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cecal, mice, gnotobiotic, activity, that, mucosa., Veterinary, Tokyo, microflora, mucosa, intestinal, mouse, with, Japan, Life, Animal, Science, 180–8602,, 1–1–1, University,, Tokyo,, University, 1–7–1, 2)Laboratory, Kyonan-cho,, Sciences,, Agricultural, School, Graduate, Health,


NOTE Laboratory Animal Science
Screening for Intestinal Microflora Influencing Superoxide Dismutase Activity
in Mouse Cecal Mucosa
Yuu DOBASHI1), Kikuji ITOH 2), Atsushi TOHEI1) and Hiromi AMAO1)*
1)Laboratory of Experimental Animal Science, Nippon Veterinary and Life Science University, 1–7–1 Kyonan-cho, Musashino,
Tokyo 180–8602, Japan
2)Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
1–1–1 Yayoi, Bunkyo-ku Tokyo 113–8657, Japan
(Received 26 June 2013/Accepted 30 October 2013/Published online in J-STAGE 13 November 2013)
ABSTRACT. We have suggested that intestinal microflora reduces the activity of the antioxidant enzyme superoxide dismutase (SOD) in the
mouse cecal mucosa. In this study, gnotobiotic mice were used to examine the species of intestinal microflora influencing SOD activity
in the cecal mucosa. The total SOD activity in the cecal mucosa of each germ-free (GF), gnotobiotic mouse with Escherichia coli, Lactobacillus and Bacteroides was significantly higher than that in the cecal mucosa of gnotobiotic mice with chloroform-treated feces (CHF),
conventionalized (CVz) mice and conventional (CV) mice (P<0.05). In addition, CuZnSOD mRNA expression showed similar tendencies.
Our results suggest that the antioxidant defense status in the cecal mucosa is influenced by CHF inoculation.
KEY WORDS: cecal mucosa, chloroform-treated feces, germ-free, superoxide dismutase.
doi: 10.1292/jvms.13-0329; J. Vet. Med. Sci. 76(3): 453–456, 2014
Antioxidant enzymes play an important role in preventing oxidative stress by acting on reactive oxidative species
(ROS) generated in vivo. While physiological concentrations of ROS in vivo have beneficial effects involving cell
signaling pathways and the killing of invading pathogens,
the unbalanced and elevated levels of ROS may contribute
to the development of various diseases, such as cancer,
hypertension, diabetes, atherosclerosis, inflammation and
premature aging [18]. Superoxide dismutases (SODs) [11]
are the most important antioxidant enzymes in the antioxidant defense system against ROS of superoxide (O2•−). At
present, three distinct SOD isoforms have been identified in
mammals. One SOD isoform has Cu and Zn in its catalytic
center (CuZnSOD) and exists in the intracellular cytoplasmic compartment; the second isoform has manganese (Mn)
in its catalytic center (MnSOD) and exists in the mitochondria of aerobic cells; and the third isoform is an extracellular
SOD [18]. SODs are the first line of defense for dismutation
of excess O2•−, which causes tissue disorders, because SODs
convert O2•− to molecular oxygen and H2O2 [16]. Of the different SOD enzymes, CuZnSOD is the most abundant and
widely distributed enzyme in many tissues [10].
We have reported that upregulation of total SOD and
CuZnSOD activities and CuZnSOD mRNA expression occurs in the mucosa of germ-free (GF) mice [3, 4]. In addition, total SOD and CuZnSOD activities in the duodenal,
jejunal, ileal, cecal and colonic mucosae of GF mice were
significantly higher than those in the mucosa of conventional
(CV) mice [4]. Consistent with these results, the total SOD
activity in conventionalized (CVz) mice decreased to the
level of the total SOD activity observed in the ceca of CV
mice [3]. These results suggested that the antioxidant defense
system in the five sites of intestinal mucosa is influenced by
intestinal microflora, which downregulates SOD activity [3,
4]. Screening for specific intestinal microflora influencing
SOD activity in the mouse cecal mucosa is critical for understanding this event. In this study, we examined the intestinal
microflora influencing SOD activity and CuZnSOD mRNA
expression in the cecal mucosa using gnotobiotic mice.
All experiments were performed using 9-week-old male
GF or CV IQI mice, which were bred in our animal facility
and originated from CLEA Japan, Inc. (Tokyo, Japan). GF
mice were maintained under GF conditions in flexible vinyl
isolators. Cages, bedding and water for GF mice were sterilized either in an autoclave or with chlorine dioxide (Exspor;
Alcide Co., Redmond, WA, U.S.A.). A commercial diet sterilized with 50-kGy gamma irradiation (CMF; Oriental Yeast
Co., Ltd., Tokyo, Japan) and water were provided ad libitum
to all mice. CV mice were kept in clean racks and were confirmed to be free of serum antibodies against Clostridium
piliforme, Mycoplasma pulmonis, HVJ and MHV. They were
also negative for Pseudomonas aeruginosa, Salmonella
spp., Pasteurella pneumotropica, Citrobacter rodentium,
Corynebacterium kutscheri, Mycoplasma spp., Dermatophytes, Giardia spp., Spironucleus muris and Syphacia spp.
GF and CV mice were maintained in the same room under
controlled conditions of temperature (23–25°C), relative humidity (40–70%) and light (12 hr, 7:00–19:00 hr). Mice were
treated according to the provisions for animal welfare of the
Nippon Veterinary and Life Science University, which fol*CorrespondenCe to: Amao, H., Laboratory of Experimental
Animal Science, Nippon Veterinary and Life Science University,
1–7–1 Kyonan-cho, Musashino, Tokyo 180–8602, Japan.
e-mail: amao@nvlu.ac.jp
©2014 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-ncnd) License .
lows the Guidelines for Animal Experimentation issued by
the Japanese Association for Laboratory Animal Science [9].
Gnotobiotic mice were produced by the inoculation
of Escherichia coli E-17, Lactobacillus, Bacteroides or
chloroform-treated feces (CHF) into GF IQI mice. The fact
that GF animals have enlarged ceca is well known [8], and
the cecal size of GF mice has been reported to be shrunk
to less than that of CV mice by inoculation with a mixture
of clostridia obtained from the CHF of CV mice [6, 13].
Clostridium species of CHF are dominant in the mouse intestine, and they play an important role in morphological and
physiological normalization of ceca. Because the CHF was
viable but non-culturable (VNC) bacteria, murine CHF was
prepared from freshly voided feces of CV IQI mice. A 1:100
fecal suspension was prepared with anaerobic trypticase soy
broth without dextrose (TSB) (BBL, Sparks, MD, U.S.A.)
supplemented with 0.5% of agar, 79 mM of Na2CO3 and 33
mM of L-cysteine·HCl·H2O−1, adjusted to pH 7.2. Chloroform was added to the suspension at a final concentration
of 3%. After vigorous shaking and incubation at 37°C for 1
hr, chloroform was evaporated by CO2 gas bubbling [6, 13].
The culture of Lactobacillus(6.0 × 107 CFU/300 μl/mice) [L.
acidophilus strain 129 (L. johnsonii), L. murinus strain 91
and, L. fermentum strain 106 (L. reuteri)] [7], Bacteroides
(3.6 × 108 CFU/300 μl/mice) [B. vulgatus 1 strains and B.
acidifaciens 6 strains isolated from the cecum of mice] [12]
or CHF (300 μl/mice) was orally inoculated into GF mice
that had previously been administered E. coli E-17 (3.6 ×
108 CFU/300 μl/mice). CVz (1:100 fecal/300 μl/mice) mice
monoassociated with E. coli E-17 (3.6 × 108 CFU/300 μl/
mice) for 2 days were inoculated through the fresh feces
of CV IQI mice. At 3 weeks after inoculation, these mice
were sacrificed by decapitation, and the cecal mucosa was
rapidly frozen in liquid nitrogen followed by storage at
−80°C until assay. Similar treatments were performed on
12-week-old GF and CV IQI mice for control of gnotobiotic
mice. Samples were homogenized by sonication in a sucrose
buffer solution (10 mM Tris–HCl, pH 7.4, 0.25 M sucrose
and 1 mM EDTA). The total SOD activity was determined
in the supernatant obtained after centrifugation at 20,000 g
for 20 min at 4°C using the SOD Assay Kit-WST (Dojindo
Molecular Technologies Ltd., Kumamoto, Japan). First, the
SOD concentration (U/ml) that produced 50% inhibition of
the WST reaction was determined (IC50) using a standard
SOD concentration (MP Biomedical, LLC, Solon, OH,
U.S.A.). Following this, the dilution rate of the mouse cecal
mucosal extract that established IC50 was determined, and
the unit concentration (U/ml) of the extract was calculated.
Each sample was analyzed in duplicates, and the results were
expressed as enzyme activity per mg protein. The protein
concentration was determined by the Bradford method [2]
using bovine serum albumin as the standard.
Total RNA was extracted from frozen cecal mucosa using
Trizol reagent (Invitrogen Co., Carlsbad, CA, U.S.A.). The
amount of total RNA was measured by spectrophotometry.
Total RNA (500 ng) was reverse transcribed in a solution of
10 µl of 1 × PrimeScript buffer, 25 pmol of oligo dT primer,
50 pmol of random 6-mer primer and 0.5 µl of PrimeScript
RT Enzyme Mix 1 at 37°C for 15 min (Takara Bio Inc., Otsu,
Japan). The reaction product was subjected to quantitative
real-time polymerase chain reaction (PCR) performed following the instructions for the 7500 Real-Time PCR System
(Applied Biosystems, Foster City, CA, U.S.A.). After cDNA
denaturation at 95°C for 30 sec, PCR was performed according to the following thermal cycling protocol: 95°C for 5
sec and 60°C for 34 sec in 20 µl of buffer containing SYBR
Premix Ex Taq (Takara Bio Inc.) and 0.8 µM each of forward
CuZnSOD (5′-GGGTTCCACGTCCATCAGT-3′) and reFig. 1. Comparison of total SOD activities in cecal mucosa of gnotobiotic mice or GF, CVz and
CV mice. Each group of animals consisted of 4–8 male animals. Values represent the means
± SD. ■: GF animals; : gnotobiotic animals; : CVz animals; □: CV animals. ab: With
significant difference between different marks P<0.05.
primers [3, 4]. The cDNA sequence was obtained from GenBank (accession number, NM_011434), and forward and reverse primers were designed to span different exons to avoid
genomic DNA amplification. Quantitative measurements
were performed by establishing a linear amplification curve
from serial dilutions of cloned mouse CuZnSOD mRNA
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA [14]. The amount of CuZnSOD mRNA was normalized to the amount of GAPDH mRNA.
The total bacterial count in the cecal content of 3 weeks
after inoculation E.coli-, Lactobacillus- or Bacteroidesinoculated mice was not significantly different compared
with those in CVz IQI mice (data not shown). The total SOD
activity in the cecal mucosa of GF and E. coli-, Lactobacillus- or Bacteroides-inoculated mice was significantly higher
than that in the cecal mucosa of CHF-treated, CVz and CV
mice (P<0.05, Fig. 1). In addition, CuZnSOD mRNA expression in the cecal mucosa of GF and E. coli-treated mice
was significantly higher than that in the cecal mucosa of
CHF-treated and CV mice (P<0.01, Fig. 2). These results
clearly demonstrate that the total SOD activity and CuZnSOD mRNA expression in the cecal mucosa of CHF-treated
mice were significantly lower than those in the cecal mucosa
of GF mice. In the cecal mucosa of CHF mice, the total SOD
activity and CuZnSOD mRNA expression decreased to the
level of the values observed in CV mice. These findings
are in agreement with the results of our previous report on
the total SOD activity of the entire cecal body in CVz mice
[3]. The observation that GF animals have enlarged ceca
is well known [8]; however, the enlarged ceca are reduced
in size in GF mice inoculated with a mixture of clostridia
obtained from CHF of CV mice [6, 13]. Similar results were
obtained in this study (data not shown), although the interaction between SOD activity and cecal size remains unknown.
Thus, our data suggest that SOD activity of the mouse cecal mucosa is influenced by CHF of colonization, i.e., CHF
downregulates SOD activity of the mouse cecal mucosa. On
the other hand, the mechanism by which bacterial colonization, components or metabolite suppresses SOD activity in
the cecal mucosa remains unclear.
The phenotypes of intraepithelial lymphocytes in GF mice
were changed to those in CV mice after the inoculation of
CHF [15]. Clostridia, which are one of the most prominent
gram-positive and spore-forming bacteria indigenous to the
murine gastrointestinal tract [13], are major components
of CHF. Moreover, Clostridium clusters IV and XIVa (also
known as the C. leptum and coccoides groups, respectively)
have been implicated in the maintenance of mucosal homeostasis and prevention of inflammatory bowel disease
(IBD) [5, 17]. In a recent study, Clostridium spp. induced
to expand Foxp3+ regulatory T-cells (Treg) [1]. Identifying
these metabolites and the molecular mechanisms underlying the CHF–cecal mucosa crosstalk will provide invaluable
information toward understanding how the gut microbiota
regulates antioxidant enzymes and may suggest potential
therapeutic options for treating IBD.
In conclusion, we suggest for the first time that SOD
activity in the cecal mucosa is downregulated by CHF inoculation.
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Fig. 2. Comparison of CuZnSOD mRNA expression in cecal mucosa of gnotobiotic mice or GF
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means ± SD. ■: GF animals; : gnotobiotic animals; □: CV animals. ab: With significant
difference between different marks P<0.01.
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