Am J Physiol Gastrointest Liver Physiol 286: G1081–G1089, 2004;
10.1152/ajpgi.00457.2003.
Medium-chain triglycerides enhance secretory IgA expression in rat intestine
after administration of endotoxin
Hiroshi Kono,1 Hideki Fujii,1 Masami Asakawa,1 Akira Maki,1
Hidetake Amemiya,1 Yu Hirai,1 Masanori Matsuda,1 and Masayuki Yamamoto2
1
First Department of Surgery, University of Yamanashi, Yamanashi 409-3898;
and 2Department of Surgery, Shinko Byoin Hospital, Hyogo 651-0072, Japan
Submitted 24 October 2003; accepted in final form 20 January 2004
saturated fat; inflammatory cytokines; endotoxemia; chemokines;
Peyer’s patch
the “motor” responsible for
multiple organ failure in critical illnesses (18). The gut mucosa
is the site in which various acute phase proteins (23, 36, 37),
gut hormones (38), and cytokines (22) are produced that affect
the mucosa as well as the function and integrity of remote
organs and tissues. Thus the role of the intestinal mucosa in
inflammatory and metabolic responses to sepsis and endotoxemia has become more apparent during the past decade.
We reported that a daily supplement of medium-chain triglycerides prevented an increase in gut permeability and intestinal injury after endotoxin administration (11, 12). We speculated that medium-chain triglycerides help protect the gut
during endotoxemia or sepsis. Proinflammatory cytokines such
as TNF-␣ are involved in triggering a vicious cycle in infec-
tious insults via the redox-sensitive transcriptional factor,
NF-␬B (14). These cytokines upregulate chemoattracting factors, which subsequently increase the number of leukocyte into
the host tissue. Thus inflammatory cytokines and chemokines
are involved in organ injury. Alternatively, secretory IgA is
synthesized within the lamina propria through the interaction
of T and B cells and then transported by overlying epithelial
cells onto the mucosal surface using secretory components.
There, it binds to specific antigens on bacteria (16, 31), viruses,
and other toxic molecules and prevents them from attaching to
the mucosal surface. It also plays a role in eliminating infectious agents that have penetrated epithelial cell layers. Intestinal secretory IgA levels correlate inversely with bacterial
overgrowth, bacterial translocation, and changes in intestinal
permeability in animal models (4). Thus secretory IgA plays an
important role in protecting against infection in the intestinal
immune system (20, 21). The purpose of this study was to
determine whether medium-chain triglycerides enhance the
production of secretory IgA and inhibit expression of inflammatory mediators in the intestine. The production of secretory
IgA is controlled by cytokine-producing T cells within the
gut-associated lymphoid tissue and possibly by cytokines released from the mucosa. T cells, which are major producers of
cytokines, are classified into two distinct subsets, Th1 and Th2,
based on the pattern of cytokines they secrete. Th1 cytokines
such as IFN-␥ and TNF-␤ downregulate IgA production,
whereas Th2 cytokines such as IL-4, IL-5, IL-6, and IL-10
upregulate IgA production (15). A balance between Th1 and
Th2 cytokines may be necessary to maintain a normal IgA
immune response. Therefore, mRNA expression of IFN-␥ and
IL-10 in the ileum and Peyer’s patches was also investigated.
THE GUT MUCOSA HAS BEEN CALLED
Address for reprint requests and other correspondence: H. Kono, First Dept.
of Surgery, Univ. of Yamanashi, 1110 Shimokato, Tamaho, Nakakoma,
Yamanashi 409-3898, Japan (E-mail: [email protected]).
http://www.ajpgi.org
MATERIALS AND METHODS
Animals and treatments. Male Sprague-Dawley rats (250 –300 g
body wt; Japan SLC, Shizuoka, Japan) were used in this study. They
were housed under barrier-sustained conditions and allowed to recover for at least 5 days after arrival before being used. During this
acclimation period, rats were fed Purina rat chow (cat. no. 5001;
Dyets, Bethlehem, PA). The experimental protocol followed the
institutional and the National Research Council criteria for the care
and use of laboratory animals in research. Furthermore, all rats
received humane care in compliance with institutional guidelines.
Rats were given medium-chain triglycerides (saturated fat, trioctanoin 8:0; 5 g/kg, Nihon-Yushi, Tokyo, Japan) or the same dose of
corn oil (polyunsaturated fat) by gavage daily for 1 wk (11), and
received food and water ad libitum throughout the study. Previous
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0193-1857/04 $5.00 Copyright © 2004 the American Physiological Society
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Kono, Hiroshi, Hideki Fujii, Masami Asakawa, Akira Maki,
Hidetake Amemiya, Yu Hirai, Masanori Matsuda, and Masayuki
Yamamoto. Medium-chain triglycerides enhance secretory IgA expression in rat intestine after administration of endotoxin. Am J
Physiol Gastrointest Liver Physiol 286: G1081–G1089, 2004;
10.1152/ajpgi.00457.2003.—The purpose of this study was to determine whether medium-chain triglycerides (MCTs) modulate the inflammatory immune response to LPS and enhance the expression of
secretory IgA in the rat intestine. Rats were given either corn oil or
MCTs by gavage daily for 1 wk, and LPS or saline vehicle was
administered via the tail vein. They were then killed, and serum and
sections from the gut were collected for further analysis. Western blot
analysis for secretory IgA revealed that MCTs significantly enhanced
its expression in the ileum compared with corn oil in rats administered
saline. After LPS challenge, expression of secretory IgA was decreased in the corn oil group but not in the MCTs group. The mRNA
expression of IL-6 was assessed by real-time RT-PCR, because IL-6
regulates secretory IgA in the intestine. The expression was significantly greater in the MCTs group than in the corn oil group after LPS
injection. Increases in expression of proinflammatory cytokines or
chemokines such as TNF-␣, IL-18, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 in the ileum were
significantly blunted by MCTs. In addition, the mRNA expression of
the Th2 IgA-stimulating cytokine IL-10 in the ileum and Peyer’s
patches was significantly greater in the MCTs than the corn oil group.
In contrast, the mRNA expression of the Th1 IgA-inhibiting cytokine
interferon-␥ was blunted by MCTs. As a result, intestinal injury was
significantly reduced. Therefore, MCTs protect the gut by modulating
the immune response to LPS and enhancing secretory IgA expression.
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MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY
secretory IgA. Samples of bile and intestinal crude particulate fractions were diluted in nonreducing Laemmli buffer. Extracted proteins
(20 ␮g) were separated by 100 g/l SDS-PAGE and transferred to
polyvinylidine fluoride membranes. The membranes were blocked
with Tris-buffered saline-Tween 20 containing 50 g/l skim milk and
probed with polyclonal goat anti-rat secretory IgA antibodies (ICN) or
␤-actin (Sigma) followed by horseradish peroxidase-conjugated secondary antibody when appropriate. They were then incubated with an
enhanced chemiluminescent substrate (ECL reagent; Amersham Life
Science, Arlington Heights, IL) and exposed to X-OMAT film (Eastman Kodak, Rochester, NY). Densitometric analysis of the image was
performed on a Macintosh computer using NIH image 1.54 analysis
software.
Real-time RT-PCR for mRNA expression of IL-6, TNF-␣, and IL-18
in mucosa from the ileum; IFN-␥, and IL-10 in the ileum; and Peyer’s
patches. mRNA was quantified by real-time RT-PCR (TaqMan; PE
Applied Biosystems, Foster City, CA). Real-time RT-PCR was performed by using a GeneAmp 5700 Sequence Detection System (PE
Applied Biosystems). Predeveloped TaqMan assay reagents (PE Applied Biosystems) were used for sequencing specific primers for
IFN-␥ (GenBank accession no. AF010466), IL-10 (GenBank accession no. X60675; L02926), IL-6 (GenBank accession no. M26744),
TNF-␣ (GenBank accession no. NM 012676) and IL-18 (GenBank
accession no. AJ222813). 18s rRNA was used as an internal control.
Four hours after LPS or saline injection, sections of the ileum and
Peyer’s patches near the terminal ileum were collected by using
scissors for measurement of the mRNA expression of IFN-␥ or IL-10.
Nine hours after LPS or saline administration, sections of mucosa
from the ileum were collected to measure the mRNA expression of
IL-6, TNF-␣, and IL-18. Samples were stored at ⫺80°C until assayed.
Total RNA was isolated from 25-mg pieces of liver tissue using a
RNA purification kit (Qiagen, Hilden, Germany). Reverse transcription of total RNA (2 ␮g) was performed in a final volume of 100 ␮l
containing 1⫻ TaqMan RT buffer, 5.5 mM MgCl2, 500 ␮M of each
dNTP, 2.5 ␮M random hexamers, 0.4 ⫻ 106 U/l RNase inhibitor, and
1.25 ⫻ 106 U/l multiscribe RT. cDNA samples (2 ␮l) were used for
quantitative RT-PCR (a 10-min step at 95°C, followed by 40 cycles of
15 s at 95°C, and 1 min at 60°C in the presence of specific forward and
reverse primers on a TaqMan Universal PCR Master Mix; PE Applied
Biosystems). mRNA levels were calculated by using the comparative
parameter threshold cycle (Ct) method (6) and normalized to rRNA.
To confirm the amplification specificity, the PCR products were
subjected to a melting-curve analysis.
Semiquantative RT-PCR for the mRNA expression of macrophage
inflammatory protein-2 and monocyte chemoattractant protein-1 in
the ileum. Macrophage inflammatory protein (MIP-2) and monocyte
chemoattractant protein (MCP-1) are chemoattractant factors predominantly produced by macrophages. They increase the number of
macrophages/monocytes and neutrophils recruited to the inflammatory focus and cause tissue injury. Because predeveloped primer and
probe for MIP-2 and MCP-1 were not commercially available, the
mRNA expression was assessed by semiquantitative RT-PCR. Intestinal tissue samples were collected from animals killed 4 or 9 h after
LPS or saline injections. Total RNA was isolated from 25-mg pieces
of liver tissue using a RNA purification kit (Qiagen) according to the
manufacturer’s instructions and used for PCR assay to detect the
mRNA expressions of MIP-2 and MCP-1.
Reverse transcription of total RNA was performed by using the
method described above. PCR primers for MIP-2 (10), MCP-1 (33),
and GAPDH contained the following sequences: MIP-2 sense (5⬘CAGAGCTTGAGTGTGACG-3⬘) and antisense (5⬘-TCGTACCTGATGTGCCTC-3⬘); MCP-1 sense (5⬘-TCCACCACTATGCAGGTCTC-3⬘) and antisense (5⬘-TGGACCCATTCCTTATTGGG-3⬘);
and GAPDH sense (5⬘-TGAAGGTCGGAGTCAACGGATTTGGT3⬘) and antisense (5⬘-CATGTGGGCCATGAGGTCCACCAC-3⬘).
Aliquots (5 ␮l) of synthesized cDNA were added to 45 ␮l of PCR
mix containing 5 ␮l of 10⫻ PCR buffer, 1 ␮l of each deoxynucleotide
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studies (30) with rats of the same strain, age, and weight showed that
their ad libitum intake of chow was 307 kcal 䡠 kg⫺1 䡠 d⫺1. The calorie
intake from the daily fat supplement was 45 kcal 䡠 kg⫺1 䡠 d⫺1. During
this experiment, a steady increase in weight was observed, and there
were no significant differences compared with untreated rats that were
also fed chow. No complications were observed in rats given mediumchain triglycerides. After 1 wk, rats were administered either a saline
vehicle or LPS (10 mg/kg, Escherichia coli serotype 0111:B4; Sigma,
St. Louis, MO) via the tail vein, and survival was assessed over the
following 24 h (n ⫽ 8 in each group).
Blood sampling and measurement of serum secretory IgA levels. In
separate experiments, rats were given medium-chain triglycerides (5
g 䡠 kg⫺1 䡠 d⫺1) or the same dose of corn oil for 1 wk (12). Then, either
LPS (10 mg/kg) or a saline vehicle was administered via the tail vein.
Blood samples were collected from the aorta 9 h later and centrifuged
at 1,200 g for 10 min at 4°C. Serum was stored at ⫺80°C until assayed
for secretory IgA. Serum secretory IgA was measured by an ELISA
using polyclonal goat anti-rat secretory IgA antibody (ICN, Costa
Mesa, CA), and data were corrected for each dilution.
Pathology and histological measurement. Sections of gut, liver,
and lung were collected 9 h after LPS administration and stained with
hematoxylin-eosin to assess inflammation and necrosis. Pathology
was evaluated in a blind manner by one of the authors and by an
expert in rodent pathology. Furthermore, an observer unaware of the
treatment status of the animals from which the tissues were harvested
measured 15 villi per slide under a microscope using a micrometer.
Immunohistochemistry of secretory IgA in gut. Paraffin-embedded
sections of jejunum, ileum, and colon were deparaffinized, rehydrated,
and stained immunohistochemically with polyclonal goat anti-rat
secretory IgA antibody (ICN) by sequential incubation with polyclonal antibodies (StressGen Biotechnologies, Victoria, BC, Canada)
in PBS (pH 7.4) containing 10 g/l Tween 20 and 10 g/l BSA (13).
Peroxidase-linked secondary antibody and diaminobenzidine (peroxidase envision kit; DAKO, Carpinteria, CA) were used to detect
specific binding. The slides were rinsed twice with PBS-1 g/l Tween
20 between each incubation, and sections were counterstained with
hematoxylin as described elsewhere (5). Sections from the same rats
were processed without the primary antibody and then examined
using the procedure detailed above as a control for nonspecific
binding of the secondary antibody.
Preparation of samples and immunoblot analysis of IgA. To collect
bile as a positive control (2, 8, 28), the bile duct was cannulated with
a small length of polyethylene-10 tubing, and bile samples were
collected and immediately placed on ice. Cold nonreducing Laemmli
buffer (62.5 mM Tris 䡠 HCL, pH 6.8, containing 20 g/l SDS and 0.1 l/l
glycerol) containing 12.5 mM benzamidine and 21 mM leupeptin
(Sigma) was added to the bile (9:1 vol buffer/bile). The resulting
mixture was stored at ⫺80°C.
Tissue samples of the ileum, jejunum, and colon were collected
from rats 9 h after saline or LPS administration. Intestinal tissue was
washed free of its contents using cold PBS, blotted on filter paper, and
weighed. A crude particulate fraction of the intestine was prepared
according to the method of Perez et al. (26) with some modifications.
Briefly, the organ was homogenized by using a homogenizer (Caframo, Wiarton, ON, Canada). Ten volumes of cold buffer (pH 7.4)
were used containing in mM: 10 TES (Sigma), 1 MgCl2, 12.5
benzamidine, and 21 leupeptin plus 0.2 M sucrose. A small sample of
homogenate was kept to assay the protein concentration. The remainder was centrifuged at 1,000 g for 10 min at 4°C after which the
supernatant was ultracentrifuged at 100,000 g for 60 min at 4°C. The
resulting pellet was resuspended in TES-sucrose buffer containing 10
ml/l Triton X-100, 12.5 mM benzamidine, 21 mM leupeptin, and 1
mM PMSF (Boehringer-Mannheim, Mannheim, Germany). This procedure yielded a crude particulate fraction rather than a pure membrane fraction. It was stored at ⫺80°C.
Western blot analysis of secretory IgA in the gut. Intestinal tissues
were collected 9 h after LPS or saline administration for expression of
MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY
Table 1. Effect of LPS and MCT on mortality
Saline, 1 ml/kg
Mortality
LPS, 10 mg/kg
CO
(5 mg/kg)
MCT
(5 mg/kg)
CO
(5 mg/kg)
MCT
(5 mg/kg)
0
0
100*
0†
Values are % mortality of 8 rats given 5 mg/kg of corn oil (CO) or
medium-chain triglycerides (MCT). Mortality after LPS administration was
determined as described in MATERIALS AND METHODS. *P ⬍ 0.05 compared
with rats given a saline vehicle; †P ⬍ 0.01 compared with rats given CO with
LPS by Fisher’s exact test.
RESULTS
Effect of lipid type and LPS on mortality. All animals
survived 24 h after saline administration in both groups (Table
1). However, all animals given corn oil appeared moribund 9 h
after LPS injection and died within 24 h after LPS adminis-
tration (Table 1). Mortality was absent in rats given mediumchain triglycerides.
Effect of lipid type and LPS on the gut. Complications were
not observed in rats given either medium-chain triglycerides or
corn oil for 1 wk. Furthermore, there were no significant
differences in body weight between the two groups. Morphological changes were not observed in the gut sections from rats
treated with corn oil for 1 wk after saline injection (Fig. 1). In
contrast, medium-chain triglycerides significantly increased
the length of villi (normal rats, 755 ⫾ 92 ␮m; rats given corn
oil, 762 ⫾ 82 ␮m; and rats given medium-chain triglycerides,
802 ⫾ 88 ␮m, P ⱕ 0.05). Furthermore, the number of goblet
cells in the gut also increased, which was consistent with the
results of a previous study (12). After LPS administration, the
ileum from rats given corn oil showed hemorrhagic changes in
the submucosal layer and necrosis of the epithelial cells.
However, these changes were almost completely prevented by
medium-chain triglycerides. Furthermore, liver (12) and lung
injury was also prevented by medium-chain triglycerides (data
not shown).
Effect of lipid type and LPS on serum secretory IgA levels.
Serum secretory IgA was not significantly different between
the two groups 9 h after saline administration (Fig. 2). After
LPS injection, values were decreased by ⬃40% in rats given
corn oil. However, levels increased ⬃1.5-fold in rats given
medium-chain triglycerides.
Effect of lipid type and LPS on intestinal secretory IgA
levels. Secretory IgA expression was assessed in the gut by
immunohistochemistry (Fig. 3). In rats administered saline, the
expression was minimal in each intestinal section. Although
LPS administration increased the expression in each gut sec-
Fig. 1. Effect of lipid type and LPS on the
gut. Sections of the distal ileum and colon
from rats given a saline vehicle (VEH) [A,
corn oil (CO); B, medium-chain triglycerides
(MCT)] and LPS; (C, CO; D, MCT) are
shown. Black arrows indicate submucosal
hemorrhage and white arrows indicate necrosis. Original magnification, ⫻400. Representative photomicrographs from 6 rats/group.
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(1 mM each), 0.5 ␮l of sense and antisense primers (0.15 mM), and
0.25 ␮l of DNA polymerase (Gene Amp PCR kit; Perkin Elmer Cetus,
Norwalk, CT).
The amplified PCR products were electrophoresed at 100 volts
through a 2% agarose gel (BRM, Sigma) for ⬃30 min. The gels were
stained with 0.5 mg/ml ethidium bromide Tris-borate-EDTA buffer
(ICN) and photographed with Type 55 Polaroid positive/negative film.
Densitometric analysis of the captured image was performed on a
Macintosh computer using NIH image 1.54 analysis software. The
area under the curve was normalized for GAPDH content.
Statistics. Data are expressed as the means ⫾ SE. ANOVA with
Bonferroni’s post hoc test or the Student’s t-test was used to determine significance when appropriate. A P value ⬍ 0.05 was considered
significant.
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Fig. 3. Immunohistochemical detection of sIgA in the gut. Nine hours after LPS administration, sections from jejunum, ileum, and
colon from rats given corn oil or medium-chain triglycerides were stained by using a mouse anti-rat sIgA antibody (brown staining)
as detailed in MATERIALS AND METHODS. Original magnification, ⫻200. Representative photomicrographs.
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Fig. 2. Effect of lipid type and LPS on serum secretory IgA (sIgA). Serum
samples were collected 9 h after LPS or saline vehicle administration and sIgA
was measured by ELISA as detailed in MATERIALS AND METHODS. Values are
the means ⫾ SE (n ⫽ 8). *P ⬍ 0.05 compared with rats given corn oil with
a saline vehicle; and #P ⬍ 0.05 compared with rats given medium-chain
triglycerides with vehicle by ANOVA with Bonferroni’s post hoc test.
tion in both treatment groups, it was greatest in the ileum.
Furthermore, elevated expression was observed in all intestinal
sections from rats given medium-chain triglycerides (Fig. 3).
To confirm these results, Western blot analyses of secretory
IgA and ␤-actin were performed on intestinal samples (Fig. 4,
A and B). The expression was greatest in the ileum, followed
by the colon, in untreated normal rats (Fig. 4B). Further
analysis was carried out by using samples from the ileum.
After saline injection, the expression did not change in rats
given corn oil. However, in rats given medium-chain triglycerides, it was significantly increased ⬃1.2-fold compared with
the corn oil group. Furthermore, after LPS administration, the
expression decreased significantly by ⬃60% in rats given corn
oil but not in those given medium-chain triglycerides.
mRNA expression of IL-6, TNF-␣, and IL-18 in the mucosa
of the ileum. The mRNA expression of IL-6, TNF-␣, and IL-18
was assessed in the ileum from rats treated with corn oil or
medium-chain triglycerides (Fig. 5). Nine hours after saline
administration, the mRNA expression of IL-6 was greater in
rats given medium-chain triglycerides than in those given corn
oil. Although LPS administration significantly increased the
mRNA expression of IL-6 in both groups, it was significantly
greater in rats given medium-chain triglycerides compared
with those given corn oil.
After saline administration, the mRNA expression of TNF-␣
and IL-18 was not significantly different between the two
groups. However, the expression of both cytokines increased
approximately twofold in rats given corn oil after LPS administration. The expression was significantly blunted in the medium-chain triglyceride group.
MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY
Effect of lipid type and LPS on MIP-2 and MCP-1 mRNA
expression in the ileum. After saline administration, the mRNA
expression of MIP-2 was significantly greater in the ileum of
rats given corn oil compared with those given medium-chain
triglycerides (Fig. 6). After LPS administration, the expression
significantly increased in rats given corn oil, whereas the
increase was significantly blunted in rats treated with mediumchain triglycerides.
After saline administration, the mRNA expression of MCP-1
was not detected in either group. In contrast, LPS increased
significantly the expression of MCP-1 in rats given corn oil,
and the increase was significantly blunted in the medium-chain
triglyceride group. The same results were observed in samples
from rats administered LPS after 9 h (data not shown).
mRNA expression of IFN-␥ and IL-10 in the ileum and
Peyer’s patches. The mRNA expression of Th1 IgA-inhibiting
cytokine IFN-␥ and Th2 IgA-stimulating cytokine IL-10 was
assessed in the ileum and Peyer’s patches from rats treated with
corn oil or medium-chain triglycerides (Fig. 7). Nine hours
after saline administration, the mRNA expression of IFN-␥
was significantly less in rats given medium-chain triglycerides
compared with those given corn oil. In contrast, LPS administration significantly increased expression of IFN-␥ in rats
given corn oil, and this increase was blunted by ⬃30% by
medium-chain triglycerides.
Medium-chain triglycerides significantly increased the
mRNA expression of IL-10 in the ileum but not in the Peyer’s
patches of rats administered saline. After LPS administration,
the expression did not change significantly in rats given corn
oil. However, it increased significantly ⬃1.5- to 2-fold in rats
given medium-chain triglycerides.
DISCUSSION
Effects of medium-chain triglycerides on the intestinal inflammatory immune response in endotoxemia. Macrophages
are an important part of the immunological and inflammatory
Fig. 5. The mRNA expressions of IL-6 (A), TNF-␣ (B), and IL-18 (C) in the ileum. Tissue samples were collected 4 h after LPS
or saline administration and the mRNA expression of cytokines was measured by real-time RT-PCR as detailed in MATERIALS AND
METHODS. mRNA levels were calculated by using the comparative parameter threshold cycle (Ct) method and normalized to
ribosomal RNA. Data represent the means ⫾ SE (n ⫽ 6). *P ⬍ 0.01 compared with rats given corn oil with a saline vehicle; #P ⬍
0.05 compared with rats given corn oil with LPS by ANOVA with Bonferroni’s post hoc test.
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Fig. 4. Effect of LPS and lipid type on the expression of sIgA in the gut.
Protein extracts (50 ␮g) from the gut were analyzed by Western blotting using
a mouse anti-rat sIgA antibody. A: specific bands for sIgA. B: densitometric
analysis (n ⫽ 4 in each group). *P ⬍ 0.01 compared with rats given corn oil
with a saline vehicle; #P ⬍ 0.05 compared with rats given corn oil with LPS
by ANOVA with Bonferroni’s post hoc test.
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MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY
responses. A large population of these cells resides in the
normal intestinal mucosa. In inflammatory bowel disease, the
mucosal macrophage population increases and is derived from
circulating monocytes. They are phenotypically different from
the resident population and play a major role in mediating the
mucosal inflammation (19). They predominantly secrete proinflammatory cytokines, such as TNF-␣ and IL-18, which are
involved in intestinal injury caused by inflammation (24).
IL-18 was a primary mediator of the inflammation associated
with dextran sulfate sodium-induced colitis in mice (29). In
this study, increases in the mRNA expression of TNF-␣ and
IL-18 were blunted by ⬃50% by medium-chain triglycerides
(Fig. 5). Among the inflammatory mediators expressed by
proinflammatory cytokines are chemoattractant factors, such as
MIP-2 and MCP-1. They control the nature and magnitude of
inflammatory cell infiltration to the site of inflammation, and
they are also involved in organ injury (32). Therefore, the
mRNA expression of chemokines was assessed by RT-PCR.
Medium-chain triglycerides also inhibited the mRNA expression of both MIP-2 and MCP-1 after LPS administration (Fig.
6). Thus medium-chain triglycerides may inhibit infiltration
and activation of inflammatory cells in the gut, leading to
decreases in inflammatory cytokine expression during endotoxemia.
Among the cytokines produced in the intestinal mucosa
during inflammation, IL-6 is important because of its multiple
biological effects in the intestine as well as other organs and
tissues. IL-6 is an integral part of the inflammatory response to
sepsis and endotoxemia. Under different conditions, IL-6 may
exert pro- or anti-inflammatory effects. In conditions of “uncontrolled” inflammation, high IL-6 levels contribute to morbidity and mortality (3). In a previous study, systemic IL-6
levels gradually increased up to 6 h after a lethal dose of LPS,
and this level correlated with pathophysiology and mortality
(12). Furthermore, levels increased significantly in rats given
corn oil and correlated with organ injury and mortality (12).
Medium-chain triglycerides, however, prevented these events.
In addition to the biological roles of systemic levels of IL-6, it
has important biological effects on the intestinal mucosa (27).
Mucosal levels of IL-6 regulate enterocyte acute-phase protein
synthesis (23), protein synthesis in the mucosa (35), and
intestinal secretory IgA production (1). IL-6 is also an important regulator of secretory IgA production by B cells in the
Peyer’s patches. In this study, the mRNA expression of IL-6
was enhanced by medium-chain triglycerides before and after
LPS administration (Fig. 5). Furthermore, intestinal and serum
secretory IgA increased significantly in rats given mediumchain triglycerides compared with those given corn oil after LPS
administration (Figs. 2– 4). Thus medium-chain triglycerides increase the expression of intestinal IL-6, which possibly correlates
with increases in expression of secretory IgA in the gut.
The role of intestinal secretory IgA and mucosal immunology during endotoxemia. IgA, but not IgG, antibodies against
fecal endotoxins increased significantly in patients with alco-
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Fig. 6. Effect of lipid type and LPS on the mRNA expression of
macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 in the ileum. Tissue samples
were collected 4 h after saline or LPS administration, and the
mRNA expression of chemokines was measured by semiquantitative RT-PCR as detailed in MATERIALS AND METHODS. A and
B: mRNA signals expressed as a ratio to GAPDH mRNA as
measured by densitometric analysis. C: amplification of each
mRNA transcript. Samples shown are representative of 4 samples/group. Data represent the means ⫾ SE (n ⫽ 6). *P ⬍ 0.01
compared with rats given corn oil with a saline vehicle; #P ⬍
0.05 compared with rats given corn oil with LPS by ANOVA
with Bonferroni’s post hoc test.
MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY
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hol-induced liver disease (25). IgA antibodies against fecal
endotoxin correlated closely with the plasma concentrations of
alanine aminotransferase, ␥-glutamyl transferase, and C-reactive protein in patients with alcoholic liver disease. Because
medium-chain triglycerides increased serum and intestinal secretory IgA (Figs. 2– 4) and prevented intestinal (Fig. 1) and
liver injury (12) after LPS administration, elevated production
of secretory IgA by medium-chain triglycerides contributes to
the inactivation of endotoxin. Also, secretory IgA located in
body tissue was shown to suppress the inflammatory process,
thereby reducing its damaging effects on multiple organs (25).
Peyer’s patches are the principal sites responsible for sensitization of secretory IgA. In humans, most of the Peyer’s
patches are located in the distal small intestine, which contains
microflora similar to the colon due to backwash from the colon
through the ileocecal valve (25). The Peyer’s patches were also
predominantly located in the distal ileum in rats in this study
(data not shown). mRNA expression of Th2 IgA-stimulating
anti-inflammatory cytokines such as IL-10 was significantly
induced in this region by glutamine after LPS administration
(7). These cytokines stimulate IgA production in vitro by cells
of the gut-associated lymphoid tissue. Thus intestinal secretory
IgA production is associated with the expression of Th2 IgAstimulating cytokines (17). The mRNA expression of IL-10
was significantly increased in the ileum and Peyer’s patches in
rats given medium-chain triglycerides compared with those
given corn oil after LPS administration (Fig. 7). Furthermore,
the mRNA expression of the Th1 IgA-inhibiting cytokine
INF-␥ also decreased in rats given medium-chain triglycerides.
Therefore, the Peyer’s patches play an important role as an
intestinal lymphoid organ in endotoxemia. Medium-chain triglycerides also affect the expression of secretory IgA, which
possibly reduces the inflammatory immune response against
endotoxin in the gut.
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Fig. 7. The mRNA expressions of IFN-␥ (A, C) and IL-10 (B,
D) in the ileum (C, D) and Peyer’s patches (A, B). Tissue
samples were collected 4 h after LPS or saline vehicle administration and the mRNA expression of cytokines was measured
by RT-PCR as detailed in MATERIALS AND METHODS. The mRNA
levels were calculated by using the comparative Ct method and
normalized to ribosomal RNA. Data represent the means ⫾ SE
(n ⫽ 6). *P ⬍ 0.01 compared with rats given corn oil with a
saline vehicle; #P ⬍ 0.05 compared with rats given corn oil
with LPS by ANOVA with Bonferroni’s post hoc test.
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MEDIUM-CHAIN TRIGLYCERIDES IMPROVE INTESTINAL INJURY
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Clinical implications. It is proposed that medium-chain
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