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Citation: Mun, C.; Cai, J.; Hu, X.;
Zhang, W.; Zhang, N.; Cao, Y.
Clostridium butyricum and Its Culture
Supernatant Alleviate the Escherichia
coli-Induced Endometritis in Mice.
Animals 2022,12, 2719. https://
doi.org/10.3390/ani12192719
Academic Editor: Tadaaki Satou
Received: 16 September 2022
Accepted: 3 October 2022
Published: 10 October 2022
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animals
Article
Clostridium butyricum and Its Culture Supernatant Alleviate
the Escherichia coli-Induced Endometritis in Mice
Cholryong Mun , Jiapei Cai, Xiaoyu Hu, Wenlong Zhang, Naisheng Zhang * and Yongguo Cao *
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University,
Changchun 130062, China
*Correspondence: znaisheng1961@163.com (N.Z.); ygcao82@jlu.edu.cn (Y.C.)
Simple Summary:
This study evaluated the therapeutic effect of Clostridium butyricum and its
culture supernatant on Escherichia coli-induced endometritis in mice. We infused E. coli into the uterine
cavity of mice and induced endometritis. After 48 h, Clostridium butyricum or its culture supernatant
was infused into the uterine cavity of mice. After 48 h, physiological indicators, uterine morphology,
histopathology and uterine bacterial load were examined. The results showed that both Clostridium
butyricum and its culture supernatant relieved uterine inflammation. In addition, we evaluated the
reproductive performance of mice treated with Clostridium butyricum and its culture supernatant,
and the results showed that they improved the reproductive performance of mice with endometritis.
Finally, we have investigated the effect of Clostridium butyricum and its culture supernatant on the
NF-
κ
B signaling pathway, and the results show that they inhibit the activation of NF-
κ
B signaling
pathway. In conclusion, Clostridium butyricum and its culture supernatant have a potential therapeutic
effect on Escherichia coli-induced endometritis.
Abstract:
Endometritis is a disease with a high incidence in dairy cows and causes great economic
loss to milk production. This study examined the therapeutic effects of Clostridium butyricum and
its culture supernatant on Escherichia coli-induced endometritis in mice. The results showed that
Clostridium butyricum and its culture supernatant effectively suppressed inflammatory responses of
uterine tissues, such as uterine morphological changes, pathological damage, and the production of
inflammatory cytokines. Clostridium butyricum and its culture supernatant significantly decreased
uterine microbial loads. In addition, Clostridium butyricum and its culture supernatant restored
reproduction outcomes in Escherichia coli-induced endometritis mice. Western blot analysis showed
that Clostridium butyricum and its culture supernatant suppressed the NF-
κ
B signaling pathway.
Therefore, the anti-inflammatory mechanism of Clostridium butyricum and its culture supernatant
may occur through the anti-bacterial activity and regulation of the expression of NF-
κ
B in the uterus.
The anti-inflammatory effect of the culture supernatant of C. butyricum was slightly better than that
of viable C. butyricum. Therefore, our experimental results showed that Clostridium butyricum culture
supernatant may be an effective drug for treating endometritis.
Keywords:
endometritis; Clostridium butyricum; culture supernatant; NF-
κ
B signaling pathway;
reproductive outcome; Escherichia coli
1. Introduction
Endometritis is an infectious disease that occurs frequently in humans and animals
and is an important disease that causes a decrease in fertility and consequently a decrease
in milk productivity, especially in cows [
1
,
2
]. Escherichia coli (E. coli), Trueperella pyogenes,
Fusobacterium necrophorum,Prevotella, and Bacteroides species are the primary pathogens
isolated from the uterus of cows with endometritis [
3
–
6
]. In particular, E. coli is one of the
most common pathogens in the uterine secretion of cows with clinical endometritis [
7
].
E. coli stimulates endometrial epithelial cells to produce inflammatory cytokines such as
Animals 2022,12, 2719. https://doi.org/10.3390/ani12192719 https://www.mdpi.com/journal/animals
Animals 2022,12, 2719 2 of 13
TNF-
α
, IL-1
β
, and IL-6 and destroys the tight junction between epithelial cells, resulting in
inflammation of the uterine tissue [
8
]. The common treatment methods for endometritis
currently used in clinical practice are systemic antibiotics and hormone therapy, and in
severe cases, drug disinfection of the uterus is used. Using antibiotics has a long history and
has a relatively good therapeutic effect. Clinically, antibiotics such as ceftiofur, cephapirin,
and ampicillin are used in the treatment of endometritis in dairy cows. Ampicillin is a
β
-lactam antibiotic that inhibits the synthesis of peptidoglycan and other components of
the bacterial cell wall [
9
]. Ampicillin received approval in 1998 for use in dairy cattle in the
United States (Center for Veterinary Medicine, Food and Drug Administration new animal
drug application 200–180; FDA, 1998), and it is indicated for the therapy of infections
caused by E. coli [
10
,
11
]. Intramuscular injection of ampicillin at a dose of 11 mg/kg
body weight once a day achieved therapeutic concentrations in the milk, lochial fluid, and
endometrial tissue of postpartum dairy cows [
12
]. Intrauterine infusion of the drug is
also a recommended treatment for uterine diseases. It has been reported that intrauterine
treatment of ampicillin and cloxacillin plus intramuscular injection of ampicillin resulted
in a good therapeutic effect [
13
]. Although the use of antibiotics has a long history, this
currently limits its use because of problems such as the emergence of resistant bacteria and
food safety [
14
]. Hormone therapy is an adjuvant treatment method that is effective for
uterine involution after delivery. Representative hormone agents are oxytocin, estrogen,
and prostaglandin [15].
Probiotics are symbiotic microorganisms that have beneficial effects on the host’s life
activities, such as activating the immune system [
16
] and anti-inflammatory action [
17
].
Recently, some studies have reported the therapeutic effect of probiotics on endometritis in
dairy cows. For example, a researcher reported that lactic acid bacteria reduced metritis
prevalence and inflammation of the bovine endometrium [18,19].
Clostridium butyricum (C. butyricum) is a Gram-positive anaerobic rod bacterium that
ferments carbohydrates such as glucose, sucrose, fructose, and starch to synthesize or-
ganic acids such as butyric acid, acetic acid, and lactic acid [
20
]. C. butyricum exhibits
anti-pathogenic activity in the digestive tract of humans and animals. It has beneficial
activity against other beneficial bacteria [
21
]. In addition, this bacterium exerts various ben-
eficial effects such as intestinal epithelial cell protective activity [
22
] and anti-inflammatory
effects [
23
]. Butyric acid, the most important metabolite of this bacterium, also exhibits
various beneficial effects such as intestinal epithelial protection, anti-inflammatory effects,
and anticancer effects [
24
]. However, the effect of C. butyricum on endometritis has not
been reported yet. Thus, the present study investigated the potential protective effects and
mechanisms of C. butyricum on E. coli-induced endometritis.
2. Materials and Methods
2.1. Animals
Eight-week-old female BALB/c mice (22–25 g) were purchased from Liaoning Chang-
sheng Biotechnology Co., Ltd. (Benxi, China). The mice were housed in a room with a
temperature of 24
±
1
◦
C and a relative humidity of 40–80%. Food and water were supplied
ad libitum. All animal procedures were performed under the Guidelines for Care and
Use of Laboratory Animals of Jilin University, and the experiments were approved by the
Animal Ethics Committee of Jilin University.
2.2. Materials
Escherichia coli MTCC 1652 were obtained from Inner Mongolia Agricultural University,
China, and cultured at 37
◦
C in LB broth with continuous shaking. Clostridium butyricum
BNCC 337239 was purchased from BeNa Culture Collection (BNCC), Kunshan, China, and
cultured at 37
◦
C in a reinforced clostridial medium with an anaerobic condition. Mouse
monoclonal antibodies, NF-
κ
B p-p65, and p-I
κ
B
α
were purchased from Cell Signaling
Technology Inc. (Danvers, MA, USA).
Animals 2022,12, 2719 3 of 13
2.3. Preparation of the Culture Supernatants of C. butyricum
C. butyricum was cultured in a reinforced clostridial medium (RCM) under an anaer-
obic condition for 24 h at 37
◦
C. The bacterial concentration of this culture broth is
1×109
CFU/mL. The culture broth was centrifuged at 5000
×
gfor 10 min at 4
◦
C, and the
supernatants were filtered through a 0.2-
µ
m pore size syringe filter. This supernatant is
defined as a high-concentration culture supernatant of C. butyricum (HSC), and the solution
diluted 10 or 20 times with physiological saline is defined as the medium-concentration
culture supernatant (MSC) and low-concentration culture supernatant of C. butyricum
(LSC), respectively.
2.4. Uterine Infusion
All agents were infused through the vagina into the uterine cavity. We used a yellow
pipette tip and blunt needle for uterine infusion. The tip of the yellow pipette was slightly
clipped off, and this was slowly inserted into the mouse’s vagina until the tip reached the
cervix. The 23-gauge blunt needle (50 mm length) of the micro syringe (0.1 mL) was then
passed through the yellow pipette tip and the cervix into the uterine cavities.
2.5. Experimental Design
To explore the effect of C. butyricum and its culture supernatant in repairing uterine
inflammatory injury, 60 female BALB/c mice were randomly divided into five groups:
control group (CON), E. coli group (ECO), ampicillin group (AMP), viable C. butyricum
group (VCB), and supernatant of C. butyricum group (SCB). For comparison with antibiotic
treatment, we included ampicillin as the antibiotic treatment control group. Drillich, M. et al.
reported that the uterine infusion concentration of ampicillin in cattle is 2500 mg/cattle [
13
].
Therefore, according to the drug dose ratio of body weight, 0.1 mg of ampicillin was
infused into the uterine cavity of each mouse (A cow weighs about 500 kg, and a mouse
weighs about 20 g; thus, the weight ratio is about 25,000:1). On day 1, mice in the CON
group were infused with 100
µ
L of saline, and mice in the other groups were infused with
100
µ
L of
1×109CFU/mL
E. coli. On days 3 and 5, mice in the CON and ECO groups
were infused with 100
µ
L of saline, mice in the AMP group were infused with 100
µ
L
ampicillin (1 mg/mL), mice in the VCB group were infused with 100
µ
L of
1×108CFU/mL
C. butyricum, and mice in the SCB group were infused with 100
µ
L of culture supernatant
of C. butyricum (1
×
10
8
CFU/mL). All agents were infused through the vagina into the
uterine cavity. On day 7, 6 mice from each group were euthanized, the uterus was collected,
and the remaining mice could stay together with the male for 1 week from day 12 to 18;
then, the reproduction outcome was evaluated (Figure 1A).
To examine the anti-inflammatory effect of bacterial culture supernatant on endometri-
tis according to the concentration, 40 female BALB/c mice were randomly divided into
five groups: control group (CON), E. coli group (ECO), low concentration supernatant of
C. butyricum group (LSC), medium concentration supernatant of C. butyricum group (MSC),
and high concentration supernatant of C. butyricum group (HSC). On day 1, the CON
group was infused with 100
µ
L of saline, and the other groups were infused with 100
µ
L of
1×109
CFU/mL E. coli. On day 3 and 5, CON and ECO groups were infused with 100
µ
L
of saline, and LSC, MSC, and HSC groups were infused with 100
µ
L of corresponding
culture supernatant of C. butyricum. On day 7, all mice were euthanized, and the uterus
was collected (Figure 1B).
2.6. Body Temperature and Weight Measurement
The body temperature of each mouse was measured rectally on day 1 and day 7 with
the aid of a lubricated digital probe thermometer (model Panlab-0331, Beijing, China). The
body weights of the animals were recorded on day 1 and day 7 (before sampling).
Animals 2022,12, 2719 4 of 13
Animals 2022, 12, x FOR PEER REVIEW 4 of 14
Figure 1. Timeline of animal experiments. (A) Experimental design of the effect of C. butyricum and
its culture supernatant on E. coli- induced endometritis in mice. (B) Experimental design of the effect
of different concentrations of C. butyricum culture supernatant on E. coli-induced endometritis in
mice.
2.6. Body Temperature and Weight Measurement
The body temperature of each mouse was measured rectally on day 1 and day 7 with
the aid of a lubricated digital probe thermometer (model Panlab-0331, Beijing, China). The
body weights of the animals were recorded on day 1 and day 7 (before sampling).
2.7. Histopathological Examination of the Uterine Tissues
Collected uterine tissues from each group were kept in 4% paraformaldehyde for 48
h. The samples were embedded in paraffin and cut into 4 μm slices. After dewaxing, the
sections were stained with hematoxylin and eosin for histological assessment under a light
microscope. The histopathologic scoring method of the uterine tissue is listed in Table S1,
according to the modification of a previously reported method [25]. The major histopatho-
logical indicators were evaluated by endometrial injury, inflammatory infiltrate, uterine
edema, and endometrium thickness (graded 0–3, from normal to severe, including nor-
mal, mild, moderate, and severe).
2.8. Uterine Bacterial Loads Examination
To determine the bacterial load in the uterus, the uterine cavity was lavaged with 1
mL of sterile saline using a micro syringe. Serial log dilutions were made, and 100 micro-
liters of each dilution were then plated on an LB plate and incubated at 37 °C for 24 h
Figure 1.
Timeline of animal experiments. (
A
) Experimental design of the effect of C. butyricum
and its culture supernatant on E. coli-induced endometritis in mice. (
B
) Experimental design of the
effect of different concentrations of C. butyricum culture supernatant on E. coli-induced endometritis
in mice.
2.7. Histopathological Examination of the Uterine Tissues
Collected uterine tissues from each group were kept in 4% paraformaldehyde for
48 h. The samples were embedded in paraffin and cut into 4
µ
m slices. After dewaxing,
the sections were stained with hematoxylin and eosin for histological assessment under
a light microscope. The histopathologic scoring method of the uterine tissue is listed in
Table S1, according to the modification of a previously reported method [
25
]. The major
histopathological indicators were evaluated by endometrial injury, inflammatory infiltrate,
uterine edema, and endometrium thickness (graded 0–3, from normal to severe, including
normal, mild, moderate, and severe).
2.8. Uterine Bacterial Loads Examination
To determine the bacterial load in the uterus, the uterine cavity was lavaged with 1 mL
of sterile saline using a micro syringe. Serial log dilutions were made, and 100 microliters of
each dilution were then plated on an LB plate and incubated at 37
◦
C for 24 h under aerobic
conditions. Colony-forming units were counted. Results were expressed as colony-forming
units per uterus.
Animals 2022,12, 2719 5 of 13
2.9. ELISA Assay
The uterine tissues were prepared and homogenized with cold PBS (weight/volume
ratio 1:9) on ice. The homogenates were centrifuged at 2000
×
gfor 40 min at 4
◦
C. The
levels of pro-inflammatory cytokines TNF-
α
and IL-1
β
in supernatants were detected using
ELISA kits (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions.
The read absorbance of the samples was tested at 450 nm using a microplate reader.
2.10. Evaluation of Mice Reproductive Outcomes
Reproductive outcomes were evaluated using three indicators. The pregnancy rate
refers to the percentage of pregnant mice to the number of mated mice in each group. Pups
per litter refers to the total number of pups born in each group divided by the number
of maternal mice in the group. Weight per pup is the total weight of pups in each group
divided by the number of pups in that group.
2.11. Western Blotting Assay
The total proteins were extracted from the uterine tissues using a tissue protein
extraction reagent (T-PER). The protein concentration was determined by using a BCA
protein assay kit. A total of 40
µ
g of protein was fractionated using 10% SDS-PAGE
and transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the
membranes were blocked with 5% skimmed milk powder for 2 h at room temperature on a
shaker, followed by overnight incubation at 4
◦
C with the specific primary antibodies and
were washed with TBS-T three times. After incubation with the horseradish peroxidase
(HRP)-conjugated secondary antibody for 45 min at room temperature, the membranes
were washed with TBS-T another three times and incubated with enhanced chemical
luminescence detection solution to detect the intensities of the proteins using an ECL Plus
western blotting detection system. β-actin protein served as an internal control.
2.12. Statistical Analysis
Data in the present study were analyzed by GraphPad prism 9. All values are ex-
pressed as the means
±
SEM. The difference between the mean values of normally dis-
tributed data was analyzed using one-way ANOVA (Dunnett’s t-test) and two-tailed
Student’s t-test. p< 0.05 was used as the criterion for statistical significance.
3. Results
3.1. Body Temperature and Body Weight Analysis
No significant differences were observed in the body temperature of the different
groups (Table S1). However, there was a slight decrease in the ECO group (Table S2). There
was a statistically significant difference in body weight between day 1 and day 7 in all
experimental groups, except the healthy group (Figure S1).
3.2. C. butyricum and Its Culture Supernatant Alleviated Inflammatory Response of the Uterine
Tissues Induced by E. coli
Uterine morphology, H&E staining, and uterine index were used to evaluate the
protective effect of C. butyricum and its culture supernatant on endometritis induced
by E. coli in mice. On the morphological characteristics of the uterus, severe erythema
was seen in the ECO group but not in other groups (Figure 2A). In H&E staining, the
ECO group showed severe destruction of the endometrial epithelial layer and neutrophil
infiltration, but the other groups had significant remission (Figure 2B). In addition, the
uterine histopathological score also indicated that treatment groups alleviated uterine
tissue damage induced by E. coli. In addition, the histopathological scores of the VCB group
were lower than those of the AMP and SCB groups (Figure 2D). In the uterine index, the
ECO group had the highest level, and the three treatment groups (AMP, VCB, and SCB)
recovered remarkably (Figure 2C).
Animals 2022,12, 2719 6 of 13
Animals 2022, 12, x FOR PEER REVIEW 6 of 14
than those of the AMP and SCB groups (Figure 2D). In the uterine index, the ECO group
had the highest level, and the three treatment groups (AMP, VCB, and SCB) recovered
remarkably (Figure 2C).
Figure 2. C. butyricum and its culture supernatant alleviated uterine morphological and histopatho-
logical changes induced by E. coli. (A) Uterus morphology ((a) CON; (b) ECO; (c) AMP; (d) VCB; (e)
SCB). (B) H&E staining of uterine tissue ((f) CON; (g) ECO; (h) AMP; (i) VCB; (j) SCB). (C) Uterine
index. (D) Histopathologic scoring of the uterine tissue. The values presented are the means ± SEM
(n = 6). ** p < 0.01, *** p < 0.001, and **** p < 0.0001 are significantly different from ECO.
3.3. C. butyricum and Its Culture Supernatant Alleviated Pro-Inflammatory Cytokines of the
Uterine Tissues Induced by E. coli
TNF-α and IL-1β are both representative cytokines of inflammation. The results show
that the production of TNF-α and IL-1β in the uterine tissues of the VCB and SCB groups
was significantly lower than that of the ECO group. The SCB group had slightly lower
levels of inflammatory cytokines than the VCB group (Figure 3).
Figure 2.
C. butyricum and its culture supernatant alleviated uterine morphological and histopatho-
logical changes induced by E. coli. (
A
) Uterus morphology ((
a
) CON; (
b
) ECO; (
c
) AMP; (
d
) VCB;
(
e
) SCB). (
B
) H&E staining of uterine tissue ((
f
) CON; (
g
) ECO; (
h
) AMP; (
i
) VCB; (
j
) SCB). (
C
) Uterine
index. (
D
) Histopathologic scoring of the uterine tissue. The values presented are the means
±
SEM
(n= 6). ** p< 0.01, *** p< 0.001, and **** p< 0.0001 are significantly different from ECO.
3.3. C. butyricum and Its Culture Supernatant Alleviated Pro-Inflammatory Cytokines of the
Uterine Tissues Induced by E. coli
TNF-
α
and IL-1
β
are both representative cytokines of inflammation. The results show
that the production of TNF-
α
and IL-1
β
in the uterine tissues of the VCB and SCB groups
was significantly lower than that of the ECO group. The SCB group had slightly lower
levels of inflammatory cytokines than the VCB group (Figure 3).
3.4. C. butyricum and Its Culture Supernatant Reduced the Bacterial Load of the Mouse Uterus
We evaluated the effect of Clostridium butyricum and its culture supernatant on in-
trauterine bacterial load. The intrauterine bacterial load of the AMP group was significantly
lower than that of the ECO group, and the bacterial load of VCB and SCB groups also
decreased significantly (Figure 4).
3.5. C. butyricum and Its Culture Supernatant Restore Reproduction Outcome in E. coli-Induced
Endometritis Mice
The reproduction outcome indexes, including pregnancy rate, pups per litter, and
weight per pup, were measured after giving birth. None of the CON became pregnant.
Pregnancy rates in the VCB and SCB groups were higher than in the AMP group but were
lower than in the CON group. In addition, the pregnancy rate in the SCB group was higher
Animals 2022,12, 2719 7 of 13
than that in the VCB group (Figure 5A and Table S3). The pups per litter significantly
decreased in all treatment groups compared to the CON group, but there was no statistical
difference between the VCB and SCB groups (Figure 5B and Table S4). The weight per pup
in the treatment groups was lower than that in the CON group; however, there was no
statistical difference between the AMP, VCB, and SCB groups (Figure 5C and Table S5).
Animals 2022, 12, x FOR PEER REVIEW 7 of 14
Figure 3. C. butyricum and its culture supernatant reduced the production of pro-inflammatory cy-
tokines in uterine tissues induced by E. coli. (A) Uterine tissue TNF-α. (B) Uterine tissue IL-1β. The
values presented are the means ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001
are significantly different from the ECO group.
3.4. C. butyricum and Its Culture Supernatant Reduced the Bacterial Load of the Mouse Uterus
We evaluated the effect of Clostridium butyricum and its culture supernatant on intra-
uterine bacterial load. The intrauterine bacterial load of the AMP group was significantly
lower than that of the ECO group, and the bacterial load of VCB and SCB groups also
decreased significantly (Figure 4).
Figure 4. C. butyricum and its culture supernatant reduced the uterine bacterial loads. The values
presented are the means ± SEM (n = 6). * p < 0.05, *** p < 0.001, and **** p < 0.0001 are significantly
different from ECO group.
Figure 3.
C. butyricum and its culture supernatant reduced the production of pro-inflammatory
cytokines in uterine tissues induced by E. coli. (
A
) Uterine tissue TNF-
α
. (
B
) Uterine tissue IL-1
β
. The
values presented are the means
±
SEM (n= 6). * p< 0.05, ** p< 0.01, *** p< 0.001, and ****
p< 0.0001
are significantly different from the ECO group.
Animals 2022, 12, x FOR PEER REVIEW 7 of 14
Figure 3. C. butyricum and its culture supernatant reduced the production of pro-inflammatory cy-
tokines in uterine tissues induced by E. coli. (A) Uterine tissue TNF-α. (B) Uterine tissue IL-1β. The
values presented are the means ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001
are significantly different from the ECO group.
3.4. C. butyricum and Its Culture Supernatant Reduced the Bacterial Load of the Mouse Uterus
We evaluated the effect of Clostridium butyricum and its culture supernatant on intra-
uterine bacterial load. The intrauterine bacterial load of the AMP group was significantly
lower than that of the ECO group, and the bacterial load of VCB and SCB groups also
decreased significantly (Figure 4).
Figure 4. C. butyricum and its culture supernatant reduced the uterine bacterial loads. The values
presented are the means ± SEM (n = 6). * p < 0.05, *** p < 0.001, and **** p < 0.0001 are significantly
different from ECO group.
Figure 4.
C. butyricum and its culture supernatant reduced the uterine bacterial loads. The values
presented are the means
±
SEM (n= 6). * p< 0.05, *** p< 0.001, and **** p< 0.0001 are significantly
different from ECO group.
3.6. C. butyricum Culture Supernatant Attenuates E. coli-Induced Endometritis in a
Concentration-Dependent Manner
We tested the effect of different concentrations, including low (LSC), medium (MSC),
and high (HSC) concentrations of C. butyricum culture supernatant, on endometritis in
mice. In the uterine morphology photographs, the uterus of the ECO group had severe
erythema, and the uterus of the LSC and MSC groups also had local erythema (Figure 6A).
There was no difference in uterine index between the ECO, LSC, and MSC groups, but the
HSC group was lower than the ECO group (Figure 6C). H&E staining (Figure 6B) and the
uterine histopathological score (Figure 6D) in the treatment groups were dose-dependently
alleviated according to the concentration of the C. butyricum culture supernatant.
Animals 2022,12, 2719 8 of 13
Animals 2022, 12, x FOR PEER REVIEW 8 of 14
3.5. C. butyricum and Its Culture Supernatant Restore Reproduction Outcome in E. coli-Induced
Endometritis Mice
The reproduction outcome indexes, including pregnancy rate, pups per litter, and
weight per pup, were measured after giving birth. None of the CON became pregnant.
Pregnancy rates in the VCB and SCB groups were higher than in the AMP group but were
lower than in the CON group. In addition, the pregnancy rate in the SCB group was higher
than that in the VCB group (Figure 5A and Table S3). The pups per litter significantly
decreased in all treatment groups compared to the CON group, but there was no statistical
difference between the VCB and SCB groups (Figure 5B and Table S4). The weight per
pup in the treatment groups was lower than that in the CON group; however, there was
no statistical difference between the AMP, VCB, and SCB groups (Figure 5C and Table S5).
Figure 5. C. butyricum and its culture supernatant restore reproduction outcome in E. coli-induced
endometritis mice. (A) Pregnancy rate refers to the percentage of the number of pregnant mice to
the number of mated mice in each group (the number of mated mice in each group = 6 animals). (B)
Pups per litter refers to the total number of pups born in each group divided by the number of
maternal mice in the group. The number of black dots indicates the number of pups. (C) Weight per
pup is the total weight of pups in each group divided by the number of pups in that group. * p <
0.05 and ** p < 0.01 are significantly different from the CON group.
3.6. C. butyricum Culture Supernatant Attenuates E. coli-Induced Endometritis in a
Concentration-Dependent Manner
We tested the effect of different concentrations, including low (LSC), medium (MSC),
and high (HSC) concentrations of C. butyricum culture supernatant, on endometritis in
mice. In the uterine morphology photographs, the uterus of the ECO group had severe
erythema, and the uterus of the LSC and MSC groups also had local erythema (Figure 6A).
There was no difference in uterine index between the ECO, LSC, and MSC groups, but the
HSC group was lower than the ECO group (Figure 6C). H&E staining (Figure 6B) and the
uterine histopathological score (Figure 6D) in the treatment groups were dose-de-
pendently alleviated according to the concentration of the C. butyricum culture superna-
tant.
Figure 5.
C. butyricum and its culture supernatant restore reproduction outcome in E. coli-induced
endometritis mice. (
A
) Pregnancy rate refers to the percentage of the number of pregnant mice to the
number of mated mice in each group (the number of mated mice in each group = 6 animals). (
B
) Pups
per litter refers to the total number of pups born in each group divided by the number of maternal
mice in the group. The number of black dots indicates the number of pups. (
C
) Weight per pup is
the total weight of pups in each group divided by the number of pups in that group. * p< 0.05 and
** p< 0.01 are significantly different from the CON group.
Animals 2022, 12, x FOR PEER REVIEW 9 of 14
Figure 6. C. butyricum culture supernatant relieved the uterine tissue inflammatory response in-
duced by E. coli in a concentration-dependent manner. (A) Mouse uterus morphology ((a) CON; (b)
ECO; (c) LSC; (d) MSC; (e) HSC). (B) H&E staining of uterine tissue ((f) CON; (g) ECO; (h) LSC; (i)
MSC; (j) HSC). (C) Uterine index. (D) Histopathologic scoring of the uterine tissue. (E) Uterine tissue
TNF-α. (F) Uterine tissue IL-1β. The values presented are the means ± SEM (n = 3). * p < 0.05, *** p <
0.001, and **** p < 0.0001 are significantly different from ECO group.
3.7. C. butyricum and Its Culture Supernatant Inhibited the Activation of the NF-κB Signaling
Pathway Induced by E. coli
We examined the effect of the C. butyricum and its culture supernatant on the NF-κB
signaling pathway. The results showed that C. butyricum and its culture supernatant in-
hibited the expression of the p-p65 and p-IκB proteins induced by E. coli in uterine tissues
(Figure 7).
Figure 6.
C. butyricum culture supernatant relieved the uterine tissue inflammatory response induced
by E. coli in a concentration-dependent manner. (A) Mouse uterus morphology ((a) CON; (b) ECO;
Animals 2022,12, 2719 9 of 13
(
c
) LSC; (
d
) MSC; (
e
) HSC). (
B
) H&E staining of uterine tissue ((
f
) CON; (
g
) ECO; (
h
) LSC; (
i
) MSC;
(
j
) HSC). (
C
) Uterine index. (
D
) Histopathologic scoring of the uterine tissue. (
E
) Uterine tissue
TNF-
α
. (
F
) Uterine tissue IL-1
β
. The values presented are the means
±
SEM (n= 3). * p< 0.05,
*** p< 0.001, and **** p< 0.0001 are significantly different from ECO group.
3.7. C. butyricum and Its Culture Supernatant Inhibited the Activation of the NF-κB Signaling
Pathway Induced by E. coli
We examined the effect of the C. butyricum and its culture supernatant on the NF-
κ
B signaling pathway. The results showed that C. butyricum and its culture supernatant
inhibited the expression of the p-p65 and p-I
κ
B proteins induced by E. coli in uterine tissues
(Figure 7).
Animals 2022, 12, x FOR PEER REVIEW 10 of 14
Figure 7. The supernatant of C. butyricum inhibited the activation of the NF-κB signaling pathway
induced by E. coli. Western blot was used to measure p-p65 and p-IκB proteins in uterine tissues. β-
Actin was used as a control. The values presented are the means ± SEM (n = 3). * p < 0.05 and ** p <
0.01 are significantly different from ECO group.
4. Discussion
Endometritis is a common postpartum disease and has been causing great economic
loss, especially in farming dairy cows. Probiotics have already been widely used in the
prevention and treatment of diseases in humans and animals [26,27]. Its effect is not only
in digestive tract diseases but also in the prevention and treatment of many diseases such
as diabetes [28]. Several studies have reported that the uterine infusion or vaginal infusion
of probiotics is effective in the treatment of endometritis [29–31]. C. butyricum has been
shown to have potential protective or ameliorating effects on a variety of human and an-
imal diseases, including gut-acquired infections, irritable bowel syndrome, inflammatory
bowel disease, neurodegenerative disease, metabolic disease, and colonic rectal cancer
[23,32]. However, the effect of C. butyricum on endometritis has not been reported.
In this study, we examined the therapeutic effect of the probiotic C. butyricum and its
culture supernatant on E. coli-induced mouse endometritis. Endometritis is mainly caused
by postpartum pathogen infection, and one of the main pathogens is E. coli [8,19]. There-
fore, in this experiment, E. coli was used to cause endometritis. Antibiotics are currently
the most widely used clinical treatment for endometritis. It has been reported that cows
receiving an intramuscular injection of 15 mg/kg body weight amoxicillin trihydrate plus
the intrauterine infusion of 8000 mg oxytetracycline dihydrate (200 mg/mL) showed good
therapeutic results [33]. Another study reported that the intrauterine treatment of ampi-
cillin 2500 mg and cloxacillin 2500 mg plus intramuscular injection of ampicillin 6000 mg
for three consecutive days resulted in a good therapeutic effect [13]. We also evaluated
the sensitivity of the E. coli used in the modeling process to ampicillin and confirmed that
it was very sensitive to ampicillin. Therefore, we used ampicillin as the positive treatment
control. The experimental results showed that the gross changes of the uterus (erythema)
and histological damage of the endometrium (destruction of the endometrial epithelial
layer, infiltration of neutrophils) caused by E. coli were all alleviated in the treatment
groups. However, the treatment effect of the AMP group is not as good as that of VCB
and SCB groups. The uterine index was also significantly lower in the treatment groups
Figure 7.
The supernatant of C. butyricum inhibited the activation of the NF-
κ
B signaling pathway
induced by E. coli. Western blot was used to measure p-p65 and p-I
κ
B proteins in uterine tissues.
β
-Actin was used as a control. The values presented are the means
±
SEM (n= 3). * p< 0.05,
** p< 0.01
and **** p< 0.0001 are significantly different from ECO group.
4. Discussion
Endometritis is a common postpartum disease and has been causing great economic
loss, especially in farming dairy cows. Probiotics have already been widely used in the
prevention and treatment of diseases in humans and animals [
26
,
27
]. Its effect is not only in
digestive tract diseases but also in the prevention and treatment of many diseases such as
diabetes [
28
]. Several studies have reported that the uterine infusion or vaginal infusion of
probiotics is effective in the treatment of endometritis [
29
–
31
]. C. butyricum has been shown
to have potential protective or ameliorating effects on a variety of human and animal
diseases, including gut-acquired infections, irritable bowel syndrome, inflammatory bowel
disease, neurodegenerative disease, metabolic disease, and colonic rectal cancer [
23
,
32
].
However, the effect of C. butyricum on endometritis has not been reported.
In this study, we examined the therapeutic effect of the probiotic C. butyricum and
its culture supernatant on E. coli-induced mouse endometritis. Endometritis is mainly
caused by postpartum pathogen infection, and one of the main pathogens is E. coli [
8
,
19
].
Therefore, in this experiment, E. coli was used to cause endometritis. Antibiotics are
currently the most widely used clinical treatment for endometritis. It has been reported that
Animals 2022,12, 2719 10 of 13
cows receiving an intramuscular injection of 15 mg/kg body weight amoxicillin trihydrate
plus the intrauterine infusion of 8000 mg oxytetracycline dihydrate (200 mg/mL) showed
good therapeutic results [
33
]. Another study reported that the intrauterine treatment of
ampicillin 2500 mg and cloxacillin 2500 mg plus intramuscular injection of ampicillin
6000 mg for three consecutive days resulted in a good therapeutic effect [
13
]. We also
evaluated the sensitivity of the E. coli used in the modeling process to ampicillin and
confirmed that it was very sensitive to ampicillin. Therefore, we used ampicillin as the
positive treatment control. The experimental results showed that the gross changes of
the uterus (erythema) and histological damage of the endometrium (destruction of the
endometrial epithelial layer, infiltration of neutrophils) caused by E. coli were all alleviated
in the treatment groups. However, the treatment effect of the AMP group is not as good
as that of VCB and SCB groups. The uterine index was also significantly lower in the
treatment groups compared to the ECO group, but there was no difference between the
AMP and VCB or SCB groups. TNF-
α
and IL-1
β
content increases in inflamed tissues,
including endometritis [
34
]. These inflammatory cytokines were significantly lowered in
VCB and SCB groups. The intrauterine bacterial load is one of the important indicators
used to evaluate the severity of endometritis [
35
]. To examine the antibacterial effect of
C. butyricum and its culture supernatant, the intrauterine bacterial load was measured. The
results showed that the uterine bacterial load was significantly reduced not only in the
AMP group but also in the VCB and SCB groups. This indicated that C. butyricum and
its culture supernatant had an antibacterial effect. The antibacterial effect of C. butyricum
has been reported in several studies [
32
,
36
]. Our results once again proved the data of
previous studies.
The negative effect of endometritis in cows is that it causes infertility and therefore a
decrease in milk production. Therefore, the improvement of fecundity after the treatment
of this disease is an important index to evaluate the therapeutic effect of endometritis.
According to previous studies, the use of antibiotics alleviates the clinical symptoms of
endometritis to a certain extent, but there is no good effect on restoring reproduction
outcomes [
37
]. Thus, we tested whether the C. butyricum and its culture supernatant would
be effective in restoring reproduction outcomes such as pregnancy rate, pups per litter,
and weight per pup. The results showed that these reproductive outcome indicators in
the C. butyricum and its culture supernatant treatment groups were higher than those
in the ampicillin-treated group. The mechanism of C. butyricum and its culture super-
natant improving the reproductive performance of mice with endometritis needs to be
further studied.
Through the above experiment, we found that the treatment effect of C. butyricum
culture supernatant on endometritis was slightly better than that of live bacteria. We
speculated that the reason is as follows. The culture supernatant of C. butyricum contains
abundant antibacterial and anti-inflammatory substances such as short-chain fatty acids
and other metabolites. Butyric acid inhibits pathogens and improves intestine barrier func-
tion [
38
], benefits intestinal microbiota, and stimulates immune factors [
39
,
40
]. However,
the growth activity of C. butyricum in utero was relatively weak compared with that of
the medium; thus, no sufficient effective substances were synthesized in the short time
between the infusion of bacterial suspension and the collection of uteri.
Since the culture supernatant has a better effect than live bacteria and it is more
convenient to use culture supernatant than live bacteria in clinical application, we evaluated
the therapeutic effect of different concentrations of culture supernatant. The results showed
that the C. butyricum culture supernatant exhibits a therapeutic effect in a concentration-
dependent manner.
Subsequently, we conducted an experiment to explain the treatment mechanism of
C. butyricum and its culture supernatant for E. coli-induced endometritis. One of the major
mechanisms of E. coli-induced endometritis is related to the activation of the NF-
κ
B pathway
by LPS, a major pathogenic factor [
41
]. NF-
κ
B is an important transcription factor that is
localized to the cytosol and bound to its inhibitor I
κ
B
α
in an inactive state. The NF-
κ
B p65
Animals 2022,12, 2719 11 of 13
unit is dissociated and phosphorylated from I
κ
B
α
by an inducer such as bacterial LPS. The
phosphorylated NF-
κ
B is translocated into the nucleus, resulting in the activation of the
NF-
κ
B-regulated target genes, such as TNF-
α
and IL-1
β
[
42
,
43
]. Our result showed that
C. butyricum and its culture supernatant significantly inhibited the E. coli-induced activation
of the NF-κB pathway in the uterine tissue.
Although this study indicated that C. butyricum and its culture supernatant have
therapeutic effects on E. coli-induced endometritis in mice, there is a great difference
between mouse endometritis and cow endometritis; thus, further experiments in cows are
needed.
5. Conclusions
C. butyricum and its culture supernatant alleviated E. coli-induced endometritis and
restored the reproduction outcomes of the uterus in mice. The therapeutic mechanism of
C. butyricum and its culture supernatant on endometritis may be related to the inhibition of
E. coli growth and the blocking of the NF-κB signaling pathway.
Supplementary Materials:
The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/ani12192719/s1, Figure S1: Comparison of body weight of mice before and
after the experiment; Table S1: The score criterion of uterine injury score; Table S2: Body temperature;
Table S3: Pregnancy rate; Table S4: Pups per litter; Table S5: Weight per pup.
Author Contributions:
Funding acquisition, N.Z. and Y.C.; investigation, C.M. and J.C.; methodology,
X.H. and W.Z.; software, J.C.; validation, Y.C.; visualization, C.M.; writing—original draft, C.M.;
writing—review and editing, X.H. All authors have read and agreed to the published version of
the manuscript.
Funding:
This work was supported by the National Natural Science Foundation of China (No. 31772812,
32172872).
Institutional Review Board Statement:
All animal experiments were performed according to regu-
lations of the Administration of Affairs Concerning Experimental Animals in China. The protocol
was approved by the Institutional Animal Care and Use Committee of Jilin University (20170318).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The datasets generated and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Conflicts of Interest: The authors declare that they have no conflict of interest.
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