Hindawi Publishing Corporation

BioMed Research International

Volume 2015, Article ID 412946, 12 pages
http://dx.doi.org/10.1155/2015/412946

Research Article
Neuroprotective Effects of Clostridium butyricum against
Vascular Dementia in Mice via Metabolic Butyrate

Jiaming Liu,1,2 Jing Sun,3 Fangyan Wang,2 Xichong Yu,2 Zongxin Ling,4
Haixiao Li,2 Huiqing Zhang,2 Jiangtao Jin,2 Wenqian Chen,2 Mengqi Pang,2
Junjie Yu,2 Yiwen He,2 and Jiru Xu1
1
Department of Immunology and Pathogenic Biology, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China
2
School of Environmental Science and Public Health, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
3
Department of Neurology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325027, China
4
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine,
Zhejiang University, Hangzhou, Zhejiang 310003, China

Correspondence should be addressed to Jiru Xu; [email protected]

Received 10 June 2015; Revised 1 September 2015; Accepted 20 September 2015

Academic Editor: Clara G. de los Reyes-Gavilán

Copyright © 2015 Jiaming Liu et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Probiotics actively participate in neuropsychiatric disorders. However, the role of gut microbiota in brain disorders and vascular
dementia (VaD) remains unclear. We used a mouse model of VaD induced by a permanent right unilateral common carotid arteries
occlusion (rUCCAO) to investigate the neuroprotective effects and possible underlying mechanisms of Clostridium butyricum.
Following rUCCAO, C. butyricum was intragastrically administered for 6 successive weeks. Cognitive function was estimated.
Morphological examination was performed by electron microscopy and hematoxylin-eosin (H&E) staining. The BDNF-PI3K/Akt
pathway-related proteins were assessed by western blot and immunohistochemistry. The diversity of gut microbiota and the levels
of butyrate in the feces and the brains were determined. The results showed that C. butyricum significantly attenuated the cognitive
dysfunction and histopathological changes in VaD mice. C. butyricum not only increased the levels of BDNF and Bcl-2 and
decreased level of Bax but also induced Akt phosphorylation (p-Akt) and ultimately reduced neuronal apoptosis. Moreover, C.
butyricum could regulate the gut microbiota and restore the butyrate content in the feces and the brains. These results suggest
that C. butyricum might be effective in the treatment of VaD by regulating the gut-brain axis and that it can be considered a new
therapeutic strategy against VaD.

1. Introduction An increasing number of studies have investigated the

bidirectional signaling of the gut-brain axis [6], which is
Vascular dementia (VaD), the second most common type of closely connected between the gut microbiota and brain [7,
dementia after Alzheimer’s disease, results from a reduction 8]. Accumulating evidence shows that central nervous system
in the cerebral blood supply by a blocked or diseased (CNS) disease, such as traumatic brain injury, psychological
vascular system and leads to a progressive decline in memory stress, and Parkinson’s disease, could lead to gastrointestinal
and cognitive function [1]. Clinical evidence supports that dysfunction, even causing intestinal barrier dysfunction and
chronic cerebral hypoperfusion is associated with cognitive compositional changes in gut microbiota. However, little
decline and hippocampal neuronal injury in neurodegenera- research has determined the effects of normal bacterial
tive disorders [2, 3]. The possible mechanisms leading to VaD colonization on the development and function of the brain.
are mainly oxidative stress and apoptosis [4, 5]. These recent findings on the new role of gut microbiota in
2 BioMed Research International

the gut-brain axis implicate that gut microbiota could be asso- with a 12 h light/dark cycle (lights on at 08:00 am). The mice
ciated with brain functions as well as neurological diseases via were allowed to acclimatize to the laboratory for 1 week
the gut-brain axis. Commensal microbes play critical roles in prior to beginning of the study. Animals were housed in a
brain health and disease [9], and dysbiosis may contribute specific room, and the treatment groups were separated from
to depression and anxiety [10, 11]. Lactobacillus and Bifi- each other to avoid cross contamination. All experiments
dobacterium have been reported to attenuate anxiety, prevent were performed according to animal use guidelines and
chronic psychological stress [12, 13], reduce apoptosis in sev- approved by the Animal Experimentation Ethics Committee
eral brain regions, and improve learning and memory in mice of Wenzhou Medical University.
[14, 15]; these phenomena are often accompanied by changes
in the microbial composition and active microbial metabo- 2.2. Bacterial Preparation. C. butyricum WZMC1016
lites in the gut [16, 17]. Overall, the colonization of probiotics (CGMCC 9831) was provided by the China General
may initiate signaling mechanisms affecting neuronal action. Microbiological Culture Collection Center and was cultured
Therefore, we hypothesized that commensal intestinal micro- in MRS broth (Hopebio, Qingdao, China) for 24 h in an
biotas communicate with the brain under normal conditions anaerobic chamber (5% CO2 ) at 37∘ C. C. butyricum was
and their presence influences VaD development. harvested from the MRS broth (4,500 rpm; 15 min) and
Probiotics are live bacteria that usually colonize the resuspended in sterile saline. We established the standard
gastrointestinal tract and exert beneficial effects through the curve between the absorbance and colony forming units
modulation of gut microbiota [18]. Probiotics are known (CFU) with a positive linear relationship, giving calculated
to offer protection against various chronic diseases such as bacteria counts of 5.0 × 109 CFU/mL at an absorbance of 0.25
constipation, antimicrobial-associated diarrhea, and allergies at 600 nm. The experimental final concentrations were 5.0 ×
[19]. Probiotics have been widely accepted as a safe and 106 CFU/mL, 5.0 × 107 CFU/mL, and 5.0 × 108 CFU/mL.
economical therapeutic option [20]. However, the use of
probiotics as treatment protocols is limited, primarily due
to a lack of effective data and defined mechanisms of 2.3. Vascular Dementia Mouse Model. Mice were subjected to
action [21]. Clostridium butyricum resides in the intestine permanent right unilateral common carotid arteries occlu-
of healthy animals and humans and is a probiotic that has sion (rUCCAO) as a VaD mouse model, as previously
been characterized for its beneficial effects in gastrointestinal described [26, 27]. Mice were anesthetized by an intraperi-
disease both in vitro and in vivo [22]. C. butyricum treatment toneal injection of 400 mg/kg body weight chloral hydrate
may exert its positive effects via modulating gut microbiota (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)
and their metabolic short chain fatty acids (SCFAs). SCFAs, and fixed on an operating table. The right common carotid
including acetate, propionate, and butyrate, are an important artery was isolated and sutured permanently with a small-
part of these metabolites; they have a crucial role as an energy diameter silk thread. The rectal temperature was maintained
source for intestinal epithelial cells and have effects on anti- at 37 ± 0.5∘ C with a homoeothermic pad controlled during
inflammatory properties [23]. Furthermore, butyrate is not the VaD surgical procedure. After skin closure, the mice were
restricted to the intestinal tract but can be disseminated returned to their cages and normal feeding was resumed. A
systemically and is detected in the brain [24]. Butyrate in the 30% drop in the regional cerebral blood flow was considered
brain can exert neuroprotective effects on neurodegenerative as a successful VaD mouse model [28, 29]. The sham group
disorders and improve behavioral deficits via the inhibition underwent the same surgical procedure without carotid
of histone deacetylases (HDACs) [25]. ligation.
In this study, we investigated the potential neuroprotec-
tive effects and possible mechanisms of C. butyricum in mice 2.4. Experimental Design. Mice were randomly divided into
with VaD. Moreover, we demonstrated that the administra- five groups: (1) sham control group (𝑛 = 12), which was
tion of C. butyricum led to a modulation of gut microbiota subjected to a sham operation; (2) VaD model group (𝑛 = 12),
composition and a change in SCFAs, namely, butyrate. The which was subjected to rUCCAO; and (3–5) C. butyricum
beneficial effects of C. butyricum were associated with a groups of fifteen mice each, which were subjected to rUC-
change in butyrate levels in the brain. These results support CAO and treated with a suspension of C. butyricum (1 ×
the notion that the probiotic-gut microbiota-butyrate-brain 106 CFU, 1 × 107 CFU, and 1 × 108 CFU, resp.) that was freshly
axis promotes metabolic neuroprotective effects against VaD. prepared as previously described. The sham control group
and VaD model groups were treated with physiological saline.
All animals were treated intragastrically with physiological
2. Materials and Methods saline or C. butyricum following rUCCAO at doses of 200 𝜇L
through a stainless steel gavage needle once daily for 6 weeks
2.1. Animals. Male ICR mice (20–25 g, 6 weeks old) were pur- (the initial treatment was 24 h after the rUCCAO operation).
chased from the Experimental Animal Center of Wenzhou At the sixth week, behavioral tests were run in the order of the
Medical University and maintained under specific pathogen- open field test followed by the Morris water maze. During the
free (SPF) conditions. Mice were housed in groups (3-4) behavioral evaluations, C. butyricum was administered after
in plastic cages (27 × 17 × 13 cm, 𝐿 × 𝑊 × 𝐻) with ad the tests every day. At the end of the experimental period, the
libitum access to food and water under controlled laboratory mice were euthanized and tissue samples (colon contents and
conditions of temperature (22 ± 2∘ C) and humidity (55 ± 5%) brain tissue) were collected for analysis.
BioMed Research International 3

2.5. Open Field Test. Locomotors activity of mice was a D-Code system (Bio-Rad). The sequence analysis of the
recorded by digital behavioral system-locomotors module excised DGGE bands was performed as previously described
(Digbehav-LA, ShangHai Ji-Liang Software Technology Co., [31].
Ltd., China). Chambers (40 × 40 × 60 cm) are connected to
computer system by video camera. Chambers were cleaned 2.9. Ultrastructure Analysis. After the Morris water maze,
by acetic acid and 75% alcohol 1 h before the test. The room the mice were euthanized under anesthesia, and the brains
temperature was kept at 22 ± 2∘ C and the humidity was were collected and fixed in 25% glutaraldehyde for 2 h at 4∘ C,
50 ± 5%. All tests were conducted between 9 and 12 o’clock. rinsed with PBS, and soaked in osmium tetroxide. Ultrathin
The mouse was gently placed in the center of the chamber sections were prepared and placed onto colloid coated copper
and allowed to move freely. The locomotors activity was grids and double-stained with 0.4% uranyl acetate and 2%
recorded by video tracking system for 60 min without any lead acetate. Following this process, the ultrastructure of the
disturbance. After test, the videos were analyzed by the Open neurons was observed and photographed using transmission
Field software of Digbehav-LA system. The total distance electron microscopy [33].
travelled and the total time of rest and active in center were
recorded for 25–30 min.
2.10. Histology Analysis. The hippocampal tissues were
removed and embedded in paraffin. Prepared sections (5 𝜇m)
2.6. Morris Water Maze. The Morris water maze (DigBehv- were stained with either hematoxylin and eosin (HE) or ter-
MG, Shanghai Jiliang Software Technology Co., Ltd., China) minal deoxynucleotidyl transferase dUTP nick-end labeling
consisted of a video capture system, a data analysis system, (TUNEL) reagents (TUNEL, Roche Diagnostics, Germany)
and a circular plastic water tank (Φ = 120 cm). The tank using standardized protocols and then analyzed and exam-
was filled with water at a depth of 30 cm and divided into ined by light microscopy. The positive cells were defined as
four equal hypothetical quadrants. The position of the marks brown cells with apoptotic nuclear features.
remained unchanged throughout testing. A platform (Φ =
9 cm) was placed into the water at a constant position and 2.11. Western Blot Analysis. Brain samples were rapidly dis-
submerged 1 cm below the surface of the water. The Morris sected, rinsed in 1x PBS, and homogenized in an ice-cold
water maze experiment assesses positional navigation and homogenization buffer (Beyotime Institute of Biotechnol-
spatial exploration. In training experiment, on days 1–5, mice ogy, Shanghai, China). The samples were then analyzed by
were placed in the water at 4 distinct starting quadrant points Western blot. The proteins were collected and centrifuged
and trained to find the submerged platform within 60 s. at 12,000 ×g for 15 min at 4∘ C. A BCA protein assay kit
The time spent finding the platform is termed as the escape (Beyotime Institute of Biotechnology, Shanghai, China) was
latency. On the sixth day, the platform was removed from the used to determine the protein concentration. The samples
tank and each mouse was tested in a probe trial in which the (20 𝜇g) were separated using 12% sodium dodecyl sulfate-
time spent in the former platform-containing quadrant was polyacrylamide gel electrophoresis (SDS-PAGE) and trans-
recorded. The Morris water maze testing procedure has been ferred onto a nitrocellulose membrane (Bio-Rad, Hercules,
previously described [30]. CA). The membranes were incubated in blocking solution
comprising 5% nonfat milk in TBST at room temperature for
2.7. Fecal Sample Collection and Total DNA Extraction. After 1.5 h and then incubated with the primary antibodies diluted
the behavior tests, the mice were euthanized and the cecum in blocking solution individually. The primary antibodies
contents were quickly removed. Fresh samples were collected were anti-BDNF, anti-p-Akt, anti-Akt, anti-Bcl-2, and anti-
separately into sterile centrifuge tubes and immediately Bax antibody (1 : 1000, Bioworld, USA). After incubation with
transferred to the laboratory on ice and stored at −80∘ C primary antibodies, the membranes were incubated with
within 15 min of preparation for further analysis. Then, the HRP conjugated secondary antibodies (1 : 2000, Beyotime
bacterial genomic DNA was extracted from the fecal samples Institute of Biotechnology, Shanghai, China) for 1 h at room
using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, temperature. The bound antibodies were detected using a
Germany) with the following modifications: the samples were chemiluminescence system (ECL Plus, Thermo Scientific,
agitated with 100 mg glass beads (0.1 mm) in a mini-bead and Rockford, IL). Images were scanned, and the results
beater (FastPrep, Thermo Electron Corporation) for 2 min were quantified using the National Institutes of Health ImageJ
and incubated at 56∘ C for 1 h in lysis solution containing software (Bio-Rad Laboratories, Hercules, CA, USA). 𝛽-actin
proteinase K. Finally, the DNA was eluted in 20 𝜇L elution was used as a loading control.
buffer and stored at −20∘ C for further analysis [31].
2.12. Immunohistochemistry. The hippocampal tissues were
2.8. PCR-DGGE Analysis. For amplification of the bacterial fixed, embedded in paraffin, and sectioned at 5 𝜇m. Immuno-
DNA, the universal bacterial primers 341F and 534R for the histochemistry for BDNF, p-Akt, Bcl-2, and Bax was then
V3 regions of the 16S rRNA genes were used with the reaction carried out on the sections. Slices were incubated with
conditions described by Ling et al. DGGE of the PCR prod- the primary antibodies anti-BDNF, anti-p-Akt, anti-Bcl-2,
ucts was performed as described by Muyzer et al. [32] and or anti-Bax (1 : 200, Bioworld, USA) overnight at 4∘ C. The
Ling et al. with a 35 to 50% gradient for intestinal microbiota sections were then incubated with secondary antibodies and
and a 25 to 55% gradient for ectocervical microbiota, using visualized using diaminobenzidine (DAB) as the chromagen.
4 BioMed Research International

25 800

20 ## # # #
# #
Total travel distance (m)

600 ∗ #

Total activity time (s)

∗
15
400
10

200
5

0 0
Sham VaD Cb H Cb M Cb L Sham VaD Cb H Cb M Cb L
(a) (b)
400
∗
#

300 #
Total test time (s)

##

200

100

0
Sham VaD Cb H Cb M Cb L
(c)

Figure 1: Open field test. (a) The total distance travelled over 1 h, (b) the time spent in the central area, and (c) the total rest time are presented
for each group. Sham, sham-operated group; VaD, VaD model group; 𝐶𝑏 H, C. butyricum- (1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum-
(1 × 107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum- (1 × 106 CFU-) treated group. Error bars indicate SEM; ∗ 𝑃 < 0.05 versus sham group;
# ##
𝑃 < 0.05 and 𝑃 < 0.01 versus VaD group.

Cells with brown granules were considered immunoreactive Tukey’s test. The results are expressed as mean ± standard
positive cells. The slides were observed under a microscope deviation (SD) or mean ± standard error of the mean (SEM).
and photographed. Behavioral data (i.e., Morris water maze experiment and open
field test) are presented as the mean ± SEM. Other data (i.e.,
2.13. Butyrate Assay. The cecocolic contents and brains were Western blot analysis, butyrate assay) are presented as the
quickly removed and accurately weighed. The content of mean ± SD. Statistical analyses were performed using SPSS
butyrate was measured using an ion chromatograph. An ion statistical software, version 17.0 (SPSS, Chicago, IL, USA).
chromatograph model (Dionex ICS-1100, Dionex, USA) inte- 𝑃 < 0.05 was considered significant.
grated with a Dionex IonPacAS14 chromatographic column
(4 × 250 mm) was used for the chromatographic separation 3. Results
of the butyrate. The mobile phase consisting of the eluent
bottle was delivered to a RFIC-ER to produce an ion strength 3.1. C. butyricum Improves Spatial Learning Ability. To verify
gradient that started at 0.1 mmol/L NaOH with a flow rate of the effects of sport ability on cognitive improvement, we
1.0 mL/min. The column was injected with 50 𝜇L supernatant tested locomotors activity. The total distance travelled and
of brain tissue. The typical retention time for the butyrate was activity time of the VaD group were significantly decreased
30 min. compared with the sham group (𝑃 < 0.05), whereas the C.
butyricum-treated mice showed a significant increase in these
2.14. Statistical Analysis. Data of escape latency in the Morris measures in comparison with the VaD group (𝑃 < 0.05,
water maze experiment were analyzed by two-way analysis Figures 1(a) and 1(b)). Furthermore, while the rest time of
of variance (treatment × trial day, ANOVA) with repeated the mice with VaD was significantly increased compared with
measures followed by the Bonferroni test. All other data (i.e., the sham group (𝑃 < 0.05, Figure 1(c)), the rest time of
open field test, probe trial, Western blot analysis, and butyrate the C. butyricum-treated mice was dramatically decreased
assay) were analyzed with one-way ANOVA followed by in comparison with the VaD mice (1 × 106 and 1 × 107
BioMed Research International 5

60 ∗ 25

Time spent in target quadrant (s)

20
50 ##
∗
Escape latency (s)

∗ ∗ #
15
#
40
∗∗
10
#
30 # 5

20 0
Day 1 Day 2 Day 3 Day 4 Day 5 Sham VaD Cb H Cb M Cb L
Sham Cb M
VaD Cb L
Cb H
(a) (b)

Figure 2: Morris water maze. (a) Escape latency (s). (b) The time spent in the target quadrant (s). Sham, sham-operated group; VaD, VaD
model group; 𝐶𝑏 H, C. butyricum- (1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum- (1 × 107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum-
(1 × 106 CFU-) treated group. Error bars indicate SEM; ∗ 𝑃 < 0.05 and ∗∗ 𝑃 < 0.01 versus sham group; # 𝑃 < 0.05 and ## 𝑃 < 0.01 versus VaD
group.

CFU: 𝑃 < 0.05; 1 × 108 CFU: 𝑃 < 0.01, Figure 1(c)). The this impairment in the neuronal ultrastructure in the VaD
locomotors activity was reduced in the VaD mice. All total group (1 × 106 CFU, 1 × 107 CFU, and 1 × 108 CFU: Figures
distance and total rest and active time in center were altered 3(c), 3(d), and 3(e)).
by C. butyricum significantly, indicating a neuroprotective As shown in Figure 4, by HE staining, the pyramidal neu-
effect in mice model of VaD with concomitant improvement rons in the CA1 region of the hippocampus lined up in order
of motor functions. with round or oval nuclei and clear nucleolus in the sham
The Morris water maze is conventionally used to measure group. By contrast, the mice subjected to VaD exhibited many
cognitive function. In the training trials, all of the groups morphological changes in the hippocampal CA1 region such
gradually learned to find the hidden platform and gently as a disappearance of the nucleolus and shrunken neurons
shortened their escape latencies. A two-way ANOVA analysis due to condensation of the cytoplasm and karyoplasms. C.
showed that the escape latency in the VaD group was signifi- butyricum treatment attenuated the morphological changes
cantly longer than in the sham group on days 1, 3, 4, and 5 (𝑃 < of the hippocampal granule cells of VaD mice. By TUNEL
0.05, Figure 2(a)), suggesting an impairment in spatial learn- staining, the neurons in area CA1 of the hippocampus of mice
ing of these vascular dementia mice. After treatment with C. with VaD showed obvious apoptosis, and the occurrence of
butyricum, the escape latency on day 5 was decreased signifi- apoptosis was significantly ameliorated by the C. butyricum
cantly as compared to the VaD mice (1 × 107 and 1 × 108 CFU: treatment; apoptotic features were not observed locally in the
𝑃 < 0.05, Figure 2(a)). In the probe trails, the data showed C. butyricum- (1 × 108 CFU-) treated mice.
that there was obvious reduction of the time spent in the tar-
get quadrant in the VaD group compared to the sham group 3.3. C. butyricum Treatment Activated BDNF-PI3K/Akt Path-
(𝑃 < 0.01, Figure 2(b)). The time spent in the target quadrant way-Related Proteins (BDNF, p-Akt, Akt, Bcl-2, and Bax) in
for the C. butyricum treatment group was much longer than Mice with VaD as Assessed by Western Blot and Immunohis-
in the VaD group (1 × 106 and 1 × 107 CFU: 𝑃 < 0.05; 1 × tochemistry. Using Western blot analysis, the protein level
108 CFU: 𝑃 < 0.01, Figure 2(b)), indicating that C. butyricum of BDNF in the VaD group was significantly decreased
treatment could attenuate cognitive impairment of VaD mice. compared with the sham group (𝑃 < 0.01, Figures 5(a)
and 5(b)), whereas that of the C. butyricum-treated mice was
3.2. C. butyricum Ameliorated the Morphological Changes significantly increased compared with the mice with VaD (1 ×
in the Hippocampus. As illustrated by electron microscopy 106 and 1 × 107 CFU: 𝑃 < 0.05; 1 × 108 CFU: 𝑃 < 0.01, Figures
in Figure 3, no destructive changes were observed in 5(a) and 5(b)). The ratio of p-Akt/Akt was significantly
the sham group; the neurons contained large oval nuclei decreased in the VaD group in comparison with the sham
with homogeneously distributed euchromatin and clear (𝑃 < 0.01, Figures 5(a) and 5(c)). The ratio of p-Akt/Akt
mitochondria (Figure 3(a)). Irregularly shaped nuclei were in the C. butyricum- (1 × 107 CFU: 𝑃 < 0.05; 1 × 108 CFU:
observed for neurons in the mice with VaD, along with the 𝑃 < 0.01-) treated mice was significantly increased compared
appearance of uneven chromatin and swollen mitochondria with the VaD group (Figures 5(a) and 5(c)), suggesting that
(Figure 3(b)). By contrast, C. butyricum treatment attenuated C. butyricum could activate Akt. The ratio of Bcl-2/Bax
6 BioMed Research International

(a) (b)

×40000 ×40000

(c) (d)

×40000 ×40000

(e)

×40000

Figure 3: Representative photomicrographs of the ultrastructural changes observed in brain tissue. (a) A representative nucleolus in a
hippocampal neuron of the sham-operated group. (b) A representative nucleolus of cerebral ischemia in the VaD group. (c) A representative
nucleolus in the 𝐶𝑏 L group, 𝐶𝑏 M group (d), and 𝐶𝑏 H group (e). Sham, sham-operated group; VaD, VaD model group; 𝐶𝑏 H, C. butyricum-
(1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum- (1 × 107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum- (1 × 106 CFU-) treated group.

(antiapoptotic/proapoptotic) was significantly reduced in the the number of cells positive for BDNF, p-Akt, and Bcl-2 was
VaD group compared with the sham (𝑃 < 0.05, Figures 5(a) significantly increased in comparison with the VaD group.
and 5(d)). However, the ratio in the C. butyricum- (1 × 107
and 1 × 108 CFU-) treated mice was remarkably increased 3.4. C. butyricum Increased the Diversity of Intestinal Bacteria.
compared with the VaD group (𝑃 < 0.05, Figures 5(a) and As shown in Figure 7, the PCR-DGGE profiles revealed
5(d)), whereas there was no significant difference between C. that the overall structure and diversity of the predominant
butyricum- (1 × 106 CFU-) treated mice and the VaD group fecal bacteria changed drastically after the C. butyricum
(𝑃 > 0.05, Figures 5(a) and 5(d)). treatment. There were fewer bands in the VaD group than
Using immunohistochemistry, as shown in Figure 6, few in the sham group, indicating a dramatically decreased level
Bax-positive cells could be found in the sham group, whereas of bacterial diversity. However, the C. butyricum treatment
most cells were positive for BDNF, p-Akt, and Bcl-2. In the clearly increased the bacterial diversity, particularly in C.
VaD group, there were a large number of Bax-positive cells, butyricum- (1 × 108 CFU-) treated mice. The cluster analysis
while the staining for BDNF, p-Akt, and Bcl-2 was reduced of the DGGE profiles, which was based on the similarity
compared with the sham group in the hippocampal CA1 indices, showed that samples from the sham mice and the
region. After the C. butyricum treatment, the number of C. butyricum- (1 × 107 CFU and 1 × 108 CFU-) treated mice
the Bax-positive cells was significantly decreased, whereas clustered together in one branch, whereas the samples from
BioMed Research International 7

HE Sham VaD Cb H Cb M Cb L

(a)
Sham VaD Cb H Cb M Cb L
TUNEL

(b)

Figure 4: Representative photomicrographs of the histopathological changes in area CA1 of the hippocampus in mice. (a) HE staining. (b)
TUNEL staining. Cells with a brown-stained cytoplasm are considered positive. Sham, sham-operated group; VaD, VaD model group; 𝐶𝑏 H,
C. butyricum- (1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum- (1 × 107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum- (1 × 106 CFU-) treated
group. Magnification: 400x. Scale bar = 20 𝜇m.

Sham VaD Cb H Cb M Cb L 1.5

BDNF 27
BDNF/𝛽-actin ratio (%)

p-Akt 56 ##
1.0
#
#
Akt 56 ∗∗
(kDa)

Bcl-2 26 0.5

Bax 21

𝛽-actin 0.0
43
Sham VaD Cb H Cb M Cb L
(a) (b)
1.5 1.5

##
p-Akt/Akt ratio (%)

#
Bcl-2/Bax ratio (%)

1.0 1.0
# #

∗∗ ∗

0.5 0.5

0.0 0.0
Sham VaD Cb H Cb M Cb L Sham VaD Cb H Cb M Cb L
(c) (d)

Figure 5: Western blot analysis. (a) Western blot of the expression levels of BDNF, p-Akt, Akt, Bcl-2, and Bax in mouse hippocampus. (b) A
quantitative analysis of the protein levels of BDNF from each group normalized to the loading control 𝛽-actin (c). (d) Bar graphs showing the
protein level ratios of p-Akt/Akt and Bcl-2/Bax from each group; Sham, sham-operated group; VaD, VaD model group; 𝐶𝑏 H, C. butyricum-
(1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum- (1 × 107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum- (1 × 106 CFU-) treated group. Error
bars indicate SD; 𝑛 = 8 for each group. ∗ 𝑃 < 0.01 and ∗∗ 𝑃 < 0.01 versus sham group; # 𝑃 < 0.05 and ## 𝑃 < 0.01 versus VaD group.
8 BioMed Research International

Sham VaD Cb H Cb M Cb L

BDNF

p-Akt

Bcl-2

Bax

Figure 6: Immunohistochemical staining. Representative images of the immunohistochemical staining of BDNF, p-Akt, Bcl-2, and Bax in
area CA1 of the hippocampus. Cells with a brown-stained cytoplasm are considered positive. Sham, sham-operated group; VaD, VaD model
group; 𝐶𝑏 H, C. butyricum- (1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum- (1 × 107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum- (1 ×
106 CFU-) treated group. Magnification: 400x. Scale bar = 20 𝜇m.

Dendrogram using UPGMA (dice coefficient)

0.43 0.50 0.60 0.70 0.80 0.90 1.00
A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 H1 H2 H3 H4 A2
1 C1
2 B4
A1
3 B3
4 B2
5 A3
6 7 8 B1
9 A4
10 C3
11 12 D3
13 C2
14 C4
15
16 D1
17 18 H4
19
D2
H3
H2
20 H1
D4

(a) (b)

Figure 7: PCR-DGGE analysis of the predominant fecal microbiota in mice. (a) PCR-DGGE fingerprints used to analyze the fecal microbiota
of the samples from the VaD model group (A), sham-operated group (H), and C. butyricum- (B, 1 × 106 CFU-; C, 1 × 107 CFU-; and D, 1 ×
108 CFU-) treated groups. Each lane represents one subject that was randomly selected from each group. The bands marked in the DGGE gel
were identified by cloning and sequencing to facilitate the interpretation of the figure. Bands: 1: Bacteroides sp.; 2: Hespellia sp.; 3: Clostridium
XVIII sp.; 4: TM7 genera incertae sedis; 5: TM7 genera incertae sedis; 6: Anaerostipes sp.; 7: Barnesiella sp.; 8: Barnesiella sp.; 9: Roseburia sp.;
10: Acinetobacter sp.; 11: unclassified Helicobacteraceae sp.; 12: Streptobacillus sp.; 13: Barnesiella sp.; 14: Clostridium XIVa sp.; 15: Alistipes sp.;
16: unclassified Lachnospiraceae sp.; 17: Lachnospiraceae incertae sedis Alistipes sp.; 18: Anaerostipes sp.; 19: unclassified Porphyromonadaceae
sp.; and 20: Butyricimonas sp. (b) Dendrogram of the DGGE profiles shown in (a).

the mice with VaD and the C. butyricum- (1 × 106 CFU-) of the predominant fecal bacteria in the C. butyricum- (1 ×
treated mice clustered in a separate branch, although one of 107 CFU and 1 × 108 CFU-) treated mice, which indicated the
the C. butyricum- (1 × 107 CFU-) treated sample clusters was restoration of fecal microbiota after C. butyricum treatment.
grouped with the VaD group. These results demonstrated that The bands excised from the DGGE gel represented the
there were dramatic changes in the richness and diversity predominant fecal microbiota in mice.
BioMed Research International 9

8 rUCCAO-induced VaD in mice. The beneficial metabolic

## neuroprotective effects of C. butyricum occurred via changes
#
in the microbiota population resident in the intestinal tract of
Butyrate content (𝜇mol/g)

6 C. butyricum-treated mice. We confirmed that C. butyricum

#
∗∗ treatment could ameliorate both the cognitive impairment
and histopathologic changes of area CA1 of the hippocampus
4
in mice with VaD. The altered gut microbiotas, especially
those that have the ability to influence the probiotic-gut
2
microbiota-butyrate-brain axis, stimulated the production of
butyrate that in turn elevated butyrate levels in the brain to
∗ # # protect against VaD in mice.
0 The improvement of cognitive function is a desirable
Sham VaD Cb H Cb M Cb L target for therapies against VaD. The rUCCAO-inducted VaD
mouse model is characterized by damage to the hippocampus
Fecal sample that is caused by cerebral hypoperfusion and results in cogni-
Brain tissue sample
tive deficits [27]. The role of probiotics in reducing depressive
Figure 8: Absolute concentration of butyrate (𝜇mol g−1 wet weight). and anxiety-like conditions is a region of active research
Sham, sham-operated group; VaD, VaD model group; 𝐶𝑏 H, C. [7, 13]. Our results showed that C. butyricum treatment could
butyricum- (1 × 108 CFU-) treated group; 𝐶𝑏 M, C. butyricum- (1 × improve the cognitive function and performance of mice with
107 CFU-) treated group; and 𝐶𝑏 L, C. butyricum- (1 × 106 CFU-) VaD in the Morris water maze, suggesting that C. butyricum
treated group. Error bars indicate SD; 𝑛 = 8 for each group. ∗ 𝑃 < is involved in alleviating cognitive deficits.
0.05 versus sham group, ∗∗ 𝑃 < 0.01 versus sham group, and # 𝑃 < Apoptosis is coincident with the development of ischemic
0.05 and ## 𝑃 < 0.01 versus VaD group. VaD, and protection against apoptosis plays an important role
in the recovery from VaD [34]. In this study, we observed
a reduction in the histopathologic changes and the degree
3.5. C. butyricum Increased the Level of Butyrate in the Feces. of apoptosis in C. butyricum-treated mice using electron
The absolute concentrations of the fecal butyrate significantly microscopy with HE and TUNEL staining. These find-
decreased in the mice with VaD compared with the sham ings could partially account for the neuroprotective effects
mice (𝑃 < 0.01, Figure 8), whereas C. butyricum treatment observed in C. butyricum-treated mice. To gain mechanistic
(1 × 106 CFU and 1 × 107 CFU: 𝑃 < 0.05; 1 × 108 CFU: 𝑃 < 0.01, insight, we investigated the expression patterns of BDNF-
Figure 8) resulted in a significant increase in the fecal butyrate PI3K/Akt pathway-related proteins (BDNF, p-Akt, Akt, Bcl-
level. 2, and Bax) in the brain. The PI3K/Akt signaling pathway,
which is a central mediator that regulates neuronal growth,
survival, and metabolism [35], has been widely reported to
3.6. C. butyricum Increased the Level of Butyrate in the Brain. participate in the protection against VaD. BDNF, a member
The concentration of butyrate in the brains of mice with VaD of the neurotrophin family, exerts a protective effect against
was significantly decreased compared with the sham mice ischemic brain injury via antiexcitatory amino acids, the inhi-
(𝑃 < 0.05, Figure 8). However, the concentration of butyrate bition of inflammatory reactions, and decreased apoptosis
in the brains of C. butyricum-treated mice (1 × 107 CFU and [36, 37]. Recent evidence has indicated that the reduction
1 × 108 CFU: 𝑃 < 0.05, Figure 8) was remarkably increased, of BDNF can aggravate anxiety-like behavior in mice [38].
whereas there was no significant difference between C. In this study, we found that the expression of BDNF in
butyricum- (1 × 106 CFU-) treated mice and the VaD group mice with VaD was significantly decreased compared with
(𝑃 > 0.05, Figure 8). These results indicate that higher the sham group, whereas C. butyricum treatment increased
quantitative C. butyricum could elevate butyrate levels in the BDNF levels compared with the VaD group, suggesting that
brains of mice with VaD. higher BDNF levels are related to the reduction in behavioral
deficits and apoptosis in mice with VaD. In addition, BDNF
4. Discussion can activate various intracellular signaling cascades including
the PI3K/Akt signal pathway [39]. Our study indicates that
Probiotics are dietary supplements that exert beneficial C. butyricum treatment could enhance the levels of p-Akt
effects under various clinical conditions including astric- and activate Akt in mice with VaD. The activation of Akt
tion, dysbacteriosis, and antimicrobial-associated diarrhea in suppresses apoptosis and promotes cell survival by regulating
humans and animals [18, 22]. However, the widespread use downstream targets such as Bcl-2 and Bax [40]. Bcl-2 is
of probiotics for conditions outside of the gastrointestinal an antiapoptotic factor that is important for cell survival
system is lacking, due to little mechanistic insight [21]. while Bax promotes apoptosis [41]. Our results show that C.
This study demonstrates, for the first time, that VaD is butyricum treatment significantly increased the levels of Bcl-
associated with gut microbiota. In this study, we evaluated 2 and decreased the levels of Bax in mice with VaD. Taken
the neuroprotective effects of the probiotic C. butyricum and together, these results indicate that the beneficial effects of
the production of SCFAs, namely, butyrate, which has been C. butyricum were attributable to the reduction in neuronal
widely used to modulate intestinal dysbacteriosis against apoptosis and that the BDNF-PI3K/Akt pathway could be
10 BioMed Research International

involved in this antiapoptotic effect. It is possible that C. the feces and in the brains. A rise of butyrate content
butyricum indirectly ameliorated the histopathologic changes in the brain may be another mechanism of C. butyricum
and apoptosis in mice with VaD. against VaD in mice. However, how does butyrate perform
Probiotics mainly present their beneficial effects via the neuroprotective effects? We hypothesized that the rise of
modulation of gut microbiota [7]. Some psychiatric diseases, butyrate in the brains is due to the changes in the microbiota
such as autism, depression, anxiety, and stress, had been composition, and the butyrate travels from the intestinal tract
reported to induce a varying degree of gut microbiota alter- to the brains to exert neuroprotective effects. Butyrate can
ations in animals and even humans [7, 13, 14]. In this study, cross the blood-brain barrier to modulate CNS functions
including brain development and behavior [17, 43]. Butyrate
we observed a significant increase in gut microbiota diversity
might play an important role in the CNS [24, 44]. A recent
in the C. butyricum-treated mice. A cluster analysis of DGGE
study suggests that butyrate, as an inhibitor of histone
profiles, which was based on the similarity indices, showed
deacetylase (HDAC), improves spatial learning and memory
that the samples from the sham group and the C. butyricum-
ability and can provide antiapoptotic and neuroprotective
treated mice (1 × 107 or 1 × 108 CFU) clustered together in effects against ischemic stroke [45]. Little is known regarding
one branch, whereas the other samples from the mice with the interactions between gut microbiota and their ability
VaD and the 1 × 106 CFU C. butyricum-treated mice clustered to elicit neuroprotective effects [23, 46]. Our experimental
on a different branch, although one of the 1 × 107 CFU results showed that C. butyricum treatments could modulate
C. butyricum sample clusters was grouped with the VaD the gut microbiota, and a relation was observed between
mouse model. However, it is plausible that other bacterial improvement of brain function and changes in the microbiota
populations were significantly changed upon C. butyricum composition. However, a further study to investigate this
administration, and further metagenomic studies will pro- potential mechanism is necessary to be carried out.
vide a comprehensive understanding of the microbiome
changes elicited by C. butyricum. These results demonstrated
that there were dramatic changes in the richness and diversity 5. Conclusion
of the predominant intestinal bacteria in the C. butyricum-
treated mice (1 × 107 or 1 × 108 CFU), which indicated the In summary, this study provides data to support the use
restoration of gut microbiota after C. butyricum treatment. of C. butyricum as a safe and economical therapeutic
option against VaD, especially its ability to influence the
In order to connect C. butyricum, gut microbiota activity, gut microbiota-butyrate-brain axis, to prevent and treat
and VaD, we investigated the microbiota composition as well VaD in mice. The possibility that dietary C. butyricum can
as its major products of fermentation, the SCFAs. SCFAs regulate gut microbiota and lead to changes in the fecal
and fecal butyrate in mice with VaD were significantly butyrate content that raise butyrate in the brain will further
decreased. A significant increase of fecal butyrate in C. incite exploration aimed at understanding the mechanisms
butyricum-treated mice was observed. SCFAs, such as acetate, underlying VaD recovery. These results open new avenues
propionate, and butyrate, are known bioactive metabolites of for viable therapeutic options against VaD by gut micro-
fermentation of the bacterial population residing in the gas- biota modulation. Therefore, the administration of live C.
trointestinal tract. SCFAs have diverse beneficial metabolic butyricum may become an adjuvant therapy for VaD patients.
effects [42]. The levels of SCFAs in the gastrointestinal
tract are influenced by the microbial composition [23]. As
with the occurrence of intestinal dysbiosis, the level of Conflict of Interests
fecal SCFAs in the intestinal tract may change or decrease
accordingly. Probiotics, such as C. butyricum, Lactobacilli, The authors declare that there is no conflict of interests
and Bifidobacterium, produce abundant SCFAs from the fer- regarding the publication of this paper.
mentation of fibers. Changes in SCFAs have been associated
with gut microbiota modulation in other studies [24, 43]. Authors’ Contribution
Moreover, fecal butyrate levels were significantly increased
in C. butyricum-treated mice. Our previous experimental Jiaming Liu and Jing Sun equally contributed to this work.
results have indicated that administration of sodium butyrate
significantly attenuates oxidative stress, inflammation, and Acknowledgments
neuronal apoptosis and improves neurological deficits in
mice with a cerebral I/R injury induced by bilateral common The research was supported by Zhejiang Science and
carotid artery occlusion (BCCAO). Altogether, these obser- Technology Development Funds (2015C37090) and Zhe-
vations suggest that gut-derived butyrate plays an important jiang Provincial Natural Science Foundation of China
role in the treatment of VaD. We hypothesize that an (LY13B070012).
increased colonization of butyrate-producing bacteria occurs
in mice administered C. butyricum. However, extensive
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