Phosphodiesterase inhibitors prevent NSAID enteropathy
independently of effects on TNF-
release
Brian K.
Reuter and
John L.
Wallace
Department of Pharmacology and Therapeutics, Faculty of
Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
 |
ABSTRACT |
Although the
ability of nonsteriodal anti-inflammatory drugs (NSAIDs) to injure the
small intestine has been well established in humans and animals, the
mechanism involved in this type of injury has yet to be elucidated. The
cytokine tumor necrosis factor-
(TNF-
) has recently been
demonstrated to play a critical role in the pathogenesis of
NSAID-induced gastric damage. We therefore assessed the possibility
that TNF-
is similarly involved in the pathogenesis of NSAID-induced
small intestinal injury. Administration of multiple doses
(n = 4) of diclofenac, but not a
single dose, resulted in profound macroscopic damage in the intestine
and significantly increased levels of TNF-
in intestinal tissue and
bile. Pretreatment of rats with a phosphodiesterase inhibitor,
pentoxifylline, theophylline, or rolipram, significantly attenuated the
macroscopic intestinal ulceration produced by diclofenac
administration. However, inhibition of TNF-
release with thalidomide
or immunoneutralization with a polyclonal antibody directed against
TNF-
failed to afford any protection. These results suggest that the
cytokine TNF-
does not play a critical role in NSAID-induced small
intestinal injury. Therefore, phosphodiesterase inhibitors mediate
their protective effect through a mechanism independent of TNF-
synthesis inhibition.
ulcer; inflammation; enteritis; cyclooxygenase; nonsteroidal
anti-inflammatory drugs; tumor necrosis factor-
 |
INTRODUCTION |
the ability of nonsteroidal anti-inflammatory drugs
(NSAIDs) to cause ulceration of the stomach is well known. However, in the past two decades it has become apparent that these drugs also have
the ability to produce extensive damage in the small intestine. Indeed,
the incidence of NSAID-induced small intestinal injury has been
suggested to be equivalent to, if not greater than, that of NSAID
gastropathy. Bjarnason and colleagues (5) estimated that 60-70%
of chronic NSAID users exhibit enteropathy, which is generally
characterized by low-grade blood and protein loss. Additional studies
conducted in rheumatoid arthritis patients using NSAIDs demonstrated
that ~40% had small intestinal lesions or ulcers (24, 25).
Furthermore, the incidence of lower bowel hemorrhage or perforation was
twofold greater in patients taking anti-inflammatory drugs than in
patients receiving other types of drugs (19).
Although research has been conducted to determine potential factors
responsible for NSAID-induced small intestinal injury, the mechanism
has yet to be elucidated. The majority of knowledge has been obtained
primarily through the use of animal models. It is likely that the
initial injury produced by NSAIDs is due to the topical irritant
properties, and in the longer term this injury is exacerbated by
enteric bacteria (26, 39). The role of enteric bacteria in
NSAID-induced small intestinal injury has been substantiated by the
observations that NSAID administration results in increased enteric
bacterial numbers within the lumen of the gut and that the
administration of broad-spectrum antibiotics reduces the severity of
damage (17, 26, 39). Furthermore, germ-free rats have been found to be
less susceptible to NSAID-induced injury than rats raised in
conventional housing conditions (23, 28). The mechanism responsible for
the initial intestinal injury has not been determined, but the topical
irritant properties of NSAIDs and recurrent exposure of the intestinal
mucosa to the drug through enterohepatic circulation are likely to be
important. Evidence in support of this hypothesis is as follows:
1) NSAIDs that do not undergo
enterohepatic circulation were found not to cause small intestinal
injury (21, 26); 2) interruption of enterohepatic circulation by bile duct ligation or by cholestyramine administration significantly attenuated intestinal injury (6, 37, 39);
and 3) when isolated intestinal
segments (Thiry loops) were created and an NSAID was administered, the
loops were spared of damage, whereas the anastomosed intestine was not
(6). However, Yamada et al. (39) suggested that the presence of an
NSAID in the lumen of the intestine is not in itself sufficient to
produce damage. They found that the NSAID must be associated with bile or a component found in bile in order to be toxic to cultured intestinal epithelial cells (39).
Jackson and colleagues (14, 15) demonstrated that the cytokine tumor
necrosis factor-
(TNF-
) is a constitutive protein found in bile
of various species, including rats. TNF-
may represent the biliary
component that helps potentiate NSAID-induced small intestinal injury.
TNF-
has been demonstrated to play a critical role in experimental
NSAID gastropathy (2, 32), and recently a correlation between increased
TNF-
levels and indomethacin-induced small intestinal injury has
been demonstrated (4). We therefore wished to determine whether TNF-
plays an important role in mediating NSAID-induced small intestinal
injury. We also wanted to establish whether bile is the key source of
TNF-
and, if so, whether TNF-
represents the component of bile
that combines with NSAIDs to produce the initial injury in the small
intestine after NSAID administration. To answer these questions, we
examined the effect of various TNF-
inhibitors, including
phosphodiesterase inhibitors and thalidomide, and an anti-TNF-
antibody on NSAID-induced small intestinal injury. We also determined
the effect of NSAID administration on bile and small intestinal tissue
levels of TNF-
.
 |
MATERIALS AND METHODS |
Male Wistar rats (250-300 g) were obtained from Charles River
Breeding Farms (Montreal, PQ, Canada) and fed standard rodent chow ad
libitum. All experimental procedures were approved by the Animal Care
Committee of the University of Calgary.
NSAID-induced small intestinal injury.
Small intestinal injury was induced by orogastric administration of
diclofenac (10 mg/kg, n
10).
Diclofenac was administered at 12-h intervals for 2 days (i.e., a total
of 4 doses). The NSAID was initially dissolved in DMSO (5% by final
volume) and then diluted in 0.5% carboxymethylcellulose. Twelve hours
after the final dose of diclofenac, the rats were killed by cervical
dislocation and the entire small intestine was removed. The intestine
was opened along the antimesenteric border and scored for damage by an
observer unaware of the treatment the rats had received. Scoring consisted of measuring the area of all the ulcers with digital calipers
and summing the areas to give a final damage score. We previously
showed by light microscopy that the damage produced in this model
consists of ulcers that penetrate through the muscularis mucosae (26).
To determine the effects of TNF-
blockade on diclofenac-induced
small intestinal injury, rats were pretreated with various inhibitors
of TNF-
synthesis 30 min before each dose of diclofenac. Groups of
rats (n = 4-7) received an
intraperitoneal injection of pentoxifylline (50, 100, or 200 mg/kg),
theophylline (50 mg/kg), rolipram (0.3, 1, or 3 mg/kg), or thalidomide
(10, 20, or 50 mg/kg) 30 min before each dose of diclofenac. All the
drugs have previously been shown to inhibit TNF-
release and/or
synthesis to prevent NSAID-induced gastrointestinal injury at the doses
used (2, 20, 29).
To further examine the role of TNF-
in diclofenac-induced small
intestinal injury, rats (n = 5) were
pretreated with 1.5 ml/kg ip of rabbit anti-recombinant mouse TNF-
antiserum. The antiserum has previously been shown to immunoneutralize
rat TNF-
at the dose used (2, 7, 9, 11). Control rats
(n = 4) received normal rabbit serum
(NRS), which was heat inactivated (56°C for 1 h) and adjusted to a
protein concentration equivalent to that of the anti-TNF-
antiserum.
The anti-TNF-
antibody or NRS was administered 4 h before each dose
of diclofenac (4 doses at 12-h intervals), and the rats were killed 12 h after the final dose of diclofenac. Small intestinal damage was
assessed as described above.
Bile and tissue levels of TNF-
.
Groups of rats (n = 4-7) received
a single dose or multiple doses (4 doses at 12-h intervals) of vehicle,
diclofenac (10 mg/kg), or nitrofenac (15 mg/kg). Nitrofenac is a nitric
oxide (NO)-releasing derivative of diclofenac that we previously showed
did not produce gastrointestinal injury, although it maintained its
inhibitory effects on cyclooxygenase (26, 36). Nitrofenac was used to permit a comparison between ulcerogenic and nonulcerogenic NSAIDs and
their effects on bile and tissue levels of TNF-
. Three hours after
the final dose of vehicle or test drugs, the rats were anesthetized with pentobarbital sodium and the common bile duct was ligated with
polyethylene tubing (PE-10, Clay Adams, Parsipany, NJ). Bile was
collected for 1 h and then stored at
70°C for subsequent determination of TNF-
levels by a commercially available ELISA kit
(Cytoscreen, Biosource, Camarillo, CA).
After a treatment protocol identical to that described above, groups of
rats (n = 5-11) were killed by
cervical dislocation 3 h after the final dose of vehicle or test drug
and the entire small intestine was removed. Tissue samples (~100
mg/sample) were obtained from five regions:
1) ligament of Treitz,
2) one-fourth the distance to the
ileocecal junction, 3) one-half the
distance to the ileocecal junction,
4) three-fourths the distance to the ileocecal junction, and 5) 10 cm
proximal to the ileocecal junction. Samples were immediately frozen in
liquid nitrogen and stored at
70°C. Samples were prepared
for determination of TNF-
levels following a method similar to that
described by Ribbons et al. (27). Briefly, frozen samples were ground
to a fine powder with a mortar and pestle prechilled with liquid
nitrogen. Isotonic saline was then added to the ground tissue (1 µl/mg wet tissue wt), and the suspensions were mixed on a vortex. The
samples were centrifuged at 10,000 g
for 20 min at 4°C. Supernatants were collected and stored at
70°C until TNF-
and protein concentration levels were determined.
Inhibition of TNF-
synthesis/release.
The ability of the phosphodiesterase inhibitors (pentoxifylline,
theophylline, and rolipram) and thalidomide to inhibit TNF-
synthesis/release in vivo was determined by stimulating TNF-
production in rats via administration of lipopolysaccharide (LPS). LPS
(5 mg/kg ip, Escherichia coli serotype
0127:B8) was administered to rats (n = 4-14/group) 30 min after the administration of vehicle (DMSO or
saline), pentoxifylline (50 or 200 mg/kg), theophylline (50 mg/kg),
rolipram (0.3, 1, or 3 mg/kg), or thalidomide (10, 20, or 50 mg/kg).
Three hours after LPS the rats were anesthetized with pentobarbital
sodium and 1 ml of blood was drawn from the descending aorta into a
syringe containing sodium citrate (100 µl of 3.8% wt/vol in saline).
The blood was transferred to plastic Eppendorf tubes and centrifuged at
16,000 g for 2 min. Supernatants were
collected and stored at
70°C for subsequent determination of
plasma TNF-
levels by ELISA.
TNF-
mRNA expression.
TNF-
mRNA expression was measured using RT-PCR. Samples of the small
intestine (full thickness) were taken from rats treated with a single
administration of LPS (5 mg/kg) or with a single dose or multiple doses
of vehicle or diclofenac (10 mg/kg). In rats that received multiple
doses of diclofenac to induce intestinal injury, tissue samples were
obtained from sites that exhibited macroscopically visible damage.
Intestinal samples were obtained from a similar location in the rats
without macroscopic damage (i.e., rats treated with vehicle, LPS, or a
single dose of diclofenac). The tissue samples were immediately frozen
in a 50% (wt/vol) guanidinium solution containing 26.4 mM sodium
citrate (pH 7.0), 0.528% sarcosyl, and 0.0072%
-mercaptoethanol.
For each 100 mg of tissue, 1 ml of the guanidinium solution was used.
Total RNA was isolated using the acid guanidinium isothiocyanate
method, as described previously (8).
RT-PCR was carried out following a previously established method (11).
TNF-
RT-PCR products were made using primers designed according to
the published rat TNF-
sequence (18). The TNF-
primer sequences
were as follows: 5'-CCA CCA CGC TCT TCT GTC TAC T-3'
(upstream) and 5'-CCA CAC TTC ACT TCC GGT TCC T-3'
(downstream). The expected length of this PCR product was 1,000 bp. The
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RT-PCR product was
made using primers described previously (38). Cycle tests indicated
that amplification of TNF-
with GAPDH was optimal if the
TNF-
gene was amplified for 31 cycles and the GAPDH gene was
amplified for 18 cycles (data not shown). GAPDH upstream and downstream
primers were therefore added to the PCR mixture during the hot start of cycle 14.
PCR products were run on a 1.65% agarose gel containing ethidium
bromide, and a Polaroid picture of the gel was taken under ultraviolet
light. The level of TNF-
mRNA expression was determined using a
densitometer and National Institutes of Health software. Quantities of
TNF-
were normalized according to control levels of GAPDH and
expressed as percentage of control.
Biliary excretion of diclofenac.
Bile samples were collected from rats that had been pretreated with an
intraperitoneal injection of saline or pentoxifylline (200 mg/kg) 30 min before they received a single dose or multiple (n = 4) doses of diclofenac (10 mg/kg
po). Bile was collected via cannulation of the common bile duct over a
1-h period at 1, 3, 6, and 12 h after diclofenac administration.
Samples were stored at
20°C for subsequent analysis by
reverse-phase HPLC.
Following a modified procedure that we have described previously (13),
biliary levels of diclofenac were determined by HPLC. Briefly, a
100-µl sample of bile was deconjugated by the addition of 50 µl of
2 mol/l sodium hydroxide. The sample was then neutralized by addition
of an equivalent amount of 2 mol/l hydrochloric acid. Finally, the pH
of the sample was lowered to ~4.5 by addition of 250 µl of 1 mol/l
potassium phosphate (monobasic). Mefenamic acid (25 µl, 100 µg/ml
in acetonitrile) was utilized as an internal standard. The drugs were
extracted with 1 ml of acetonitrile. Samples were then centrifuged for
30 min at 2,000 g, and the organic layer was transferred to clean glass tubes. The organic layer was
evaporated to dryness (Speed Vac, Savant Instruments, Holbrook, NY) and
then reconstituted in mobile phase consisting of acetonitrile and 0.3%
acetic acid (60:40). A 100-µl aliquot was injected onto the HPLC
system consisting of an HPLC instrument (model 1050, Hewlett-Packard,
Palo Alto, CA) with a 5-µm C18
analytical column (HP Spherisorb ODS2, 250 × 4 mm,
Hewlett-Packard). All analyses were performed at ambient temperature
and at a flow rate of 1 ml/min. Diclofenac and mefenamic acid were
measured at a detection wavelength of 275 nm and had retention times of
6 and 9 min, respectively. Concentration of diclofenac was determined
by interpolation from calibration curves. Diclofenac calibration curves
were constructed by addition of appropriate volumes of stock (100 µg/ml in methanol) or diluted stock solutions to yield final
concentrations of 1.0-100 µg/ml.
Materials.
Diclofenac sodium, mefenamic acid, pentoxifylline, and theophylline
were obtained from Sigma Chemical (St. Louis, MO). Thalidomide and
rolipram were obtained from Research Biochemicals International (Natick, MA). Nitrofenac was kindly provided by NicOx (Nice, France). The anti-TNF-
serum was kindly provided by Drs. Cory M. Hogaboam and
Steven L. Kunkel (University of Michigan, Ann Arbor, MI). All other
products were obtained from VWR Canlab (Mississauga, ON, Canada).
Statistical analysis.
Values are means ± SE. Comparison between two experimental groups
was performed using a Student's
t-test. Comparison among three or more
experimental groups was performed using a one-way ANOVA followed by a
Dunnett's multiple comparison test or a Bonferroni post hoc
test. An asociated probability
(P value) of <5% was considered significant.
 |
RESULTS |
Bile and tissue levels of TNF-
.
TNF-
levels in bile of rats treated with a single dose of diclofenac
or nitrofenac were not significantly different from those of
vehicle-treated rats (Fig. 1). However,
after multiple administrations of diclofenac, a significant increase in
the levels of TNF-
in bile was found. Diclofenac-treated rats
exhibited TNF-
levels more than twice that of the vehicle-treated
group (P < 0.01). In rats treated
with multiple doses of nitrofenac, TNF-
levels in bile were not
different from those in the vehicle group.

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Fig. 1.
Effect of nonsteroidal anti-inflammatory drug (NSAID) administration on
levels of tumor necrosis factor- (TNF- ) in rat bile. Diclofenac
(10 mg/kg) and nitrofenac (15 mg/kg; equimolar) were administered
orally at 12-h intervals, and bile was collected 3 h after a single
dose or multiple (n = 4) doses.
Results are expressed as percentage of control of vehicle-treated group
(n = 4-7/group).
 P < 0.01 vs. vehicle-treated group.
|
|
Similar results were obtained when small intestinal tissue TNF-
levels were measured (Fig. 2). Multiple
doses of diclofenac, but not nitrofenac, doubled TNF-
levels in the
small intestine compared with the vehicle group. In addition,
indomethacin (also known to produce extensive small intestinal injury
at the dose used) significantly (P < 0.01) increased tissue levels of TNF-
12 h after a single
administration of 10 mg/kg sc (192.2 ± 23.9% of control
levels).

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Fig. 2.
Effect of NSAIDs on small intestinal tissue levels of TNF- .
Diclofenac (Dicl, 10 mg/kg) and nitrofenac (Nitr, 15 mg/kg) were
administered orally at 12-h intervals for a total of 4 doses, and ileal
tissue samples were obtained 3 h after final dose. A separate group of
animals received a single administration of indomethacin (Indo, 10 mg/kg sc) 12 h before tissue collection. Data are expressed as
percentage of control of vehicle (Veh)-treated group
(n = 5-11/group).
P < 0.05;
 P < 0.01 vs. vehicle-treated group.
|
|
Small intestinal tissue TNF-
mRNA expression.
Alterations in tissue TNF-
mRNA expression were determined using
semiquantitative RT-PCR. TNF-
mRNA expression was normalized to
GAPDH mRNA expression, and results were expressed as percent control.
Figure 3 shows the TNF-
mRNA expression
for small intestinal tissue taken from rats treated with a single dose
or multiple doses of diclofenac. No changes were seen in mRNA
expression in tissue obtained from rats receiving a single dose of
diclofenac or from tissue that appeared macroscopically normal after
multiple doses of diclofenac compared with the vehicle-treated group.
Despite increased TNF-
levels in bile and tissue, no significant
changes in mRNA expression were seen in damaged tissue taken from rats treated with multiple doses of diclofenac. As a positive control, a
separate group of rats was given LPS (5 mg/kg ip), and small intestinal
tissue was collected 1 h later. LPS markedly increased small intestinal
tissue TNF-
mRNA expression over control levels (P < 0.001).

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Fig. 3.
Effect of a single dose or multiple (n = 4) doses of diclofenac (10 mg/kg) on expression of TNF- mRNA in
small intestinal tissue. Ileal tissue was collected 2 h after final
dose of diclofenac. A separate group of rats was treated with
lipopolysaccharide (LPS, 5 mg/kg ip) as a positive control, and tissue
was collected 1 h later. Results are represented as percentage of
vehicle-treated group (n = 5-14/group).
  P < 0.001 vs. vehicle-treated group.
|
|
Inhibition of TNF-
synthesis/release.
Multiple oral doses of diclofenac consistently resulted in extensive
small intestinal injury, with an average damage score of 277 ± 36 (Fig.
4A).
Rats receiving only the vehicle for diclofenac did not develop any
small intestinal damage. Pretreatment with the phosphodiesterase
inhibitors pentoxifylline and theophylline significantly attenuated the
damage produced by diclofenac (Fig. 4A). Pentoxifylline dose dependently
inhibited diclofenac-induced small intestinal injury, with a
significant reduction in damage scores at 100 and 200 mg/kg
(P < 0.05). Rats pretreated with
theophylline (50 mg/kg) also exhibited significantly less small
intestinal damage than the vehicle-treated group
(P < 0.01).

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Fig. 4.
Effect of phosphodiesterase inhibitor pretreatment on
diclofenac-induced small intestinal injury
(A) and plasma levels of TNF-
(B) after LPS administration.
Phosphodiesterase inhibitors pentoxifylline (50-200 mg/kg ip) and
theophylline (Theo, 50 mg/kg ip) were administered 30 min before
diclofenac (10 mg/kg po) or LPS (5 mg/kg ip). Values are means ± SE
(n = 4-14/group).
P < 0.05;
 P < 0.01 vs. vehicle-treated group.
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To ensure that the doses of pentoxifylline and theophylline were
sufficient to inhibit TNF-
synthesis in vivo, rats were treated with
LPS (5 mg/kg ip) to induce a significant and consistent increase in
plasma TNF-
levels. LPS administration elicited an increase in
plasma levels of TNF-
to 838 ± 254 pg/ml from plasma levels of
<10 pg/ml in untreated rats. Pentoxifylline at 200 mg/kg (225 ± 22 pg/ml, P < 0.05), but not at 50 mg/kg (583 ± 182 pg/ml), substantially attenuated
(~70% inhibition) the increase in plasma TNF-
levels induced by
LPS (Fig. 4B). Plasma TNF-
levels
in the theophylline-pretreated group were 130 ± 25 pg/ml
(P < 0.05), representing a
reduction in TNF-
levels of >80%. Thus pentoxifylline and
theophylline only reduced diclofenac-induced intestinal damage at the
doses that also significantly inhibited TNF-
synthesis.
In addition to examining the effect of pentoxifylline on LPS-induced
increases in plasma TNF-
, its ability to inhibit the increase in
bile TNF-
levels after diclofenac administration was also determined
(Fig. 5). Administration of diclofenac
significantly increased bile TNF-
levels compared with controls
(P < 0.05). Pretreatment with
pentoxifylline at 200 mg/kg, but not at 100 mg/kg, significantly
reduced the TNF-
levels (Fig. 5).

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Fig. 5.
Effect of pentoxifylline (Pentox) pretreatment on TNF- levels in
bile after diclofenac administration. Diclofenac (10 mg/kg po) was
administered at 12-h intervals for a total of 4 doses. Pentoxifylline
(100-200 mg/kg ip) was given 30 min before each dose of
diclofenac. Data are shown as percentage of control (vehicle/saline
group) and expressed as means ± SE
(n = 4-17/group).
P < 0.05 vs. vehicle/saline group.
P < 0.05 vs. diclofenac/saline group.
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Figure 6 depicts the effects of various
doses of rolipram, a specific type IV phosphodiesterase inhibitor (34),
on intestinal damage (A) and
LPS-induced plasma levels of TNF-
(B). Rolipram significantly
attenuated LPS-induced increases in plasma TNF-
at all doses tested
(Fig. 6B;
P < 0.05). Rolipram administered at
0.3, 1.0, and 3.0 mg/kg reduced plasma TNF-
levels to a similar extent (75-78%). Although all three doses of rolipram
significantly inhibited TNF-
synthesis, only the higher two doses
(1.0 and 3.0 mg/kg) protected the small intestine from
diclofenac-induced damage (Fig. 6A).

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Fig. 6.
Effect of pretreatment with type IV phosphodiesterase inhibitor
rolipram on diclofenac-induced small intestinal injury
(A) and plasma levels of TNF-
(B) after LPS administration.
Rolipram (0.3-3.0 mg/kg ip) was administered 30 min before
diclofenac (10 mg/kg po) or LPS (5 mg/kg ip). Values are means ± SE
(n = 4-14/group).
P < 0.05;
 P < 0.01 vs. vehicle-treated group.
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To further establish whether TNF-
blockade was required to afford
protection against diclofenac-induced intestinal damage, two other
studies were performed. Thalidomide is a well-characterized inhibitor
of TNF-
that is structurally unrelated to the phosphodiesterase inhibitors (31). Thalidomide, at all doses tested (10, 20, and 50 mg/kg
ip), had no protective effect in the diclofenac-induced small
intestinal injury model (Fig.
7A).
However, this compound was able to significantly inhibit LPS-induced
increases in plasma TNF-
at the higher two doses (Fig.
7B).

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Fig. 7.
Effect of thalidomide pretreatment on diclofenac-induced small
intestinal injury (A) and plasma
levels of TNF- (B) after LPS
administration. Thalidomide (10-50 mg/kg ip) was administered 30 min before diclofenac (10 mg/kg po) or LPS (5 mg/kg ip). Values are
means ± SE (n = 4-10/group).
P < 0.05 vs. vehicle-treated group.
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Similar results were obtained using an anti-TNF-
antibody (Fig.
8). Rats pretreated with the antibody
developed intestinal injury that was not significantly different, in
terms of severity, from that produced in the group pretreated with NRS.

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Fig. 8.
Effect of pretreatment with a polyclonal rabbit anti-mouse TNF-
antibody (Ab) on diclofenac-induced small intestinal injury. Polyclonal
antibody was given 4 h before each dose of diclofenac (10 mg/kg po).
Small intestinal damage was scored 12 h after final dose of diclofenac.
Values are means ± SE (n = 4-5/group).
P < 0.05;
 P < 0.01 vs. normal rabbit serum (NRS)/vehicle group; ns, not
significant.
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Biliary excretion of diclofenac.
The protection afforded by the phosphodiesterase inhibitors could have
been attributable to decreased diclofenac absorption and/or biliary
excretion. To test this hypothesis, the concentration of diclofenac in
bile was determined in rats pretreated with saline or pentoxifylline
(200 mg/kg). Pretreatment with pentoxifylline resulted in decreased
absorption of diclofenac after a single dose of drug (Fig.
9A). The
area under the curve in the pentoxifylline-pretreated group was
approximately one-half that of the saline-treated group. The major
difference in excretion profiles occurred during the first 3 h, with
the biliary levels being significantly lower
(P < 0.01) at the 3-h time point
(Fig. 9A). Although a significant difference occurred between the two groups after a single dose of drug,
this difference was not seen when bile was collected after
administration of multiple doses of diclofenac (Fig.
9B). The two diclofenac excretion
profiles were very similar, with no significant differences between the
two groups at any of the time points examined.

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Fig. 9.
Effect of pentoxifylline (200 mg/kg ip) pretreatment on biliary levels
of diclofenac after a single dose
(A) or multiple doses
(B) of NSAID. Saline or
pentoxifylline was administered 30 min before each dose of diclofenac.
Bile was collected at 1, 3, 6, and 12 h after a single dose or multiple
(n = 4) doses of diclofenac. Values
are means ± SE (n 4/time point).
 P < 0.01 vs. pentoxifylline-pretreated group.
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 |
DISCUSSION |
Santucci et al. (32) and Appleyard et al. (2) established a causal link
between TNF-
and NSAID-induced gastric damage in rats. Recently,
TNF-
has been postulated to play a role in NSAID-induced small
intestinal injury. Bertrand and colleagues (3, 4) demonstrated that
small intestinal tissue levels of TNF-
increased in rats treated
with indomethacin or flurbiprofen. In addition, no increase in small
intestinal tissue levels of TNF-
was found in animals treated with
NO-flurbiprofen, which was found not to produce small intestinal
injury. These authors also reported that the intestinal damage produced
by indomethacin could be significantly attenuated by pretreatment with
the specific type IV phosphodiesterase inhibitor RO-20-1724. From the
above evidence, Bertrand and colleagues concluded that TNF-
plays a critical role in NSAID-induced small intestinal injury.
The results of the present study confirm, to some extent, the findings
of Bertrand et al. (3, 4). We also found that rats treated with
indomethacin or diclofenac developed severe intestinal injury, which
was associated with a significant increase in small intestinal tissue
levels of TNF-
. Furthermore, administration of nitrofenac
(NO-diclofenac) did not produce small intestinal injury, as previously
shown (26), or an increase in intestinal tissue levels of TNF-
.
Finally, we demonstrated a significant attenuation of
diclofenac-induced small intestinal damage by pretreatment with the
phosphodiesterase inhibitors pentoxifylline, theophylline, and
rolipram. However, the intestine was not protected from NSAID-induced injury if the rats were pretreated with another inhibitor of TNF-
synthesis, thalidomide, or with an antibody directed against TNF-
. These results could not simply be explained by an ineffective dose of
drug, since thalidomide at 20 and 50 mg/kg was able to inhibit TNF-
synthesis induced by the administration of LPS, whereas the antibody
has previously been shown to effectively immunoneutralize TNF-
in
the rat at the dose used (2, 9, 11). Furthermore, the lowest dose of
rolipram (type IV phosphodiesterase inhibitor) was unable to attenuate
diclofenac-induced small intestinal damage, although it inhibited
TNF-
synthesis as effectively as the doses that did afford
protection (Fig. 6). We previously demonstrated (10) that rats treated
with an NO-releasing derivative of naproxen have increased plasma
levels of TNF-
but did not develop gastric or intestinal injury.
Taken together, these findings suggest that TNF-
does not play a
critical role in the pathogenesis of NSAID-induced small intestinal injury.
Our results and those of Bertrand et al. (4) suggest that the increases
in tissue levels of TNF-
occurred in parallel with the development
of damage. In the present study we saw increased levels of TNF-
in
bile and tissue only after four doses of diclofenac; that is, when
extensive intestinal damage was evident. In the study of Bertrand et
al. (4), indomethacin produced a significant increase in intestinal
tissue levels of TNF-
8 h after its administration, the same amount
of time required for a significant increase in intestinal damage to be
observed. A feasible explanation for the increase in TNF-
levels in
intestinal tissue, and possibly bile, is that the cytokine is produced
as a consequence of the damage but does not contribute to the
initiation of the damage.
Phosphodiesterase inhibitors have many effects besides inhibition of
TNF-
synthesis that might account for the beneficial effects in
experimental NSAID enteropathy. Indeed, there is renewed clinical
interest in phosphodiesterase inhibitors because of their ability to
act as vasodilators, antithrombotics, and anti-inflammatory and
immunosuppressive agents (30, 34). The vasodilatory properties of
phosphodiesterase inhibitors may be of particular relevance in the
context of NSAID enteropathy. There have been various accounts of
microcirculatory changes within the intestine of rats treated with
NSAIDs (1, 16, 23). NSAID administration has been associated with
intestinal villus shortening, decreased blood flow, and subsequent
ulceration (1, 23). It is possible that the phosphodiesterase
inhibitors are able to prevent the early ischemic event induced by
NSAID administration. Phosphodiesterase inhibitors are known to be
potent vasodilators and of clinical benefit in the treatment of
occlusive vascular diseases (30). In addition, pentoxifylline has been
shown to preserve and restore intestinal microvascular blood flow
associated with hemorrhagic shock and bacteremia (12, 33). It appears
that this effect is accomplished via a cAMP-dependent pathway. Studies
utilizing other agents (opiate antagonists or
-agonists) that
increase cAMP levels have also found protection against
indomethacin-induced small intestinal injury (1, 35).
In conclusion, TNF-
does not appear to play a critical role in the
pathogenesis of NSAID-induced small intestinal injury. Phosphodiesterase inhibitors, but not thalidomide or an anti-TNF-
antibody, were able to protect against diclofenac-induced small intestinal damage. The results of the present study also suggest that
the increase in TNF-
levels (tissue and biliary) occurs as a
consequence of the injury and the ensuing inflammatory reaction.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the Medical Research
Council of Canada (MRC). J. L. Wallace is an MRC Senior Scientist and
an Alberta Heritage Foundation for Medical Research Senior Scientist.
B. K. Reuter is supported by an MRC Studentship.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. L. Wallace,
Dept. of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1 (E-mail:
wallacej{at}ucalgary.ca).
Received 5 May 1999; accepted in final form 14 July 1999.
 |
REFERENCES |
1.
Anthony, A.,
A. K. Bahl,
I. G. Oakley,
C. F. Spraggs,
A. P. Dhillon,
M. A. Trevethick,
C. K. Piasecki,
R. E. Pounder,
and
A. J. Wakefield.
The
-adrenoceptor agonist CL316243 prevents indomethacin-induced jejunal ulceration in the rat by reversing early villous shortening.
J. Pathol.
179:
340-346,
1996[Medline].
2.
Appleyard, C. B.,
D. M. McCafferty,
A. W. Tigley,
M. G. Swain,
and
J. L. Wallace.
Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G42-G48,
1996[Abstract/Free Full Text].
3.
Bertrand, V.,
R. Guimbaud,
P. Sogni,
A. Lamrani,
C. Mauprivez,
J. P. Giroud,
D. Couturier,
L. Chauvelot-Moachon,
and
S. Chaussade.
Role of tumour necrosis factor-
and inducible nitric oxide synthase in the prevention of nitro-flurbiprofen small intestine toxicity.
Eur. J. Pharmacol.
356:
245-253,
1998[Medline].
4.
Bertrand, V.,
R. Guimbaud,
M. Tulliez,
C. Mauprivez,
P. Sogni,
D. Couturier,
J. P. Giroud,
S. Chaussade,
and
L. Chauvelot-Moachon.
Increase in tumor necrosis factor-
production linked to the toxicity of indomethacin for the rat small intestine.
Br. J. Pharmacol.
124:
1385-1394,
1998[Abstract].
5.
Bjarnason, I.,
J. Hayllar,
A. J. MacPherson,
and
A. S. Russell.
Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans.
Gastroenterology
104:
1832-1847,
1993[Medline].
6.
Brodie, D. A.,
P. G. Cook,
B. J. Bauer,
and
G. E. Dagle.
Indomethacin-induced intestinal lesions in the rat.
Toxicol. Appl. Pharmacol.
17:
615-624,
1970[Medline].
7.
Chensue, S. W.,
D. G. Remick,
C. Shmyr-Forsch,
T. F. Beals,
and
S. L. Kunkel.
Immunohistochemical demonstration of cytoplasmic and membrane-associated tumor necrosis factor in murine macrophages.
Am. J. Pathol.
133:
564-572,
1988[Abstract].
8.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
9.
Cooper, A. L.,
S. Brouwer,
A. V. Turnball,
G. N. Luheshi,
S. J. Hopkins,
S. L. Kunkel,
and
N. J. Rothwell.
Tumor necrosis factor-
and fever after peripheral inflammation in the rat.
Am. J. Physiol.
267 (Regulatory Integrative Comp. Physiol. 36):
R1431-R1436,
1994[Abstract/Free Full Text].
10.
Davies, N. M.,
A. G. Roseth,
C. B. Appleyard,
W. McKnight,
P. del Soldato,
A. Calignano,
G. Cirino,
and
J. L. Wallace.
NO-naproxen vs. naproxen: ulcerogenic, analgesic and anti-inflammatory effects.
Aliment. Pharmacol. Ther.
11:
69-79,
1997[Medline].
11.
Ferraz, J.-G. P.,
A. W. Tigley,
C. B. Appleyard,
and
J. L. Wallace.
TNF-
contributes to the pathogenesis of ethanol-induced gastric damage in cirrhotic rats.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G809-G814,
1997[Abstract/Free Full Text].
12.
Flynn, W. J.,
H. G. Cryer,
and
R. N. Garrison.
Pentoxifylline restores intestinal microvascular blood flow during resuscitated hemorrhagic shock.
Surgery
110:
350-356,
1991[Medline].
13.
Guimbaud, R.,
V. Bertrand,
L. Chauvelot-Moachon,
G. Quartier,
N. Vidon,
J. P. Giroud,
D. Couturier,
and
S. Chaussade.
Network of inflammatory cytokines and correlation with disease activity in ulcerative colitis.
Am. J. Gastroenterol.
93:
2397-2404,
1998[Medline].
14.
Hamilton, L. J.,
Y. Dai,
and
G. D. F. Jackson.
Bile regulates the expression of major histocompatibility complex class II molecules on rat intestinal epithelium.
Gastroenterology
113:
1901-1905,
1997[Medline].
15.
Jackson, G. D. F.,
Y. Dai,
C. Fung,
and
P. G. C. Hansen.
Tumour necrosis factor-
a constitutive protein of bile.
In: Advances in Mucosal Immunology, edited by J. Mestecky,
C. Blair,
and P. L. Ogra. New York: Plenum, 1995, p. 1083-1086.
16.
Kelly, D.,
C. Piasecki,
A. Anthony,
A. P. Dhillon,
R. E. Pounder,
and
A. J. Wakefield.
Reversal and protection against indomethacin-induced blood stasis and mucosal damage in the rat jejunum by
3-adrenoceptor agonist.
Aliment. Pharmacol. Ther.
12:
1121-1129,
1998[Medline].
17.
Kent, T. H.,
R. M. Cardelli,
and
F. W. Stamler.
Small intestinal ulcers and intestinal flora in rats given indomethacin.
Am. J. Pathol.
54:
237-249,
1969[Medline].
18.
Kirisits, M. J.,
D. Vardimon,
H. W. Kunz,
and
T. J. Gill III.
Mapping of the TNFA locus in the rat.
Immunogenetics
39:
59-60,
1994[Medline].
19.
Langman, M. J. S.,
L. Morgan,
and
A. Worrall.
Use of anti-inflammatory drugs by patients admitted with small or large bowel perforations and haemorrhage.
Br. Med. J.
290:
347-349,
1985[Medline].
20.
Lifrak, E. T.,
R. A. Erickson,
L. Halbur,
and
E. Quimbo.
Role of tumor necrosis factor-
(TNF-
) in NSAID-induced intestinal ulceration (Abstract).
Gastroenterology
108:
A149,
1995.
21.
Melarange, R.,
C. Gentry,
M. Durie,
C. O'Connell,
and
P. R. Blower.
Gastrointestinal irritancy, anti-inflammatory activity, and prostanoid inhibition in the rat: differentiation of effects between nabumetone and etodolac.
Dig. Dis. Sci.
39:
601-608,
1994[Medline].
22.
Melarange, R.,
G. Moore,
P. R. Blower,
M. E. Coates,
F. W. Ward,
and
V. Ronaasen.
A comparison of indomethacin with ibuprofen on gastrointestinal mucosal integrity in conventional and germ-free rats.
Aliment. Pharmacol. Ther.
6:
67-77,
1992[Medline].
23.
Miura, S.,
M. Suematsu,
S. Tanaka,
H. Nagata,
S. Houzawa,
M. Suzuki,
I. Kurose,
H. Serizawa,
and
M. Tsuchiya.
Microcirculatory disturbance in indomethacin-induced intestinal ulcer.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G213-G219,
1991[Abstract/Free Full Text].
24.
Morris, A. J.,
R. Madhok,
R. D. Sturrock,
H. A. Capell,
and
J. F. MacKenzie.
Enteropscopic diagnosis of small bowel ulceration in patients receiving non-steroidal anti-inflammatory drugs.
Lancet
337:
520,
1991[Medline].
25.
Morris, A. J.,
L. A. Wasson,
and
J. F. MacKenzie.
Small bowel enteroscopy in undiagnosed gastrointestinal blood loss.
Gut
33:
887-889,
1992[Abstract].
26.
Reuter, B. K.,
N. M. Davies,
and
J. L. Wallace.
Nonsteroidal anti-inflammatory drug enteropathy in rats: role of permeability, bacteria, and enterohepatic circulation.
Gastroenterology
112:
109-117,
1997[Medline].
27.
Ribbons, K. A.,
J. H. Thompson,
X. P. Liu,
K. Pennline,
D. A. Clark,
and
M. J. S. Miller.
Anti-inflammatory properties of interleukin-10 administration in hapten-induced colitis.
Eur. J. Pharmacol.
323:
245-254,
1997[Medline].
28.
Robert, A.,
and
T. Asano.
Resistance of germ-free rats to indomethacin-induced intestinal lesions.
Prostaglandins
14:
333-341,
1977[Medline].
29.
Salcedo, C.,
J. Puig,
J. M. Palacios,
and
A. G. Fernandez.
Phosphodiesterase 4 inhibition, a new mechanism to maintain and promote gastric and intestinal integrity against NSAID-induced injury in the rat (Abstract).
Gastroenterology
114:
A274,
1998.
30.
Samlaska, C. P.,
and
E. A. Winfield.
Pentoxifylline.
J. Am. Acad. Dermatol.
30:
603-621,
1994[Medline].
31.
Sampaio, E. P.,
E. N. Sarno,
R. Galilly,
Z. A. Cohn,
and
G. Kaplan.
Thalidomide selectively inhibits tumour necrosis factor production by stimulated human monocytes.
J. Exp. Med.
173:
699-703,
1991[Abstract].
32.
Santucci, L.,
S. Fiorucci,
M. Giansanti,
P. M. Brunori,
F. M. Di Matteo,
and
A. Morelli.
Pentoxifylline prevents indomethacin-induced acute gastric mucosal damage in rats: role of tumor necrosis factor-
.
Gut
35:
909-915,
1994[Abstract].
33.
Steeb, G. D.,
M. A. Wilson,
and
R. N. Garrison.
Pentoxifylline preserves small-intestine microvascular blood flow during bacteremia.
Surgery
112:
756-763,
1992[Medline].
34.
Thompson, W. J.
Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function.
Pharmacol. Ther.
51:
13-33,
1991[Medline].
35.
Waisman, Y.,
H. Marcus,
M. Ligumski,
and
G. Dinari.
Modulation by opiates of small intestinal prostaglandin E2 and 3',5'-cyclic adenosine monophosphate levels and of indomethacin-induced ulceration in the rat.
Life Sci.
48:
2035-2042,
1991[Medline].
36.
Wallace, J. L.,
B. Reuter,
C. Cicala,
and
G. W. McKnight.
A diclofenac derivative without ulcerogenic properties.
Eur. J. Pharmacol.
257:
249-255,
1994[Medline].
37.
Wax, J.,
W. A. Clinger,
P. Varner,
P. Bass,
and
C. V. Winder.
Relationship of the enterohepatic cycle to ulcerogenesis in the rat small bowel with flufenamic acid.
Gastroenterology
58:
772-780,
1970[Medline].
38.
Wong, H.,
W. D. Anderson,
T. Cheng,
and
K. T. Riabowol.
Monitoring mRNA expression by polymerase chain reation: the "primer-dropping" method.
Anal. Biochem.
223:
251-258,
1994[Medline].
39.
Yamada, T.,
E. Deitch,
R. D. Specian,
M. A. Perry,
R. B. Sartor,
and
M. B. Grisham.
Mechanisms of acute and chronic intestinal inflammation induced by indomethacin.
Inflammation
17:
641-662,
1993[Medline].
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