Synergistic synbiotic containing fructooligosaccharides and Lactobacillus delbrueckii CIDCA 133 alleviates chemotherapy-induced intestinal mucositis in mice

Abstract

Intestinal mucositis is a commonly reported side effect in oncology patients undergoing chemotherapy and radiotherapy. Probiotics, prebiotics, and synbiotics have been investigated as alternative therapeutic approaches against intestinal mucositis due to their well-known anti-inflammatory properties and health benefits to the host. Previous studies showed that the potential probiotic Lactobacillus delbrueckii CIDCA 133 and the prebiotic Fructooligosaccharides (FOS) alleviated the 5-Fluorouracil (5-FU) chemotherapy-induced intestinal mucosa damage. Based on these previous beneficial effects, this work evaluated the anti-inflammatory property of the synbiotic formulation containing L. delbrueckii CIDCA 133 and FOS in mice intestinal mucosa inflammation induced by 5-FU. This work showed that the synbiotic formulation was able to modulate inflammatory parameters, including reduction of cellular inflammatory infiltration, gene expression downregulation of Tlr2, Nfkb1, and Tnf, and upregulation of the immunoregulatory Il10 cytokine, thus protecting the intestinal mucosa from epithelial damage caused by the 5-FU. The synbiotic also improved the epithelial barrier function by upregulating mRNA transcript levels of the short chain fatty acid (SCFA)-associated GPR43 receptor and the occludin tight junction protein, with the subsequent reduction of paracellular intestinal permeability. The data obtained showed that this synbiotic formulation could be a promising adjuvant treatment to be explored against inflammatory damage caused by 5-FU chemotherapy.

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World Journal of Microbiology and Biotechnology (2023) 39:235
https://doi.org/10.1007/s11274-023-03679-0
RESEARCH
Synergistic synbiotic containing fructooligosaccharides
andLactobacillus delbrueckii CIDCA 133 alleviates
chemotherapy‑induced intestinal mucositis inmice
LaísaMacedoTavares1· LuísCláudioLimadeJesus1· VivianeLimaBatista1· FernandaAlvarengaLimaBarroso1·
AndriadosSantosFreitas1· GabrielaMunisCampos1· MoniqueFerraryAmérico1· TalesFernandodaSilva1·
NinaDiasCoelho‑Rocha1· GiovannaAngeliBelo1· MarianaMartinsDrumond2,3· PamelaMancha‑Agresti2,3·
KátiaDuarteVital4· SimoneOdíliaAntunesFernandes4· ValbertNascimentoCardoso4· AlexanderBirbrair5,6,7·
EnioFerreira5· FlavianoSantosMartins8· JulianaGuimarãesLaguna1· VascoAzevedo1
Received: 14 December 2022 / Accepted: 15 June 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023
Abstract
Intestinal mucositis is a commonly reported side effect in oncology patients undergoing chemotherapy and radiotherapy. Pro-
biotics, prebiotics, and synbiotics have been investigated as alternative therapeutic approaches against intestinal mucositis due
to their well-known anti-inflammatory properties and health benefits to the host. Previous studies showed that the potential
probiotic Lactobacillus delbrueckii CIDCA 133 and the prebiotic Fructooligosaccharides (FOS) alleviated the 5-Fluorouracil
(5-FU) chemotherapy-induced intestinal mucosa damage. Based on these previous beneficial effects, this work evaluated the
anti-inflammatory property of the synbiotic formulation containing L. delbrueckii CIDCA 133 and FOS in mice intestinal
mucosa inflammation induced by 5-FU. This work showed that the synbiotic formulation was able to modulate inflammatory
parameters, including reduction of cellular inflammatory infiltration, gene expression downregulation of Tlr2, Nfkb1, and
Tnf, and upregulation of the immunoregulatory Il10 cytokine, thus protecting the intestinal mucosa from epithelial damage
caused by the 5-FU. The synbiotic also improved the epithelial barrier function by upregulating mRNA transcript levels of
the short chain fatty acid (SCFA)-associated GPR43 receptor and the occludin tight junction protein, with the subsequent
reduction of paracellular intestinal permeability. The data obtained showed that this synbiotic formulation could be a promis-
ing adjuvant treatment to be explored against inflammatory damage caused by 5-FU chemotherapy.
Keywords Chemotherapy· Intestinal damage· Synbiotic· Immunomodulation· Intestinal barrier
Laísa Macedo Tavares and Luís Cláudio Lima de Jesus have
contributed equally to this work.
* Vasco Azevedo
vascoariston@gmail.com
1 Department ofGenetics, Ecology, andEvolution, Federal
University ofMinas Gerais, BeloHorizonte, Brazil
2 Federal Center forTechnological Education ofMinas Gerais,
Department ofBiological Sciences, BeloHorizonte, Brazil
3 Federal Center forTechnological Education ofMinas
Gerais, Materials Engineering Post- Graduation Program,
BeloHorizonte, Brazil
4 Department ofClinical andToxicological Analysis, Federal
University ofMinas Gerais, BeloHorizonte, Brazil
5 Department ofGeneral Pathology, Federal University
ofMinas Gerais, BeloHorizonte, Brazil
6 Department ofDermatology, University
ofWisconsin-Madison, Madison, WI, USA
7 Department ofRadiology, Columbia University Medical
Center, NewYork, NY, USA
8 Department ofMicrobiology, Federal University ofMinas
Gerais, BeloHorizonte, Brazil

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Introduction
5-Fluorouracil (5-FU) is an antimetabolite drug widely
used for neoplasia treatment, including head, neck, breast,
and colorectal cancers (Longley etal. 2003; Sougiannis
etal. 2021). Due to its non-specificity, many patients
undergoing treatment with 5-FU develop mucositis as
the main oncological therapy adverse effect (Sonis 2004;
Crombie and Longo 2016). Mucositis is a debilitating
condition characterized by an inflammation of the gas-
trointestinal tract (GIT), which leads to symptoms such
as diarrhea, bleeding, malnutrition, electrolytic misbal-
ance, and infections (Sonis 2004; Touchefeu etal. 2014).
These adverse effects occur due to the compromising effect
of chemotherapy on the intestinal mucosa architecture
(Soares etal. 2008; Savassi etal. 2021), and onintestinal
microbiota composition (van Vliet etal. 2010; Pedroso
etal. 2015; Li etal. 2017).
There is currently no clinical procedure recommended
to prevent or treat mucositis. The palliative options are
based only on analgesics and antibiotics, which shortly
alleviate the symptoms (Kostler etal. 2001). Probiotics,
prebiotics, and synbiotic have been explored as therapeutic
approaches to ameliorate mucositis because they improve
intestinal barrier function and promote systemic and
local host immune system responses (Batista etal. 2020).
These beneficial biotics are also able to regulate the dys-
biotic microbiota and provide bioactive compounds (e.g.,
vitamins, peptides, short-chain fatty acids-SCFAs) with
potential benefits to the host health (Trindade etal. 2018;
Chang etal. 2018; Galdino etal. 2018; Yeung etal. 2020;
Carvalho etal. 2021; Savassi etal. 2021).
Among probiotics, the Lactobacillus delbrueckii subsp.
lactis CIDCA 133 (CIDCA 133) strain has been character-
ized as a promising health-promoting microorganism (de
Jesus etal. 2021b). This strain demonstrated an invitro
immunomodulatory response against Citrobacter roden-
tium and Bacillus cereus infections, stimulating phagocy-
tosis and pathogen clearance by eukaryotic cells (Rolny
etal. 2016; Hugo etal. 2017). Invivo tests demonstrated
that this strain in a fermented milk formulation (107 CFU/
mL), administrated continuous feeding, was able to pre-
vent mucosal damage caused by 5-FU in mice, decreasing
inflammatory infiltrate, preventing goblet cell loss, and
reducing intestinal permeability (De Jesus etal. 2019).
Similar results were observed after heat-inactivation of the
strain (109 CFU/mL, via gavage) or by its cell-free super-
natant (CFS) (Batista etal. 2022), demonstrating that pos-
sibly bacteria cell surface components or secreted products
derived from CIDCA 133 are responsible for its anti-
inflammatory properties, as previously predicted through
a probiogenomics study (de Jesus etal. 2021a, b). The
molecular mechanisms associated with CIDCA 133 (107
CFU/mL, continuous feeding) beneficial effects include
inflammatory NF-κB signaling pathway modulation and
upregulation of the immunoregulatory Il10 cytokine and
epithelial barrier markers (mucin 2, claudin 1, zonulin, and
junctional adhesion molecule gene expression) (Barroso
etal. 2022). Additionally, it has been reported that CIDCA
133 (107 CFU/mL, continuous feeding) presented safety
levels for consumption (de Jesus etal. 2021a, b).
Prebiotics are selectively utilized substrates by host
microorganisms conferring a health benefit. Most are non-
digestible oligosaccharides including fructans, such as the
well-known Fructooligosaccharides (FOS) (Gibson etal.
2017; Davani-Davari etal. 2019). Biotherapeutics appli-
cations of FOS (75–240mg) on intestinal inflammatory
conditions (e.g., colitis and intestinal mucositis) have been
reported, such as its ability to increase SCFA production,
reduce inflammatory cell infiltration, improve epithelial bar-
rier function, attenuate the intestinal mucosa damage and
promote immunological and GIT microbiota modulation
(Capitán-Cañadas etal. 2016; Galdino etal. 2018; Liao etal.
2021; Carvalho etal. 2021).
Prebiotics, when associated with probiotic microorgan-
isms, results in synbiotic formulations that confer a health
benefit on the host (Markowiak and Śliżewska 2017). The
synbiotic can be categorized as complementary, composed
of a probiotic combined with a prebiotic, which is designed
to target autochthonous microorganisms; or as synergistic,
in which the substrate is designed to be selectively utilized
by the co-administered microorganism(s) (Swanson etal.
2020). Beneficial effects of putative synbiotic have been
mainly reported for intestinal inflammatory conditions
(Trindade etal. 2018; Sheng etal. 2020), metabolic diseases
(Soleimani etal. 2019), and enteric infections (Pourmasoumi
etal. 2019), among others, with promising beneficial results.
Thus, based on previous reports in which CIDCA 133 and
FOS were individually able to prevent the mucosal inflam-
mation caused by 5-FU, this study investigated the addi-
tive efficacy of synergistic synbiotic containing CIDCA 133
plus FOS on the 5-FU-induced intestinal mucositis in mice
model.
Materials andmethods
Bacterial growth condition
Lactobacillus delbrueckii subsp. lactis CIDCA 133 strain
(culture collection of CIDCA Center from the National Uni-
versity of La Plata, Argentine) was cultured in MRS broth
(de Man, Rogosa Sharp) (Kasvi, São José dos Pinhais, Bra-
zil) at 37°C for 18h, under microanaerobiosis conditions.

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Page 3 of 14 235
Then, before mice administration, the bacteria concentration
was adjusted to 108 colony-forming units (CFU)/mL.
Fructooligosaccharides (FOS) andsynbiotic
preparation
The prebiotic supplement FOS NewNutrition (Nutratec,
Ribeirão Preto, Brazil) was used at 240mg/animal/day,
according to Galdino etal. (2018). Before mice administra-
tion, the prebiotic was diluted in modified MRS broth [10g
peptone and 10g meat extract (Kasvi, São José dos Pinhais,
Brazil); 5g yeast extract (Sigma-Aldrich, St. Louis, USA);
1 mL Tween 80, 2g ammonium citrate and 5g sodium
acetate (Synth, Diadema, Brazil); 0.05g manganese sulfate
(Dinâmica, Indaiatuba, Brazil), 0.1g magnesium sulfate and
2g monopotassium phosphate (Vetec, Duque de Caxias,
Brazil) diluted in 1000 mL of distilled water; pH 6.5].
To evaluate the CIDCA 133 property to metabolize FOS,
this probiotic strain was grown on a modified MRS broth
supplemented with FOS (2%) at 37°C for 18h under micro-
anaerobiosis conditions, with its growth rate measured by
absorbance at optical density (600nm), and compared to
its growth on commercial MRS broth (Kasvi, São José dos
Pinhais, Brazil), which contains 2% glucose in its formu-
lation. Then, when CIDCA 133 cultures (grown in MRS
or modified MRS) reached 108 CFU/mL, the probiotic and
synbiotic formulation was administrated to mice. CIDCA
133 (108 CFU/mL) dose was determined by growth curves
using counting colony formation-unit (CFU).
Mice trial protocol
The Ethics Committee in the Use of Animals (CEUA-
UFMG, Protocol n º 34/2021) approved all animal experi-
ment protocols. The Bioterism Center (CEBIO) from the
Federal University of Minas Gerais (UFMG, Brazil) sup-
plied the thirty-six male BALB/c mice (6–8 weeks old)
used in this study. The animals were kept in ventilated poly-
carbonate cages at room temperature (25 ± 2°C), with a
12-hour light/dark cycle, and free access to chow and filtered
water for 24h before experiments.
The animals were divided into six groups (n = 6 animals):
Control (NC), Synbiotic (S); mucositis (MUC); and mucosi-
tis treated with CIDCA 133 (BAC), Fructooligosaccharides
(FOS) or Synbiotic (ST), respectively.
Mice from the NC and MUC groups were fed with MRS
broth, whereas the BAC group was fed with CIDCA 133
(108 CFU/mL) previously grown on MRS. Mice from the
FOS group received modified MRS broth containing the
prebiotic FOS (2%). On the other hand, mice from the S
and ST groups were fed with CIDCA 133 (108 CFU/mL)
previously grown on modified MRS broth containing FOS
(2%). All mice were continuously feeding (ad libitum) for
13 days and bottles (100 mL/day) were changed every 24h.
Mice’s body weight was evaluated daily throughout the
experimental period.
For induction of intestinal inflammation, on the 11th day,
mice in the MUC, BAC, FOS, and ST groups received an
intraperitoneal injection of 5-Fluorouracil drug (300mg/
kg) (Fauldfluor®, Libbs, São Paulo, Brazil) (De Jesus etal.
2019). Mice in the control group (NC) received a saline
solution (NaCl 0.9%) (Vetec, Rio de Janeiro, Brazil). After
72h (14th experimental day), all mice were anesthetized with
a ketamine (80mg/kg) and xylazine (16mg/kg) mixture
(Syntec, Tamboré, Brazil) for blood collection, and eutha-
nized by cervical dislocation, with the small intestine being
collected for further analysis (Fig.1a).
Histopathological andmorphometric analysis
The entire small intestine length was measured, and the
ileum section was collected and prepared for histomor-
phology analysis. For this, the tissues were washed with
phosphate-buffered saline (PBS) 0.1M, and the rolls were
prepared and fixed in 10% buffered formaldehyde solution
(Neon, Suzano, Brazil) for 24h. The material was paraff-
ined and sections of 4-µm thickness were mounted on glass
slides and stained with hematoxylin and eosin. A pathologist
performed the histopathological analysis using a modified
histological score previously determined by Howarth etal.
(1996). For each parameter, a score was given according to
the severity of the lesion in the ileum: absent (0), mild (1),
moderate, (2), and severe (3). For analyses of villus heights
and crypt depth, ten images of the ileum section of each ani-
mal were captured by a BX41 optical microscope (Olympus,
Tokyo, Japan), and analyzed with ImageJ 1.51j.8 software
(National Institute of Health, Bethesda, USA).
Myeloperoxidase andeosinophil peroxidase activity
assays
The recruitment of neutrophils and eosinophils to the ileum
mucosa was measured by their respective enzymes activity:
myeloperoxidase (MPO) and eosinophilic peroxidase (EPO)
(Strath etal. 1985; Souza etal. 2000). Briefly, the tissues
were homogenized and submitted to hypotonic lysis, and
processed with three cycles of freeze-thaw in liquid nitrogen,
respectively, followed by the collection of the supernatant
for enzymatic colorimetric assay.
For EPO, the supernatant (75 µL) was added to 75 µL
of o-phenylenediamine 1.5 mM (Sigma-Aldrich, St. Louis,
MO, USA) previously diluted in Tris–HCl 0.075 mM (pH 8)
plus hydrogen peroxide 6.6 mM (Synth, Diadema, Brazil).
The plate was incubated at 20°C for 30min, and the reaction
was stopped with sulfuric acid 1M (Vetec, Rio de Janeiro,
Brazil). For MPO, the supernatant (25 µL) was added to 25

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235 Page 4 of 14
µL of 3,3,5,5′-Tetramethylbenzidine1.6 mM (Sigma-Aldrich,
St. Louis, MO, USA) diluted in dimethyl sulfoxide (DMSO)
(Sigma-Aldrich, St. Louis, MO, USA). After adding 100 µL
of hydrogen peroxide 0.5mM (Synth), the plate was incu-
bated at 37 ºC for 5min and stopped with sulfuric acid 1M.
The enzymatic assays were measured at 450nm (MPO) and
492nm (EPO) on a microplate spectrophotometer (Bio-
Rad 450 model, Bio-Rad Laboratories). The results were
expressed as EPO or MPO arbitrary units/mg of tissue based
on absorbance.
Intestinal secretory IgA (sIgA) levels
To evaluate the sIgA levels, mice’s intestinal contents were
placed in PBS 0.1M (pH 7.2) supplemented with phenyl-
metanossulfonil fluoride 1 µM (PMSF) (Sigma-Aldrich,
St. Louis, MO, USA). Afterward, the samples were centri-
fuged (5000rpm for 30min at 4°C), and the supernatant
was collected and tested by enzyme-linked immunosorbent
assay (ELISA) using goat anti-mouse IgA (A-4789, Sigma-
Aldrich). The assay absorbance was measured at 492nm
and the concentration of sIgA was expressed in µg/mL of
intestinal contents (Barroso etal. 2021b).
Intestinal permeability
On the 14th experimental day, intestinal permeability was
evaluated by determining radioactivity in the mice’s blood
after oral gavage of 0.1 mL of diethylenetriaminepentaacetic
acid (DTPA) solution labeled with 18.5 megabecquerel
(MBq) of technetium-99m (99mTc-DTPA) (Viana etal.
2010; Carvalho etal. 2021). Four hours after the 99mTc-
DTPA administration, the mice’s blood was collected and
the radioactivity was determined in the gamma radiation
counter (Wizard 1480, PerkinElmer/Wallac, Turku, Fin-
land). The results were expressed as the percentage of the
dose injected per gram (% ID/g) of blood, following: [counts
per minute (blood)/(the standard dose)] × 100 (Viana etal.
2010).
Cytokines andepithelial barrier markers gene
expression
The profile of cytokines and genetic markers involved with
epithelial barrier function was evaluated through relative
gene expression. For this, ileum’s total RNA isolation was
carried out using Trizol (Ludwig Biotec, Alvorada, Bra-
zil), according to the manufacturer’s recommendations.
The RNA was evaluated qualitatively and quantitatively in
1.5% agarose gel and NanoDrop® 2000 spectrophotometer
(Thermo Scientific, Waltham, MA, USA), respectively.
Residual genomic DNA was removed using the Turbo DNA-
free™ Kit (Carlsbad, CA, USA). Complementary DNA was
obtained from 2µg of RNA using the High-Capacity cDNA
Reverse Transcription kit (Applied Biosystems™, Ther-
moFisher, Waltham, MA, USA), according to the recom-
mended protocol.
Gene expression analysis was performed using Pow-
erUp™ SYBR® Green Master Mix (ThermoFisher®) on
Applied Biosystems 7900HT Fast Real-Time PCR System
under the following conditions: 95°C for 10min, and 40
cycles of 95°C for 15s and 60°C for 1min. The gene-
specific primers are listed in Table1. All samples were ana-
lyzed in duplicate, and Gapdh (glyceraldehyde 3-phosphate
dehydrogenase) and Actb (actinbeta) genes were used as
endogenous references. Data were analyzed according to
the relative expression using the 2–ΔΔCT method (Livak and
Schmittgen 2001).
Statistical analysis
The Shapiro-Wilk test was used to evaluate data normality.
Data were analyzed using one-way ANOVA followed by
Tukey’s post-test (parametric data) or Kruskal-Wallis’ test
followed by Dunn’s post-test (non-parametric data) using the
GraphPad Prism 8.0 software. Statistically significant find-
ings were marked according to the p < 0.05 cutoffs.
Results
Lactobacillus delbrueckii CIDCA 133 metabolizes FOS
The data in Fig.1b showed that, besides glucose, CIDCA
133 is also able to metabolize the prebiotic FOS as a carbon
source. However, its growth rate was lower (Fig.1b). After
this investigation, the therapeutic effect of this formula-
tion was analyzed in mice with mucositis induced by the
5-Fluorouracil.
5-FU induced body weight loss in the MUC group
(− 16.06 ± 1.80%) compared to the control group (NC)
(0.93 ± 2.10%) (p < 0.05), but no protective effect was
observed on this parameter after treatments with probiotic
CIDCA 133 (BAC), prebiotic FOS (FOS) and synbiotic (ST)
(p > 0.05) (Fig.1c). After 24h of administration, it was pos-
sible to observe that CIDCA 133 reached a maximum count
of 1010 CFU/mL in both MRS and MRS modified medium,
with a pH of 4.13 and 3.93, respectively.
Synbiotic modulates thegene expression ofinflammatory
cytokines
Mice in the MUC group exhibited gene expression upregu-
lation of Tlr2 (p < 0.0001) (Fig.2a) and Nfkb1 (p < 0.0001)
(Fig.2b), the cytokines Tnf (p < 0.0001) (Fig. 2c), Il1b
(p < 0.05) (Fig. 2d) and Tgfb1 (p < 0.001) (Fig. 2g), and

World Journal of Microbiology and Biotechnology (2023) 39:235
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downregulation of immunoregulatory cytokine Il10
(p < 0.0001) (Fig.2f) compared to the control group (NC).
On the other hand, non-inflamed mice that consumed the
synbiotic (S) presented upregulation of the gene expression
of Il1b (Fig.2d), Il12 (Fig.2e), and Il10 (Fig.2f) cytokines
compared to the control group (p < 0.001).
The treatment with probiotic (BAC) or prebiotic (FOS)
reduced the mRNA expression of Tlr2 (Fig.2a), Nfkb1
(Fig.2b), Tnf (Fig.2c), Il12 (Fig.2e), and Tgfb1 (Fig.2g)
(p < 0.0001). Treatment with prebiotic (FOS) was also
effective to upregulate the transcripts levels of Il10 (Fig.2f)
(p < 0.0001). Synbiotic (ST) downregulated the gene expres-
sion of Tlr2 (Fig.2a), Nfkb1 (Fig.2b), Tnf (Fig.2c), and
Tgfb1 (Fig.2g) (p < 0.0001) and upregulate the transcripts
levels of the immunoregulatory cytokine Il10 (Fig.2f)
(p < 0.0001). Nevertheless, this treatment was more effec-
tive for modulating only the relative gene expression of
Il1b (Fig.2d), Il12 (Fig.2e), and Il10 (Fig.2f) cytokines
(p < 0.001) than probiotic and prebiotic treatments.
Synbiotic contributes tothemaintenance
oftheintestinal epithelial barrier
Mice in the MUC group (0.065 ± 0.01% ID/g) exhibited
increased intestinal permeability (p < 0.001) (Fig. 3a)
and gene expression downregulation of the tight junction
protein occludin (Fig.3b) compared to the control group
(NC) (0.005 ± 0.000095% ID/g) (p < 0.001). Probiotic
(BAC) (0.01 ± 0.0050% ID/g) (p < 0.0001), prebiotic (FOS)
(0.045 ± 0.0083% ID/g) (p < 0.05), and synbiotic (ST)
(0.02 ± 0.01% ID/g) (p < 0.0001) treatments significantly
reduced 5-FU-induced intestinal permeability (Fig.3a).
Additionally, prebiotic (FOS) and synbiotic treatment (ST)
upregulated the gene expression of the tight junction claudin
1 (Fig.3c) compared to the MUC group (p < 0.05). Probiotic
and synbiotic were also effective in upregulating the gene
expression of the GPR43 receptor (Fig.3d). However, the
synbiotic treatment (ST) was more effective for modulating
the occludin gene expression (p < 0.01) (Fig.3c) than the
probiotic and prebiotic individually. No difference in sIgA
levels was observed between the inflamed groups (MUC,
BAC, FOS, and ST) (p > 0.05) (Fig.3e).
Synbiotic reduces cell inflammatory infiltrate
andameliorates theintestinal epithelial
architecture
Histopathological analysis demonstrated that 5-FU induced
significant alterations (e.g., intense polymorphonuclear cell
infiltration, villus shortening, and crypt depth increase)
Table 1 List of primer
sequences used in qPCR. Gene Primer sequence (5′……. 3′) References
Actb F: GCT GAG AGG GAA ATC GTG CGTG (Volynets etal. 2016)
R: CCA GGG AGG AAG AGG ATG CGG
Gapdh F: TCA CCA CCA TGG AGA AGG C (Giulietti etal. 2001)
R: GCT AAG CAG TTG GTG GTG CA
Tlr2 F: ACA ATA GAG GGA GAC GCC TTT (Chang etal. 2020)
R: AGT GTC TGG TAA GGA TTT CCCAT
Nfkb1 F: GTG GAG GCA TGT TCG GTA GTG (Zheng etal. 2017)
R: TCT TGG CAC AAT CTT TAG GGC
Tnf F: ACG TGG AAC TGG CAG AAG AG (Song etal. 2013)
R: CTC CTC CAC TTG GTG GTT TG
Il1b F: CTC CAT GAG CTT TGT ACA AGG (Song etal. 2013)
R: TGC TGA TGT ACC AGT TGG GG
Il12p40 F: GGA AGC ACG GCA GCA GAA TA (Giulietti etal. 2001)
R: AAC TTG AGG GAG AAG TAG GAA TGG
Il10 F: GGT TGC CAA GCC TTA TCG GA (Giulietti etal. 2001)
R: ACC TGC TCC ACT GCC TTG CT
Tgfb1 F: TGA CGT CAC TGG AGT TGT ACGG (Giulietti etal. 2001)
R: GGT TCA TGT CAT GGA TGG TGC
Ocln F: ACT CCT CCA ATG GAC AAG TG (Volynets etal. 2016)
R: CCC CAC CTG TCG TGT AGT CT
Cldn1 F: TCC TTG CTG AAT CTG AAC A (Volynets etal. 2016)
R: AGC CAT CCA CAT CTT CTG
Gpr43 F: ACA GTG GAG GGG ACC AAG AT (Xu etal. 2019)
R: GGG GAC TCT CTA CTC GGT GA

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235 Page 6 of 14
in the ileum mucosa compared to the control group (NC)
(Fig.4). These data were correlated with a respective shorter
small bowel length (Fig.5a) and higher histopathological
scores (Fig.5b) (p < 0.05). Non-inflamed mice that con-
sumed the synbiotic (S) showed a normal intestinal mucosa
aspect, evidencing that this biotic did not change the intes-
tinal epithelial architecture (Fig.4).
The synbiotic treatment (ST) increased the small intes-
tine length (p < 0.001) (Fig.5a) and ameliorated the intes-
tinal architecture by improving the villus length (Fig.5c)
(p < 0.0001), the crypt depth (p < 0.0001) (Fig.5d), and the
villus height to crypt depth ratio (p < 0.0001) (Fig.5e). A
protective effect was also observed in BAC and FOS groups
to these morphometric parameters (Fig.4b, c, d, e, f). Nev-
ertheless, the synbiotic treatment (ST) was more effective in
increasing the crypt/villus ratio (Fig.5e) and the small intes-
tine length (Fig.5a) than probiotic or prebiotic treatments.
Regarding polymorphonuclear cells infiltrating into
ileum mucosa, high levels of MPO (neutrophil infil-
trate) (1.83 ± 0.60 AU/mg) (Fig.5f) and EPO (eosinophil
infiltrate) (1.06 ± 0.14 AU/mg) (Fig.5g) enzyme activity
were observed after 5-FU (MUC group) administration com-
pared to the negative control (NC) (MPO: 0.13 ± 0.06 AU/
mg; EPO: 0.71 ± 0.15 AU/mg) (p < 0.01) (Fig.5f, g). After
synbiotic treatment (ST), the activity levels of these enzymes
were reduced (MPO: 1.10 ± 0.28 AU/mg; EPO: 0.065 ± 0.01
AU/mg) (p < 0.05). A protective effect was also observed
for the group treated only with the prebiotic FOS (MPO:
0.94 ± 0.47 AU/mg; EPO: 0.29 ± 0.17 AU/mg) (p < 0.01).
However, the synbiotic treatment (ST) was more effective
in reducing EPO activity levels than probiotic and prebiotic
treatments individually. On the other hand, the BAC treat-
ment did not improve these parameters (p > 0.05) (Fig.5f, g).
Discussion
The beneficial action of synbiotic on intestinal mucosi-
tis has been reported (Smith etal. 2008; Trindade etal.
2018; Savassi etal. 2021). Thus, considering the previous
Fig. 1 Lactobacillus delbrueckii CIDCA 133 metabolizes the prebi-
otic FOS. aExperimental scheme. bGrowth curve of CIDCA 133 in
modified MRS supplemented with FOS (2%). cThe therapeutic effect
of the synbiotic on mice weight loss. Different letters (a, b) indicate
statistically significant differences (p < 0.05) by Kruskal–Wallis fol-
lowed by Dunn’s post-test. NC: control; S: synbiotic; MUC: mucosi-
tis; BAC: mucositis treated with CIDCA 133; FOS: mucositis treated
with Fructooligosaccharides; ST: mucositis treated with synbiotic

World Journal of Microbiology and Biotechnology (2023) 39:235
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Page 7 of 14 235
Fig. 2 Synbiotic modulates cytokines gene expression in mice
inflamed with 5-FU. aTlr2, b, Nfkb1, cTnf, dIl1b, eIl12, fIl10, and
gTgfb1. Different letters (a, b, c, d) indicate statistically significant
differences (p < 0.05) by ANOVA followed by Tukey’s post-test. NC:
control; S: synbiotic; MUC: mucositis; BAC: mucositis treated with
CIDCA 133; FOS: mucositis treated with Fructooligosaccharides;
ST: mucositis treated with synbiotic

World Journal of Microbiology and Biotechnology (2023) 39:235
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235 Page 8 of 14
beneficial effects of FOS (Galdino etal. 2018; Carvalho
etal. 2021) and L. delbrueckii CIDCA 133 (De Jesus etal.
2019; Barroso etal. 2022; Batista etal. 2022) on mucositis,
this study investigated the anti-inflammatory role of a syn-
biotic containing the association of CIDCA 133 and FOS in
5-FU-induced intestinal mucositis.
Our study shows that L. delbrueckii CIDCA 133, despite
glucose, can also metabolize FOS. However, the growth rate
of CIDCA 133 with FOS was lower than in a glucose-con-
taining medium. Similar results were also observed by (Cao
etal. 2019) when studying the effects of different oligosac-
charides (FOS, GOS, and MOS) and glucose on Lactiplan-
tibacillus plantarum ATCC14917 growth (Cao etal. 2019).
Therefore, based on this finding, although glucose is the
main carbohydrate source that is easily assimilated by most
species of gut microbiota (Moraes etal. 2014), FOS can
be an interesting alternative that can prevent the unwanted
growth of some bacteria and other pathogenic microorgan-
isms that require rich and facile nutritional sources (Pas-
salacqua etal. 2016).
Although some studies report that CIDCA 133 (De Jesus
etal. 2019) and FOS (Galdino etal. 2018) independently
ameliorate intestinal mucositis, in our study neither treat-
ment, including the synbiotic formulation, was effective in
reducing the 5-FU-induced mice weight loss, an important
clinical parameter evaluated on chemotherapy practice.
Similar results were obtained by (Carvalho etal. 2021) and
(Barroso etal. 2022), when using FOS (250mg/day) and
continuous feeding CIDCA 133 (107 CFU/mL). However,
in a fermented milk formulation (De Jesus etal. 2019) and
high concentrations of FOS (550mg/day) (Trindade etal.
2018), was observed that both were able to ameliorate the
mice’s weight loss. Thus, one possible explanation for this
outcome is that the beneficial effects of these biotics, either
Fig. 3 Synbiotic regulates the epithelial barrier. a Intestinal perme-
ability, b occludin, c claudin 1, dGPR43 gene expression, e sIgA
levels. Different letters (a, b, c, d, e) indicate statistically significant
differences (p < 0.05) by ANOVA followed by Tukey’s post-test.NC:
control; S: synbiotic; MUC: mucositis; BAC: mucositis treated with
CIDCA 133; FOS: mucositis treated with Fructooligosaccharides;
ST: mucositis treated with synbiotic

World Journal of Microbiology and Biotechnology (2023) 39:235
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Page 9 of 14 235
alone or in combination, depend on FOS concentration and
CIDCA 133 growth matrix type, respectively.
According to the pathobiology of mucositis, the main
histopathological changes associated with chemotherapy-
induced mucositis include villus atrophy, crypt goblet cell
reduction, inflammatory cells infiltrate in the mucosa, and
mucosal barrier dysfunction (Sonis 2004; Dahlgren etal.
2021). Cell damage after 5-FU administration is primar-
ily due to its ability to generate reactive oxygen species
(ROS) and cellular apoptosis, with subsequent production
of inflammatory cytokines derived from the activation of
signaling pathways such as NF-κB (Sonis 2004; Sougiannis
etal. 2021; Dahlgren etal. 2021). The histological analysis
performed in our study revealed that the administration of
synbiotic in inflamed mice ameliorated the intestinal epithe-
lium architecture by improving intestinal villus height, and
the villus/crypt ratio and reducing the polymorphonuclear
cell infiltrate. This ameliorative effect was also observed
macroscopically via restoration of the small bowel length.
In our study, the beneficial effects of synergistic synbiotic
to ameliorate the intestinal epithelium architecture can be
associated with its property to reduce the inflammatory pro-
cess, via inhibition of mRNA transcript levels of pro-inflam-
matory markers (e.g., Tlr2, Nfkb1, and Tnf) and upregulation
of the immunoregulatory cytokine Il10, which is essential
to decreasing the intensity of inflammatory response at the
tissue damage site (Ihara etal. 2017; Wei etal. 2020). This
process could reduce the recruitment of inflammatory cells
and the production of pro-inflammatory cytokines in the
intestinal mucosa, thus restoring intestinal homeostasis.
Other studies support these findings, in which the adminis-
tration of different synbiotic formulations alleviated chem-
otherapy-associated mucosal damage and other GIT-related
inflammatory conditions by improving the intestinal inflam-
matory process and reducing neutrophils recruitment and
secretion of pro-inflammatory cytokines (e.g., IL1b, IL17A,
TNF) (Smith etal. 2008; Trindade etal. 2018; Sheng etal.
2020; Savassi etal. 2021).
Our study also showed that 5-FU induced upregulation
of Tgfb1 and downregulation of Il10 gene expression, cor-
roborating previous studies (Barbosa etal. 2018; Wu etal.
2020; Al-Khrashi etal. 2022; Barroso etal. 2022; Batista
etal. 2022). Synbiotic treatment modulated this profile,
upregulating Il10 and downregulating Tgfb1 cytokines gene
expression. Both cytokines act as anti-inflammatory agents
(Ihara etal. 2017; Wei etal. 2020). Thus, we believe that
the high transcript levels of the TGFβ1 cytokine can suggest
a compensatory anti-inflammatory mechanism to restore
gut homeostasis disrupted by 5-FU chemotherapy, due to
reduced levels of immunoregulatory IL10 cytokine (Gomes-
Santos etal. 2017; Batista etal. 2022).
It is also important to highlight that the transcripts levels
of IL1b and IL12 cytokines and high levels of sIgA were
increased after synbiotic treatment. In general, some studies
Fig. 4 Synbiotic restores intestinal mucosa architecture altered by 5-FU

World Journal of Microbiology and Biotechnology (2023) 39:235
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235 Page 10 of 14
of chemotherapy-induced intestinal mucositis recognize
these cytokines as pro-inflammatory (Yeung etal. 2015; Li
etal. 2017; Shen etal. 2021; Quintanilha etal. 2022; Coe-
lho-Rocha etal. 2022). However, the gut secretion of IL1b
can be associated with intestinal barrier repair (Wu etal.
2022) or induction of IgA production (Jung etal. 2015).
On the other hand, IL12 secretion can promote the activa-
tion of natural killer (NK) cells and the development of Th1
cells, thus increasing the immune defense against infections
(Shida etal. 2011). Thereby, a possible explanation for the
synergistic synbiotic increased mRNA transcript of IL1b and
IL12, and sIgA levels can be linked to an immunostimula-
tory mechanism of this biotic to control pathogenic bacteria-
causing infections resulting from the dysbiosis induced by
5-FU, which in our study can be associated with high mRNA
transcript levels of TLR2. This receptor recognizes bacteria
factors as being important to the communication between
microbes and the gut (van Vliet etal. 2010).
Chemotherapy-induced intestinal damage seems to be
related to TLR2 activation (Wu etal. 2020; Wei etal. 2021).
A previous study of our research group demonstrated that
5-FU (300mg/Kg) causes alterations in the microbiota
composition (Carvalho etal. 2018). This information was
also corroborated by Andrade etal. (2023). On the other
hand, our research group has reported that this 5-FU dose
increases the gene expression of TLR2 (Barroso etal. 2021,
2022; Batista etal. 2022). Based on this information, even
though we have not directly evaluated dysbiosis in our work,
Fig. 5 Synbiotic reduces epithelial damage induced by 5-FU.aSmall
bowel length. b Histological score. c Villus length. d Crypt
depth. e Villus/crypt ratio. (f) MPO activity (g) EPO activity. Dif-
ferent letters (a, b, c, d) indicate statistically significant differences
(p < 0.05) by Kruskal–Wallis followed by Dunn’s post-test or by
ANOVA followed by Tukey’s post-test. NC: control; S: synbiotic;
MUC: mucositis; BAC: mucositis treated with CIDCA 133; FOS:
mucositis treated with Fructooligosaccharides; ST: mucositis treated
with synbiotic

World Journal of Microbiology and Biotechnology (2023) 39:235
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Page 11 of 14 235
evidence shows that this dose of 5-FU causes dysbiosis and
TLR alterations, suggesting that modulation of this receptor
may be evidence of microbiota regulation by the synbiotic
formulation. Thus, although the pathogen inhibition prop-
erty by CIDCA 133 (Rolny etal. 2016; Hugo etal. 2017)
and increased number of beneficial bacteria (e.g., SCFA-
producer Bifidobacterium) after FOS supplementation (Mao
etal. 2018; Dou etal. 2022) have been reported, we rein-
force that further studies should be conducted to evaluate the
impact of the synbiotic containing FOS plus CIDCA 133 on
microbiota regulation.
Another common feature of chemotherapy-induced
mucositis is intestinal barrier disruption due to the apoptosis
of intestinal epithelial cells, resulting in an intestinal perme-
ability increase (Nakao etal. 2012; Song etal. 2013; Wardill
etal. 2014; Li etal. 2017). Our study observed that the syn-
biotic treatment ameliorated the integrity of the epithelial
barrier disrupted by 5-FU administration via upregulation
of tight junctions’ occludin and claudin 1 gene expression
and reduction of intestinal permeability. These results are
consistent with the data obtained by(Barroso etal. 2022)
and (Carvalho etal. 2021). These authors demonstrated that
increased gene expression of tight junctions (e.g., claudin 1,
occludin, zonulin) following administration of CIDCA 133
or FOS, respectively, was associated with a reduction of the
intestinal permeability and mitigation of epithelial damage
induced by 5-FU chemotherapy (Carvalho etal. 2021; Bar-
roso etal. 2022).
The gut barrier integrity and immunomodulatory activ-
ity observed in synbiotic treatment can be also linked to
CIDCA 133 metabolism-derived SCFA production, evalu-
ated in our study through the upregulation of the Gpr43
receptor gene expression. SCFA, such as acetate, propion-
ate, and butyrate, are metabolites produced through the fer-
mentation of dietary substrates by commensal microbiota.
These compounds, mainly acetate, can improve the epithelial
barrier function through the induction of genes encoding
tight-junctions (TJ) components and the production of anti-
microbial peptides (AMPs) (e.g., RegIIIγ and β-defensins)
by intestinal epithelial cells (IECs) (Parada Venegas etal.
2019a). Regarding immunomodulation properties, their anti-
inflammatory effects in intestinal mucosa are mainly derived
from their ability to activate their specific cell surface G-pro-
tein coupled receptors (GPCR) (e.g., GPR41, GPR43, and
GPR109A) present in immune cells and intestinal epithelial
cells (Rooks and Garrett 2016; Parada Venegas etal. 2019;
Russo etal. 2019).
The beneficial impact of SCFA-producing beneficial
bacteria on intestinal inflammation has been reported. In
this context, the potential probiotic Lacticaseibacillus
rhamnosus improved inflammatory damage of the intestinal
mucosa affected by 5-FU chemotherapy by reducing NLRP3
inflammasome and enhancing the expressions of ZO-1 and
Occludin in the small intestinal mucosa (Yue etal. 2022).
These ameliorative effects were associated with Lb. rham-
nosus influence to increase the SCFA (acetate, propionate,
and butyrate) levels in mice stool (Yue etal. 2022). Similar
results were also reported for Streptococcus thermophilus
ST4 (Shen etal. 2021), which alleviated 5-FU-induced epi-
thelial damage by reducing serum levels of TNF-α, IL-1β,
and IL-6 cytokines and increasing acetic acid levels in mice
stool. Thus, we believed that the production and interaction
of SCFA-derived from CIDCA 133 metabolism with the
GPR43 receptor may have promoted the anti-inflammatory
IL10 cytokine-producing cell activation, with subsequent
suppression of pro-inflammatory markers, such as TNFα,
derived from NF-κB signaling pathway activation, being a
possible mechanism used by the synbiotic formulation to
alleviated mucositis.
Our study has some limitations that should be improved
in future related work. Firstly, our work was conducted using
a laboratory culture broth (MRS) and a continuous feed-
ing (ad libitum) route. Thus, the results may be different
when using other growth matrices of CIDCA 133 (e.g., fer-
mented milk formulation) and treatment administration route
(gavage). In addition, with continuous feeding, we have no
control over exactly how much bacteria the mice consume
throughout the day. Secondly, another aspect that deserves
attention is that if we consider translating the approach to
humans, it is important to point out that they do not con-
sume biotic formulation (probiotics, prebiotics, synbiotics)
or MRS as the only hydric source throughout the day. Fur-
thermore, comparing the effect of the synbiotic formulation
to the effects of the prebiotic and probiotic alone deserves
attention, since the probiotic was grown on another carbon
source (such as glucose), which could lead to difficulties in
interpreting the results in a continuous feeding administra-
tion. Finally, the molecular mechanisms of synbiotic in the
improvement of the epithelial barrier function, inflamma-
tory parameters (e.g., immune regulatory cell expansion, and
cytokines levels) and microbiota regulation, and the analysis
of SCFA still need to be further studied.
Notwithstanding these limitations, most importantly, our
results demonstrated that the synergistic synbiotic has par-
tially potentiated the intestinal mucosa from 5-FU-induced
intestinal mucositis compared to CIDCA 133 and FOS. Fur-
thermore, the primary mechanism used by the synbiotic to
ameliorate intestinal mucosa architecture included modula-
tion of inflammatory parameters, epithelial barrier markers,
and reduction in intestinal permeability, demonstrating to be
a promising therapeutic approach to be explored to alleviate
the intestinal inflammatory damage caused by chemotherapy.
Acknowledgements The authors would like to acknowledge the Pró-
Reitoria de Pesquisa - Universidade Federal de Minas Gerais, Con-
selho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

World Journal of Microbiology and Biotechnology (2023) 39:235
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235 Page 12 of 14
(CAPES), and Fundação de Amparo à Pesquisa do Estado de Minas
Gerais (FAPEMIG) for their financial support and fellowships. We
would also like to acknowledge the CIDCA center (Center for Research
and Development in Food Cryotechnology) and Pablo F. Pérez of the
National University of La Plata, Argentine, for bacterial strain supply.
Author contributions Conceptualization: PM-A, LCLJ, MMD, and
VA; Methodology: FALB, VLB, GMC, GAB, TFS, NDC-R, KDV, ÊF;
Formal analysis and investigation: LMT, LCLJ; Writing-original draft
preparation: LMT, MFA, ASF, GMC, LCLJ; Writing-review and edit-
ing: JGL, SOAF, VNC, MMD, PM-A, AB, EF, FSM, and VA; Super-
vision: VA; Funding acquisition: VA. All authors read and approved
the final manuscript.
Funding This research was funded by Fundação de Amparo à Pesquisa
do Estado de Minas Gerais -FAPEMIG (grant numbers: RED-00132-
16 and APQ-00593-14), and Conselho Nacional de Desenvolvimento
Científico e Tecnológico - CNPq) (grant number: 312045/2020-4).
Data availability The current study’s data are available from the cor-
responding author upon reasonable request.
Declarations
Conflict of interest The authors declare no conflict of interest.
Ethical approval The study was conducted according to the guide-
lines of the Brazilian College of Animal Experimentation (COBEA)
and approved by the Local Animal Experimental Ethics Committee
(CEUA-UFMG) (Protocol n º 34/2021).
Consent for publication All authors consent to the publication of this
article.
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