantly reduce CYP-induced intestinal
mucosal damage and improve immune function by regulating intestinal microora12. In this study, we exam-
ined the eects of B. coagulans MZY531 (MZY531) on intestinal mucosal injury, inammation, and intestinal
microora induced by CYP in mice and explored its protective mechanism. ese data provide a theoretical
basis for the development and utilization of B. coagulans and support its addition to functional foods to improve
intestinal health.
Materials and methods
Preparation of bacterial strain. B. coagulans MZY531 is a probiotic strain isolated from naturally fer-
mented kimchi and stored in China Center for Type Culture Collection (CCTCC, accession M2021622, Wuhan,
China). B. coagulans MZY531 was inoculated in LB liquid medium and cultured in a constant temperature
vibration incubator for 48h (180 r/min, 50°C). en the culture medium was centrifuged (2000×g, 10min) and
washed three times with aseptic phosphate-buered saline (PBS, pH 7.4) to remove the residual medium and
collect bacteria. Next, the bacteria were resuspended in saline solution, and the concentration was adjusted to
1.0 × 109CFU/mL, which was stored at 4°C for subsequent intragastric administration of mice.
Animals and experimental design. Immunosuppressive model was induced by CYP according to pre-
vious study14. A total of 40 7-week-old female BALB/C mice were purchased from Changchun Yisi Experi-
mental Animal Technology Co., Ltd. (Changchun, China). All mice were kept in a suitable environment with
a temperature of 22 ± 1°C, relative humidity of 50 ± 1%, and a light/dark cycle of 12h, and had free access to
water and food. All animal studies (including the mice euthanasia procedure) were done in compliance with the
regulations and guidelines of the Jilin Academy of Agricultural Sciences institutional animal care and conducted
according to the AAALAC and the IACUC guidelines.
e experimental animal protocol is shown in Fig.1A. Aer 1week of adaptation, the mice were randomly
divided into four groups (n = 10 in each group): Control group, CYP group, CYP + LH group, and CYP + MZY531
group. e body weights of the mice were measured twice every week. e CYP + LH group was given 40mg/
kg levamisole hydrochloride (LH), the CYP + MZY531 group was given B. coagulans MZY531, and the Control
and CYP groups were given the same dose of normal saline. All mice were given oral administration according
to the volume of 0.1mL/10g for 14days, once daily. e immunosuppression mouse model induced by CTX
was established according to the previous method. CYP (50mg/kg/days) was intraperitoneally injected into
the mice in CYP, CYP + LH, and CYP + MZY531 groups on days 15 and 16. Control group was administered
intraperitoneally with the same volume of physiological saline. Aer the last injection, the mice were starved
for 24h but given free access to water. e mice were sacriced by cervical dislocation, and the jejunum, ileum,
spleen, and feces were collected.
Determination of immune organ index. Before the mice were killed, the nal weight of the mice was
recorded. en, the thymus and spleen tissues were immediately dissected, washed in precooled normal saline
at 4°C, dried using lter paper, and weighed. e spleen and thymus index were calculated according to the fol-
lowing formula: spleen and thymus index = spleen and thymus weight (mg)/nal weight (g)15.
Determination of immune and inammatory factors in the ileum. e ileum of mice was quickly
collected and placed in an ice bath. An appropriate amount of ileum tissue was then selected and mixed with
normal saline according to the proportion of 1:9 to prepare 10% tissue homogenate. Next, the homogenate was
centrifuged (4000×g, 10min) at 4°C, and the supernatant was collected. e immunoglobulin (IgA, IgE, IgG,
and IgM) and inammatory factors (IL-2, IFN-γ, IL-4, and IL-10) concentrations were detected using ELISA
kits according to the instructions of Jiangsu Enzymatic Biology Co., Ltd. (Jiangsu, China). e optical density
(OD) value of the solution was measured at 450nm using an automatic microplate reader.
Pathological observation of jejunum. e fresh jejunum tissue was washed with normal saline, xed in
4% paraformaldehyde for 48h, embedded in paran, and prepared into 8-μm slices. en, samples were stained
with hematoxylin–eosin (H&E) for 5min, dehydrated, and sealed with neutral glue. e pathological changes in
the jejunum of each group were observed under light microscope (Nikon Corporation, Tokyo, Japan), and the
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villus length (V) and crypt depth (C) were recorded16. Moreover, the images were acquired at a magnication
of × 25 and × 200 (Supplementary Information).
Western blotting. e ileal tissue was lysed by RIPA kit, and the supernatant was collected. e BCA
kit determined the total protein concentration in the supernatant. Samples were then mixed with the protein
sample buer at 1:1 and boiled in a water bath for 8min to collect the proteins, which were then separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene uo-
ride (PVDF) membrane. e membrane was blocked for 60min in TBST solution containing 3% bovine serum
albumin (BSA) and then incubated with rabbit anti-ZO-1, Occludin, Claudin-1, TLR4, MyD88, NF-κB, IKBα,
and β-actin overnight at 4°C. Samples were then incubated with horseradish peroxidase (HRP) labeled second-
ary antibody for 60min at room temperature. Samples were then washed with TBST three times, and the protein
expression was detected by an enhanced chemiluminescence reagent. e gray values of the bands were detected
by Image Quant LAS 4000 (Shanghai, China) and standardized by β-actin.
Gut microbial analysis. Fresh fecal samples of the cecum were immediately frozen in liquid nitrogen and
stored at − 80°C. QIAamp Fast DNA kit was used to extract total DNA from feces. According to the previous
study17, the V3 and V4 regions of 16S rDNA were amplied by universal primers using polymerase chain reac-
tion (PCR). en the amplied products were sequenced by Illumina MiSeq, and the sequences of high quality
with 97% similarity were incorporated into a taxon on QIIME soware, and the diversity of gut microbiota was
analyzed. e Chao1, Shannon, Simpson, and Pielou-e indices were used to investigate α diversity. e principal
coordinate method of weighted UniFrac phylogenetic distance matrix was used to analyze β diversity; the rela-
tive abundance at the gate level was used to indicate the dierence in bacterial colony structure among groups,
and the heat map analysis showed the dierence in dierent microorganisms at the genus level. In addition,
Spearman’s analysis revealed the correlation between gut microbiota and immune and inammatory levels in
mice. e original data and sequencing sample data obtained in this study can be obtained from the National
Center forBiotechnology Information (NCBI) database with the registration number: PRJNA884309.
Statistical analysis. All the experimental data were expressed as mean ± standard deviation (SD). SPSS20.0
and Origin8.0 were used for data processing and analysis. e overall signicant dierence was evaluated by
single factor analysis of variance (ANOVA) and Tukey multiple comparisons. A P value < 0.05 was considered to
be statistically signicant.
Institutional review board statement. e animal experiment procedures were approved by the Com-
mittee of Animal Experimental Ethical Inspection of Laboratory Animal Centre, Yanbian University (approved
number: SCXK-2020-0001).
ARRIVE guidelines. All the research methods contained in the manuscript are carried out in accordance
with the requirements of ARRIVE.
Figure1. A schedule of experimental procedures (A) and eects of B. coagulans MZY531 on spleen (B) and
thymus (C) index in immunosuppressed mice. All data were statistically analyzed using a one-way analysis of
variance and Tukey multiple comparison. #P < 0.05 and ##P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs.
CYP group.
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Results
B. coagulans MZY531 increases the immune organ index of intestinal injury mice. As shown in
Fig.1, the intervention of CYP signicantly decreased the spleen (Fig.1B) and thymus (Fig.1C) index of mice
compared with the control group, while the treatment of LH and B. coagulans MZY531 signicantly increased
the spleen and thymus index (all P < 0.01). Aer B. coagulans MZY531 treatment, the spleen and thymus index
increased by 71.36% and 69.39%, respectively, compared with the CYP group (all P < 0.01). ese results indi-
cate that B. coagulans MZY531 could eectively alleviate immune organ atrophy induced by CYP. Additionally,
the spleen and thymus indexes of the B. coagulans MZY531 group were higher than in the CYP group, with the
indexes being close to those of the LH group. ese ndings suggest that B. coagulans MZY531 plays a crucial
role in preventing the atrophy of immune organs.
B. coagulans MZY531 increases the level of immune protein in the ileum of intestinal injury
mice. e results showed that (Fig.2A–D), the induction of CYP signicantly decreased the levels of IgG,
IgM, IgA, and IgE by 47.75%, 46.92%, 38.15%, and 39.50%, respectively, compared with the control group (all
P < 0.01). e levels of IgG, IgM, IgA, and IgE in the B. coagulans MZY531 treatment were signicantly higher
than those in the CYP group (P < 0.05), approaching the values of the control group. Aer the B. coagulans
MZY531 treatment, the levels of IgG and IgM were similar to those of the positive control LH group (P > 0.05).
ese results showed that the treatment of B. coagulans MZY531 could reverse the decrease of immune protein
level induced by CYP and improve the immunity of mice.
B. coagulans MZY531 increases the level of anti-inammatory cytokines in the ileum of intes-
tinal injury mice. As shown in Fig.3A–D, the levels of IFN-γ, IL-2, IL-4, and IL-10 in the CYP group were
signicantly lower than those in the control group (P < 0.05), indicating that CYP could signicantly inhibit
the production of anti-inammatory cytokines. Compared with the CYP group, the treatment of B. coagu-
lans MZY531 signicantly increased the levels of IFN-γ, IL-2, IL-4, and IL-10 by 18.86%, 89.03%, 29.81%, and
51.14%, respectively (P < 0.05). e above results also showed that B. coagulans MZY531 could improve the
anti-inammatory ability of CYP-induced intestinal injury model mice. Furthermore, B. coagulans MZY531,
in particular, signicantly improved the secretion of IL-10. ese results indicated that B. coagulans MZY531
could improve inammatory responses by increasing the secretion of anti-inammatory cytokines in ileum of
CYP-induced immunosuppressed mice.
Figure2. Eects of B. coagulans MZY531 on the levels of immune proteins level in ileum of mice with
intestinal injury induced by CYP. IgG (A); IgM (B); IgA (C) and IgE (D). All data were statistically analyzed
using a one-way analysis of variance and Tukey multiple comparison. #P < 0.05 and ##P < 0.01 vs. control group;
*P < 0.05 and **P < 0.01 vs. CYP group.
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B. coagulans MZY531 improves the histomorphological changes of jejunum in intestinal injury
mice. e results of HE staining of the jejunum of mice are shown in Fig.4A, while no obvious pathologi-
cal changes in jejunum were found in the blank group. In the CYP group, the jejunal villi were shortened and
exfoliated (black arrow), the intestinal epithelium of the local mucous layer was missing, the lamina propria was
exposed (yellow arrow), slight edema could be seen locally, and the gap between the intestinal epithelium and
lamina propria was seen (red arrow). However, the villi length increased, and the intestinal epithelial structure
was signicantly recovered in LH and B. coagulans MZY531 groups. In addition, the treatment of MZY531
signicantly increased villus length (Fig.4B), crypt depth (Fig.4C), and the V/C ratio (Fig.4D), which were
153.85%, 26.44%, and 101.37%, respectively, higher than those of the CYP group (all P < 0.01). ese results
also suggested that B. coagulans MZY531 could improve the pathological intestinal damage in CYP-induced
intestinal injury model mice.
B. coagulans MZY531 improves intestinal barrier function in intestinal injury mice. e eect
of B. coagulans MZY531 on proteins (ZO-1, occludin, and claudin-1) of the intestinal barrier pathway in the
ileum of mice was evaluated by Western blotting. As shown in Fig.5, compared with the blank group, the induc-
tion of CYP signicantly decreased the protein levels of ZO-1, occludin, and claudin-1 (all P < 0.05). Compared
with CYP group, Intervention with B. coagulans MZY531 signicantly increased the protein expression of ZO-1,
occluding, and claudin-1 by 61.87% (P < 0.01), 47.80% (P < 0.05), and 17.32% (P < 0.05), respectively. Based on
this result, B. coagulans MZY531 could repair the intestinal barrier damage induced by CYP in mice.
B. coagulans MZY531 inhibits the level of inammation in intestinal injury mice. e results
presented in Fig.6 shows that the expression of TLR4 inammatory pathway protein in mouse jejunum. e
induction of CYP signicantly upregulated the levels of TLR4, MyD88, NF-κB, and IKBα. On the contrary,
the treatment of LH and B. coagulans MZ531 signicantly inhibited the expression of TLR4, MyD88, NF-κB
and IKBα, and the level of MZY531 group was down-regulated by 34.55% (P < 0.01), 29.95% (P < 0.05), 20.71%
(P < 0.01), and 52.36% (P < 0.01) compared with CYP group, respectively. us, these results revealed that the
treatment of B. coagulans MZY531 markedly resisted the expression of intestinal inammation induced by CYP
through downregulation of TLR4/MyD88/NF-κB inammatory signaling pathways.
B. coagulans MZY531 remodels the intestinal microora of intestinal injury mice. We evalu-
ated the eect of B. coagulans MZY531 on gut microbiota by 16SrRNA high-throughput sequencing. e results
of α diversity showed that (Table1), the indexes of Chao1, Shannon, Simpson, and Pielou-e in the B. coagulans
Figure3. Eects of B. coagulans MZY531 on the levels of anti-inammatory factors in ileum of
immunosuppressed mice. IFN-γ (A); IL-2 (B); IL-4 (C) and IL-10 (D). All data were statistically analyzed
using a one-way analysis of variance and Tukey multiple comparison. #P < 0.05 and ##P < 0.01 vs. control group;
*P < 0.05 and **P < 0.01 vs. CYP group.
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MZY531 group were signicantly higher than those in the CYP group (all P < 0.01), which suggested that the
intervention of B. coagulans MZY531 increased the richness and diversity of gut microbiota. Venn diagram
(Fig.7A) further showed that the four groups shared 273 OTU, while the number of unique OTU in the blank
group, CYP group, LH group, and MZY531 group was 451, 549, 783, and 666, respectively, indicating that the
treatment of B. coagulans MZY531 increases the number of OTU induced by CYP. In addition, in PCoA analysis
(Fig.7B), the CYP group was far away from the blank group, LH group, and MZY531 group, while the treatment
of LH and B. coagulans MZY531 made the diversity of gut microbiota of mice more inclined to the blank group.
In order to further evaluate the specic changes in gut microbiota, we investigated the relative abundance of gut
microbiota (Fig.7C) at the gate level. We found that B. coagulans MZY531 treatment increased Firmicutes but
decreased Bacteroidetes abundance. In genus-level thermographic analysis (Fig.7D), the intervention of B. coag-
ulans MZY531 increased the abundance of probiotic, including Lactobacillus, Prevotella and Bidobacterium,
and decreased the level of harmful bacteria Odoribacter and Shigella. ese results showed that the intervention
of B. coagulans MZY531 could reshape the structure of gut microbiota, increase the abundance of probiotics, and
reduce the level of pathogenic bacteria.
Next, Spearman analysis was used to analyze the correlation between single strains of gut microbiota and
immune and anti-inammatory proteins in mice (Fig.7E), revealing that Prevotella and Bidobacterium were
positively correlated with immune proteins, including IgG, IgM, IgA, and IgE, and anti-inammatory factors,
including IFN-γ, IL-2, IL-4, and IL-10, while Bacteroidetes and Odoribacter was negatively correlated with
immune proteins and anti-inammatory factors. ese results further reveal that the intervention of B. coagu-
lans MZY531 could increase the immunity and anti-inammatory ability of mice by increasing the abundance
of probiotics.
Discussion
CYP is a widely used chemotherapeutic drug for cancer treatment. Yet, CYP can seriously damage the body’s
immunity and induce the disorder of intestinal microora, thus increasing the risk of immune deciency and
intestinal injury diseases18. It has been found that probiotics can improve the function of intestinal microora
by regulating the value of specic microora in the intestinal tract, thus having a benecial eect on the body19.
Figure4. Eects of B. coagulans MZY531 on the histomorphological changes of jejunum in immunosuppressed
mice. e pathological changes of jejunum (magnication × 25 and × 200) (A). Black arrows indicated the
shorterning of intestinal villi, yellow arrows indicated exposed lamina propria, and black arrows indicated mild
edema and enlarged spaces. Villus height (B), Crypt depth (C). e ratio of villus height to crypt depth (D). All
data were statistically analyzed using a one-way analysis of variance and Tukey multiple comparison. #P < 0.05
and ##P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs. CYP group.
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erefore, we speculate that probiotics may be a new therapeutic way to alleviate CYP-induced intestinal injury.
is study focused on the protective eect of B. coagulans MZY531 on intestinal injury induced by CYP.
As important immune organs, spleen and thymus have an important role in regulating systemic immune
function20. Our study showed that the intervention of B. coagulans MZY531 signicantly increases the spleen and
thymus index of mice induced by CYP. Coincidentally, Awad etal. reported that the use of probiotics increases the
spleen and thymus index of broilers21; similar results were obtained by Kabir etal.22. In addition, the occurrence
of immune dysfunction seems to be regulated by IFN-γ and IL-4 levels, and the decrease of their levels may lead
to impaired immune function23. Studies have shown that CYP, as an eective immunosuppressant, can induce the
decrease of IFN-γ and IL-4 levels, thus destroying immune homeostasis and leading to immunosuppression24.
It is worth noting that B. coagulans MZY531 treatment signicantly increases the levels of IFN-γ and IL-4 and
increases the expression of immune-related cytokines, including IgG, IgM, IgA, and IgE. Furthermore, Bomko
etal. found that B. coagulans had normalised both the quantitative parameters of the immune system and the
cells’ functional activity by decreasing the level of immune-related cytokines25. is study further suggests that
treating B. coagulans MZY531 can resist the immune function damage induced by CYP by improving the func-
tion of immune organs and the level of immune protein.
As the main food digestive organ most easily aected by foreign antigens or microorganisms, the intesti-
nal tract is the rst line of defense against pathogenic microorganisms26. A harmful environment may induce
oxidation and inammation in the intestinal tract, damaging intestinal mucosal. Studies have shown that CYP
can induce the shortening and shedding of intestinal villi and, in turn, lead to intestinal mucosal damage27. In
this study, the pathological results showed that the treatment of B. coagulans MZY531 signicantly reduces the
shedding of intestinal villi, increases the crypt depth, and reduces the edema of intestinal endothelial cells, which
indicates that the intervention of B. coagulans MZY531 could alleviate the intestinal mucosal injury induced by
CYP. It is worth noting that the impairment of intestinal mucosal barrier function seems to be the main inducing
factor of intestinal mucosal injury28. erefore, increasing intestinal barrier function seems to be a good means
to prevent or treat intestinal injury. As intestinal tight junction proteins, ZO-1, occludin, and claudin-1 have
an important role in maintaining intestinal barrier permeability and constitute intestinal mucosal barrier with
intestinal epithelial cells29,30. According to previous studies, B. coagulans SCC-19 improves the intestinal barrier
Figure5. Eects of B. coagulans MZY531 on ZO-1 intestinal barrier pathway in ileum of immunosuppressed
mice. e protein expression of Claudin-1, Occludin and ZO-1 in the ileal were detected by western blot (A).
e ratios of ZO-1/β-actin (B), Occludin/β-actin (C) and Claudin-1/β-actin (D) protein bands for each region
were quantied using densitometry and presented in the graph. All data were statistically analyzed using a one-
way analysis of variance and Tukey multiple comparison. #P < 0.05 and ##P < 0.01 vs. control group; *P < 0.05 and
**P < 0.01 vs. CYP group.
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of carp induced by heavy metal cadmium (Cd) by up-regulating the mRNA expression of ZO-1, occluding,
and claudin-131. In addition, Zhou etal. conrmed that NCU116 extracellular polysaccharides of Lactobacillus
plantarum could increase the expression of the ZO-1 tight junction protein pathway by promoting the binding
of STAT3 to occludin and ZO-1 promoters, thus repairing the intestinal mechanical barrier function induced by
dextransodiumsulfate (DSS)32. Importantly, our study also found that MZY531 activates the expression of ZO-1,
occluding, and claudin-1 proteins, thus restoring intestinal mucosal barrier function. erefore, we speculate that
MZY531 may improve intestinal permeability by activating the expression of intestinal tight junction protein,
thus resisting intestinal mucosal injury induced by CYP.
In recent years, increasing evidence has shown that intestinal injury can also induce intestinal leakage (leaky
gut), causing bacteria and their metabolites to translocate to the blood, releasing inammatory factors, includ-
ing LPS and TNF-α33. In addition, the accumulation of pro-inammatory mediators can break the balance of
anti-inammatory and pro-inammatory factors and further aggravate the inammatory cascade and intestinal
injury34. As a specic anti-inammatory factor, IL-10 has an important role in enhancing the anti-inammatory
ability of the body35. It has been reported that probiotics can reduce advocated inammatory expression by acti-
vating IL-10-mediated immune pathways36. Interestingly, similar results were obtained in this study. erefore,
enhancing the expression of the anti-inammatory factor IL-10 may be one of the keys to controlling intestinal
Figure6. Eects of B. coagulans MZY531 on TLR4/MyD88 inammatory pathway in ileum of
immunosuppressed mice. e protein expression of IKBα, NF-κB, MyD88 and TLR4 in the ileal were detected
by western blot (A). e ratios of TLR4/β-actin (B), MyD88/β-actin (C), NF-κB/β-actin (D) and IKBα/β-actin
(E) protein bands for each region were quantied using densitometry and presented in the graph. All data
were statistically analyzed using a one-way analysis of variance and Tukey multiple comparison. #P < 0.05 and
##P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs. CYP group.
Table 1. Eects of MZY531 on α-diversity of gut microbiota in mice with intestinal injury induced by CYP.
All data were statistically analyzed using a one-way analysis of variance and Tukey multiple comparison.
# P < 0.05 and ##P < 0.01 vs. control group; *P < 0.05 and **P < 0.01 vs. CYP group.
Groups Chao1 Shannon Simpson Pielou_e
Control 516.75 ± 112.82** 5.57 ± 0.66** 0.9326 ± 0.027** 0.6286 ± 0.05*
CYP 341.79 ± 82.56## 4.33 ± 0.52## 0.8397 ± 0.04## 0.5254 ± 0.05#
CYP + LH 548.66 ± 39.71** 5.83 ± 0.49** 0.9488 ± 0.024** 0.651 ± 0.05**
CYP + MZY531 615.62 ± 72.18** 6.00 ± 0.41** 0.9516 ± 0.02** 0.658 ± 0.04**
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inammation. Nevertheless, some studies have reported that the expression of IL-10 is aected by the TLR4
pathway. As a type I transmembrane protein expressed on the cell membrane, TLR4 has an important role in
regulating the balance of inammation37. e NF-κB pathway is a downstream signal transduction pathway
dependent on TLR4/MyD88 pathway. When the body is in normal homeostasis, NF-κB will bind to I-κB and
remain static38. However, external stimulation can activate the expression of the TLR4/MyD88 pathway and
further induce the activation of the I-κB complex, thus regulating the expression of target genes, including TNF-
α, IL-1β, IL-6, and IL-1039. It is reported that B. coagulans TL3 inhibits intestinal inammation induced by LPS
through TLR4/MyD88, which suggests that the TLR4/MyD88 signal pathway may be the signal transduction
mechanism of B. coagulans inhibiting intestinal inammation expression13. In addition, some scholars have
reported that the combined treatment of several probiotics, including B. coagulans, suppresses DSS-induced
colitis by up-regulating the level of IL-1040. Interestingly, it has been reported that E5564, an antagonist of TLR4,
can competitively bind to TLR4-MD2, further inhibit the activation of downstream NF-κB and promote the
release of anti-inammatory factor IL-1041. In this study, we found that MZY531 down-regulates the expression
of the TLR4/MyD88 pathway and increases the level of IL-10. is further suggests that B. coagulans MZY531
may be an inhibitor of TLR4, i.e., it increases the level of IL-10 in the intestine of mice by inhibiting the TLR4/
MyD88 pathway, thus improving the anti-inammatory ability of mice and nally improving the intestinal
inammatory injury induced by CYP.
B. coagulans can regulate the disorder of intestinal microora, which has a benecial eect on the host42. Stud-
ies have shown increased abundance and diversity of probiotics in feces collected from elderly taking B. coagulans
GBI-30 and 6086 for 28 days43. Our study also obtained consistent results; we found that the intervention of B.
coagulans MZY531 increases the total number of bacteria in the feces of mice with intestinal injury induced by
CYP, which also indicates that B. coagulans MZY531 restores the abundance of intestinal ora. In addition, we
also found that B. coagulans MZY531 can increase the abundance of probiotics (Bidobacterium, Prevotella and
Firmicutes) and reduce the number of bacteria causing inammation (Bacteroides and Shigella). Of note, it has
also been reported that taking B. coagulans 13002 increases intestinal damage induced by cyclophosphamide by
increasing the abundance of probiotics12. In addition, Xie etal. reported that taking L. plantarum NCU116 may
increase the number of Bidobacteria in the feces of mice, and further reduce the disorder of gut microbiota,
thus improving the intestinal mucosal damage induced by CYP8. is indirectly conrms that B. coagulans
MZY531 has an important role in improving the intestinal injury induced by CYP. Interestingly, we examined
the correlation between intestinal ora and intestinal immune function and anti-inammatory ability by Spear-
man analysis, nding that Bidobacterium and Prevotella were positively correlated with immune proteins and
anti-inammatory factors in the intestinal tract; similar conclusions were reached by Li etal.44. is further
Figure7. Eects of B. coagulans MZY531 on the changes of gut microbiota in immunosuppressed mice.
Venn diagram (A); PCoA analysis (B). e species compositions analysis at phylum level (C). e heat map at
genus level (D). e correlation analysis between gut microbiota and intestinal immune proteins and anti-
inammatory factors at genus level by Spearman analysis (E). #P < 0.05 and ##P < 0.01 vs. control group; *P < 0.05
and **P < 0.01 vs. CYP group.
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suggests that the intervention of B. coagulans MZY531 can improve the immune and anti-inammatory ability
of the intestine by increasing the abundance of probiotics in the intestine (Fig.8), thus reducing the intestinal
injury caused by intestinal inammation and immunosuppression caused by CYP.
Conclusion
To sum up, B. coagulans MZY531 treatment improves intestinal barrier function and inammatory expression
in CYP-induced immunosuppressive mice, and its possible mechanism is related to the ZO-1 intestinal barrier
pathway and TLR4/MyD88 inammatory pathway. In addition, B. coagulans MZY531 also improves the disorder
of intestinal microora by increasing the abundance of probiotics in the intestine and further improving the
immune function and anti-inammatory ability of mice. erefore, this study provides a new research idea for
treating intestinal injury in CYP-induced immunosuppressive mice and a solid theoretical basis for the develop-
ment and utilization of B. coagulans.
Data availability
e data presented in this study are available on request from the corresponding author.
Received: 10 March 2023; Accepted: 7 July 2023
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Acknowledgements
is work was sponsored by 2022 Jilin Province Science and Technology Development Plan, Natural Science
Foundation of Jilin Province (20220101310JC).
Author contributions
Methodology, Soware, Formal analysis, Investigation, Writing—original dra, Writing—review & editing,
Visualization, Z.Z.; Methodology, Supervision, Writing—review & editing, M.S.; Project administration, Data
curation, Writing-review & editing, X.C.; Writing—review & editing, J.C.; Conceptualization, Methodology,
C.L.; Project administration, Resources, Formal analysis, Investigation, Writing—original dra, Writing—review
& editing, Funding acquisition, Supervision, X.Z. All authors have read and agreed to the published version of
the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 38379-0.
Correspondence and requests for materials should be addressed to X.Z.
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