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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
-methyl-2-naphthaleneacetic Acid 4-(Nitrooxy)butyl Ester]) Interactions with Aspirin in Gastric Mucosa of Arthritic Rats Reveal a Role for Aspirin-Triggered Lipoxin, Prostaglandins, and NO in Gastric ProtectionClinica di Gastroenterologia ed Epatologia, Dipartimento di Medicina Clinica e Sperimentale, Faculty of Medicine, Università degli Studi di Perugia, Perugia, Italy (S.F., B.R., S.Fa., A.M.); and Dipartimento di Farmacologia Sperimentale, Faculty of Pharmacy, Università degli Studi di Napoli, Napoli, Italy (A.D.L., G.C.)
Received June 15, 2004; accepted July 23, 2004.
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Abstract |
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-methyl-2-naphthaleneacetic acid
4-(nitrooxy)butyl ester], a nitric oxide (NO)-releasing derivative of
naproxen, exacerbates gastric mucosal injury in arthritic rats administered
low doses of ASA. Our results demonstrated that while treating arthritic rats
with a dose of 30 mg/kg/day ASA causes detectable mucosal injury, but had no
effect on arthritis score and interleukin-6 plasma levels, coadministration of
naproxen (10 mg/kg/day) and celecoxib (30 mg/kg/day), in combination with ASA
from day 7 to day 21, attenuates arthritis development (P < 0.01
versus arthritis alone), but markedly enhanced gastric mucosal damage caused
by ASA (P < 0.01 versus ASA alone). In contrast, coadministration
of HCT-3012 (15 mg/kg/day) significantly attenuated arthritis development,
because HCT-3012 was equally or more effective than naproxen and celecoxib in
attenuating local and systemic inflammation (P > 0.001 versus
arthritis) without exacerbating gastric mucosal injury caused by ASA.
Arthritis development associates with gastric COX-2 induction, mRNA and
protein, and enhanced gastric prostaglandin E2 (PGE2)
synthesis (P < 0.01 versus control rats). Although all treatments,
including celecoxib, were effective in reducing gastric PGE2
synthesis, administering arthritic rats with ASA resulted in a significant
increase in gastric content of aspirin-triggered lipoxin (ATL), a
COX-2-derived lipid mediator that regulates proinflammatory responses at the
neutrophils/endothelial interface. Administering arthritic rats with naproxen
and celecoxib abrogates ATL formation induced by ASA although enhanced
neutrophils accumulate into the gastric mucosa (P < 0.01 versus
ASA alone). In contrast, whereas HCT-3012 inhibited ATL formation, it did not
increase neutrophil recruitment into the gastric microcirculation.
Collectively, these data indicate that HCT-3012 derived from NO has the
potential to compensate for inhibition of PGE2 and ATL and to
protect the gastric mucosa by limiting the recruitment of neutrophils. These
data suggest that HCT-3012 might be a safer alternative to nonsteroidal
anti-inflammatory drugs and coxibs in rheumatic patients that take low doses
of ASA.
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Administration of low-dose acetylsalicylic acid (ASA) to reduce the risk of
myocardial infarction and stroke is of proven efficacy
(Patrono, 1994
). Low-dose ASA
profoundly suppresses platelet thromboxane (TX)A2 biosynthesis,
although largely sparing endothelial prostacyclin generation, resulting in a
net antithrombotic effect (Patrono,
1994; Funk, 2001).
In addition to its antithrombotic activity, ASA also impacts lipoxin
(LX)-sensitive response (Claria and Serhan,
1995). Thus, acetylation of cyclooxygenase (COX)-2 by ASA modifies
its activity to generate 15R-hydroxyeicosatetraenoic acid
(15R-HETE), which can be oxygenated to produce
15-epi-LXA4, also termed aspirin-triggered LX or ATL
(Claria and Serhan, 1995;
Serhan and Oliw, 2001;
Serhan et al., 2002). Similar
to endogenous LXA4, ATL exerts potent anti-inflammatory actions
acting as a braking signal to limit neutrophil chemotaxis and transmigration
across endothelial cell layers (Serhan and
Oliw, 2001) and by inducing nitric-oxide (NO) synthesis
(Paul-Clark et al., 2004).
Local generation of ATL and NO mediate some of the anti-inflammatory
activities of ASA.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to treat pain
and inflammation in OA and RA. A major limitation of their use, however, is
the potential for severe gastrointestinal side effects, such as bleeding and
perforation (Patrono et al.,
2001). Unlike ASA that prevents arachidonic acid (AA) access to
the catalytic core of COX isoenzymes by a nonreversible modification
(acetylation) of a serine residue near the COX-active site
(Roth et al., 1975;
Lecomte et al., 1994;
Loll et al., 1995;
Mancini et al., 1997;
FitzGerald, 2003), NSAIDs
deplete platelet TXA2 formation for only a limited amount of time
and therefore do not reduce the risk of a first myocardial infarction
(Rahme et al., 2001;
Cleland, 2002). Selective COX-2
inhibitors, coxibs, a newer class of anti-inflammatory agents
(FitzGerald, 2003), spare
COX-1 and produce gastrointestinal ulcer complications at about half the rate
of conventional NSAIDs (Bombardier et al.,
2000; Silverstein et al.,
2000). However, in contrast to conventional NSAIDs, coxibs not
only are devoid of antiplatelet activity
(FitzGerald, 2003), but their
use has been linked to an increased risk of nonfatal myocardial infarction
(Bombardier et al., 2000)
raising the question of whether cardioprotection should be recommended to
patients with cardiovascular risk factors that take a coxib. Although the use
of low doses of ASA has been recommended
(Bombardier et al., 2000),
human studies suggest that coadministration of a coxib with ASA increases the
risk of gastrointestinal injury
(Silverstein et al., 2000;
Wallace et al., 2000; Fiorucci
et al., 2002,
2003b).
NO, a ubiquitous signaling molecule
(Cirino, 2003), is increasingly
recognized as a key mediator of gastrointestinal mucosal integrity. NO
protects gastric epithelial cells against injury caused by exposure to NSAIDs
in vitro (Fiorucci et al.,
1999,
2001) and attenuates
gastrointestinal injury in rodent models of NSAID gastropathy
(Lopez-Belmonte et al., 1993).
NO-releasing NSAIDs (NO-NSAIDs) are a family of anti-inflammatory drugs that
inhibit COX activities while releasing NO
(Fiorucci et al., 2001).
HCT-3012 [(S)-6-methoxy--methyl-2-naphtalene-acetic acid
4-(nitrooxy)butyl ester] is the NO-releasing derivative of naproxen, which
similar to the parent drug, inhibits formation of COX-1- and COX-2-derived
prostanoids and exerts NO-mediated activities
(Wallace and Cirino, 1994;
Wallace et al., 1994;
Cicala et al., 2000;
Muscarà et al., 2000).
Thus, in contrast to naproxen, HCT-3012 modulates T cell reactivity in a
rodent model of RA (Cicala et al.,
2000) and reduces mean arterial blood pressure in hypertensive
rats (Muscarà et al.,
1998). Furthermore, the administration of HCT-3012 to healthy
human volunteers associates with significantly fewer endoscopic lesions than
naproxen (Hawkey et al.,
2003). It is unknown whether or not HCT-3012 will be proven safe
for the gastric mucosa when administered in combination with ASA.
In the present study, we have investigated the anti-inflammatory activity and gastrointestinal safety of administering HCT-3012 in combination with low doses of ASA to rats rendered arthritic by the administration of Freund's complete adjuvant (FCA).
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Materials and Methods |
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).
Polyclonal rabbit antibody against COX-2 was from Cayman Chemical (Ann Arbor,
MI), and the donkey anti-rabbit IgG secondary antibody conjugated to
horseradish peroxidase was from Amersham Biosciences UK, Ltd. (Little
Chalfont, Buckinghamshire, UK). The IL-6 ELISA kit was from Pierce Endogen
(Rockford, IL).
Induction of Freund's Adjuvant Arthritis. Arthritis
(Pearson et al., 1961;
Cicala et al., 2000) was
induced by subcutaneous injection at the base of the tail of 100 µl of FCA
(mineral oil containing 6 mg/ml of heat-killed M. butirricum) to 7-
and 8-week-old male Lewis rats (Harlan Nossan, Milan, Italy). Seven days
later, FCA-treated rats were randomized to receive one of the treatments
listed in Table 1. All drugs
were suspended in 1% carboxymthyl cellulose. Rats (6–8 per group) were
administered daily by gavage from day 7 to 21 after arthritis induction.
Control animals received 300 µl of 1% carboxymthyl cellulose by oral
gavage. The severity of arthritis was assessed by measuring the paw volume
(edema) by a hydroplethysmometer (Ugo Basile, Milan, Italy) immediately before
arthritis induction (basal value) and 3, 7, 11, 14, 17, and 21 days thereafter
for both hindpaws. The hindpaw swelling was expressed as the ratio between the
hindpaw volume (measured in milliliters) and the animal weight (expressed in
grams). To assess the severity of arthritis, the number of tail nodules was
also counted through the study. Animals were sacrificed on day 21 after RA
induction, and gastric mucosal injury was scored as previously described by an
operator that was unaware of the treatment that the animals had received
(Fiorucci et al., 1999,
2002). Briefly, the length (in
millimeters) of all erosive/hemorrhagic lesions was measured with a digital
caliper, and a "gastric damage score" was calculated for each
stomach by summing these values. After scoring the damage, a sample of the
corpus region of each stomach was excised and processed for measurement of
myeloperoxidase (MPO) activity (Fiorucci
et al., 1999), prostaglandin E2 (PGE2)
synthesis, and ATL content (Fiorucci et
al., 2002). The remainder of the stomach was fixed in formalin and
processed by routine methods for light microscopy.
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Gastric Eicosanoids. The corpus mucosa was isolated, weighed, and
added to a tube containing 100% ethanol plus 100 µM indomethacin to prevent
further synthesis of prostaglandins. Then, samples were homogenized with a
polytron homogenizer and centrifuged at 12,000 rpm for 10 min at 4°C.
After the supernatant of each sample had been evaporated under a nitrogen
stream, the residue was resolved in an assay buffer solution and used for
determination of PGE2. The concentration of PGE2 was
measured using an enzyme immunoassay (Cayman Chemical). LXA4
content was measured using commercially available ELISA kits (Neogen,
Lexington, KY). The antibody used in this assay specifically recognizes
15(R)-epi-LXA4 and has been characterized previously
(Chiang et al., 1998;
Fiorucci et al., 2002). Plasma
levels of TXB2 were quantified with commercially available ELISA
(Cayman Chemical) according to the manufacturer's protocol.
RT-PCR Analysis. Stomachs were removed and immediately snap-frozen
in liquid nitrogen and stored at –80°C until used. Total RNA from
stomach specimens were prepared using TRIzol reagent (Invitrogen, Milan,
Italy) as described (Fiorucci et al.,
2002). Reverse transcription of total RNA (1 µg) was performed
with random hexamers and Superscript II (Invitrogen) 50 min at 42°C. The
resulting single-strand cDNA was used as a template for the subsequent PCR
amplification reaction. PCR experiments were carried out with 2 µl of the
first-strand DNA (cDNA) in a 20-µl mixture containing: 2 µlof PCR buffer
10x (200 mM Tris-HCl pH 8.4, 500 mM KCl), 200 µM dNTP, 1.5 mM
MgCl2, 1 µM specific primers pair, and 1 U of Platinum
Taq DNA polymerase (Invitrogen) and RNase-free water to a 20-µl
final volume using iCycler (Bio-Rad, Hercules, CA). The sequence of the sense
and antisense primers for rat COX-1 (167 bp) were 5'
GCCTCGACCACTACCAATGT 3' and 5' AGGTGGCATTCACAAACTCC 3' and
rat COX-2 (214 bp) 5' TACCCGGACTGGATTCTACG 3' and 5'
AAGTTGGTGGGCTGTCAATC 3'. PCR was carried out for 30 and 36 amplification
cycles for COX-1 and COX-2, respectively, as follows: 30 s at 94°C, 30 s
at 55°C, and 45 s at 72°C. A final extension step was then performed
by heating at 72°C for 5 min. The integrity of cDNA samples was confirmed
using rat β-actin (198 bp) specific primers: 5'
TCACACTGGCATTGTGATGG 3' for the sense and 5' TTAATGTCACGCACGGATTC
3' for the antisense. Control PCR reactions also were performed on
nonreverse-transcribed RNA to exclude any contamination by genomic DNA. The
amplified products were detected by electrophoresis on 1.8% agarose gel
stained with 0.5 µg/ml ethidium bromide. The fragment size was assessed by
comparison with a 100-bp DNA ladder (Invitrogen). The gel was photographed
under ultraviolet transillumination, images were then digitalized, and a
semiquantitative analysis was performed using Kodak Digital Science ID image
analysis software. Each assay was carried out in quadruplicate. Results were
normalized and expressed as ratio of pixel density units for specific mRNA to
β-actin mRNA.
Gastric COX-1 and COX-2 expression was also assessed by Western blotting analysis. For immunoblot analysis, 20 µg of protein obtained from gastric mucosal lysates were electrotransferred onto nitrocellulose filters (Bio-Rad). The membrane was then rinsed briefly with 20 mM Tris-HCl, 150 mM NaCl, pH 7.8 TBS, blocked with 5% w/v skim milk in TBST (TBS 0.05% Tween 20, pH 7.8) for 30 min at room temperature to prevent nonspecific binding and then probed overnight at 4°C with a 1:1000 dilution of monoclonal mouse anti-sheep COX-1 and COX-2 polyclonal rabbit anti-mouse (Cayman Chemical). Membranes were washed three times in TBST for 10 min each at room temperature and then incubated for 60 min with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-hamster secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After further washes in TBST at room temperature, the blots were developed with the enhanced chemiluminescent detection reaction (ECL Western blotting kit; Amersham Biosciences UK, Ltd.) and exposed to Kodak Biomax film. Images where then digitalized and densitometric analysis of COX-1 and COX-2/β-actin immunoprecipitates were carried out using a specific software (Kodak Digital Science ID image analysis software).
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25% of body weight in comparison
with naive rats (n = 8–12/group; P < 0.001 versus
naive).
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IL-6 is a marker of systemic inflammation in RA. Consistent with this concept, a 3-fold increase in IL-6 plasma levels was documented in arthritic rats in comparison with naive animals (n = 8–12; P < 0.01 versus naive). Administration of HCT-3012, as well as celecoxib, reduced IL-6 plasma levels (Fig. 3a), the inhibitory effect being insensitive to the addition of ASA (n = 6; P < 0.01 versus FCA). In contrast, administering rats with naproxen, alone or in combination with ASA, had no effect on IL-6 plasma levels (n = 6; P > 0.05 versus FCA). TXB2 generation, a measure of platelet COX-1 activity, was significantly reduced by the administration of ASA, naproxen, and HCT-3012 and by the combination of these agents (Fig. 3b). Coadministration of HCT-3012 and naproxen with ASA (n = 6; both treatment P < 0.01 versus arthritic rats) did not cause further suppression of TXB2 generation. As expected, a significant reduction of TXB2 plasma levels was observed in arthritic rats administered ASA alone (n = 8; P < 0.01 versus arthritic rats). Confirming the fact that platelet's COX-1 is the main source of plasma TXB2, administering rats with celecoxib alone failed to reduce TXB2 plasma levels (n = 6; P > 0.05 versus arthritic rats), although inhibition was observed in arthritic rats treated with the combination of celecoxib and ASA (n = 6; P > 0.05 versus FCA alone).
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Gastric Mucosal Lesions. Although a minor mucosal injury was detected in rats treated with FCA alone (Fig. 4a), administering FCA-injected rats with naproxen (10 mg/kg) resulted in extensive gastric damage with a mean gastric mucosal injury score of 22.1 ± 4.6 mm (n = 8–12; P < 0.01 versus arthritic rats). Administration of 15 mg/kg HCT-3012, i.e., equimolar with the dose of naproxen, caused significantly less injury than naproxen [6.8 ± 0.9 mm (n = 8–12, P < 0.01 versus naproxen)]. Although treating rats with ASA alone caused mild gastric injury [11.0 ± 1.9 (n = 8–12; P < 0.05 versus arthritic rats)], the combined administration of naproxen with ASA resulted in extensive mucosal injury that was significantly higher than damage caused by ASA alone, indicating that the two agents synergize to produce mucosal damage in arthritic rats (n = 8; P < 0.05 versus ASA alone). In contrast, coadministration of HCT-3012 in combination with ASA did not exacerbate gastric mucosal injury caused by ASA alone resulting in a gastric mucosal injury score of 11.2 ± 2.1 mm (n = 8; P > 0.05 versus ASA alone). Celecoxib caused similar damage than HCT-3012, but the gastric injury caused by this agent was significantly enhanced by the coadministration of ASA (n = 8; P < 0.01 versus ASA alone).
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As illustrated in Fig. 4b, administration of naproxen alone or in combination with ASA significantly increased gastric MPO activity, a measure of neutrophils margination within the gastric microcirculation (P < 0.01 versus FCA-injected rats). In contrast, no significant increase of gastric MPO activity was detected in animals treated with HCT-3012 alone (n = 8; P < 0.001 versus FCA alone). Cotreating arthritic rats with ASA in combination with HCT-3012 increased MPO activity significantly in comparison with HCT-3012 alone, although this figure was significantly lower than that observed in rats taking ASA in combination with naproxen (n = 8; P < 0.01 versus ASA plus naproxen). Although celecoxib alone did not increase MPO activity, its coadministration, in combination with ASA, enhanced neutrophil accumulation into the gastric mucosa (n = 8–12; P < 0.01 versus arthritic rats).
Gastric COX-1 and COX-2 Expression. As illustrated in Fig. 5a–c, although the expression of gastric COX-1, mRNA, and protein did not differ among groups, COX-2 expression, mRNA, and protein was significantly enhanced in the stomach of arthritic rats on day 21 following the FCA injection (Fig. 5, a, b, and d). COX-2 expression, mRNA, and protein were further enhanced by treating arthritic rats with ASA, naproxen, and the combination of the two. In contrast, no significant induction of COX-2 was detected in rats treated with HCT-3012 alone or in combination with ASA. Finally, whereas celecoxib alone did not up-regulate COX-2 expression, this effect was evident in rats coadministered celecoxib in combination with ASA.
Gastric Mucosal Eicosanoids. Gastric PGE2 generation was significantly enhanced in arthritic rats in comparison with naive animals (n = 8–12; P < 0.001 versus arthritic rats). Despite the fact that HCT-3012 was better tolerated than naproxen, it inhibited PGE2 generation to the same extent than its parent drug (n = 6; P < 0.001 versus FCA-injected rats). A significant inhibition of gastric PGE2 formation was documented in rats treated with celecoxib (n = 6; P < 0.001 versus arthritic rats), thereby supporting the view that in arthritic rats, gastric COX-2 contributes to a generation of mucosal prostanoids. Cotreating rats with ASA in combination with HCT-3012 or naproxen did not induce a further reduction of PGE2 concentration (n = 6; P < 0.001 versus FCA-injected rats). Administering ASA to arthritic rats resulted in a significant increase in gastric ATL content, an effect that was almost completely abrogated by cotreating rats with HCT-3012, naproxen, and celecoxib (n = 6; P < 0.001 versus ASA alone) (Fig. 6).
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Discussion |
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TopAbstractEditorial Expression of ConcernMaterials and MethodsResultsDiscussionReferences |
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). Although low
doses of ASA inhibits COX-1 in platelets, they largely spare systemic COX-2
activity, raising the question of whether they might be used in association
with selective and nonselective COX-2 inhibitors to treat pain and
inflammation in patients with RA and OA with cardiovascular risk factors
(Patrono, 1994;
FitzGerald, 2003). However,
results from a large clinical trial indicate that the use of ASA might
increase the risk of gastrointestinal bleeding in patients with RA and OA
taking celecoxib (Silverstein et al.,
2000).
In the present study, we have shown that administration of selective and
nonselective COX-2 inhibitors to arthritic rats treated with a dose of ASA
that cause 70% inhibition of TXA2 formation, exacerbates gastric
damage induced by ASA by a mechanism that involves the inhibition of formation
of mediator(s) generated by the acetylated form of COX-2
(Claria and Serhan, 1995;
Serhan and Oliw, 2001). ASA
prevents AA access to the catalytic core of COX isoenzymes by covalently
modifying a serine residue near the COX-active site of the enzyme
(Lecomte et al., 1994).
However, although acetylation of serine residue 530 of human COX-1 abolishes
the enzyme's capability to oxidize AA, the acetylation of the corresponding
serine residue in COX-2 (Ser 516) modifies the enzyme such that it performs an
incomplete reaction in which AA is converted to 15-HETE carrying its C15
alcohol in the R configuration [15(R)-HETE]
(Serhan and Oliw, 2001).
15(R)-HETE derived from ASA-acetylated COX-2 can be converted by
lipoxygenase isoforms into 15(R)epi-lipoxin A4
(15-epi-LXA4) also known as aspirin-triggered lipoxin or ATL
(Claria and Serhan, 1995;
Serhan and Oliw, 2001). ATL is
a potent counter-regulatory agent and mediates some of the anti-inflammatory
activities of ASA at the leukocytes/endothelial cell interface
(Claria and Serhan, 1995;
Fiore and Serhan, 1995; Takano
et al., 1997,
1998;
Filep et al., 1999;
Serhan and Oliw, 2001;
Fiorucci et al., 2003a).
Indeed, ATL (and its metabolically stable analogs) inhibit granulocytes
recruitment at the site of inflammation by dampening the release of
proinflammatory chemokines/cytokines, as well as by modulating the expression
of adhesion molecules on neutrophils and endothelial cells
(Jozsef et al., 2002). A
growing body of evidence indicate that, similar to other members of the
resolvins family (Serhan et al.,
2002), ATL promotes resolution of inflammation and has the
capability to inhibit the activity of transcription factors such as the
adaptor protein-1 and nuclear factor-
B
(Jozsef et al., 2002). We have
previously shown by favoring neutrophils disengagement from the gastric
microcirculation, ATL is also involved in the development of gastric
adaptation to ASA (Fiorucci et al.,
2002). The demonstration that ATL induces NO formation
(Paul-Clark et al., 2004)
further support the anti-inflammatory and gastrosparing role of ATL in animal
models of NSAID gastropathy. The present results add to this concept by
demonstrating a critical role of ATL in gastric adaptation to low doses of ASA
in a rodent model of arthritis. In this model, arthritis development
associates with induction of gastric COX-2, likely as a result of systemic
inflammation and high plasma levels of proinflammatory cytokines such as IL-6
(Davies et al., 1997; Kato et
al., 1999,
2001). Thus, although COX-2
was barely detectable in the mucosa of intact rats, its expression, mRNA, and
protein was markedly enhanced in arthritic rats, an event that correlates with
a significant increase in gastric PGE2 synthesis. Gastric COX-2
expression was even further increased in arthritic rats exposed to ASA
(Davies et al., 1997) alone or
in combination with naproxen and celecoxib, but not in rats treated with
HCT-3012 alone or in combination with ASA or celecoxib alone.
Although all NSAIDs used in this study, being selective or not for COX-2,
reduced gastric PGE2 synthesis, celecoxib was significantly better
tolerated than naproxen, suggesting that COX-1-derived prostanoids might
compensate for selective COX-2 inhibition in this experimental model. However,
when COX-1 activity was suppressed by feeding arthritic rats a low dose of
ASA, we found that administration of celecoxib, similarly to naproxen,
exacerbates mucosal injury. These data are consistent with previous studies
indicating that simultaneous inhibition of COX-1 and COX-2 is required to
cause gastric damage in rodents (Wallace
et al., 2000). Because the combination of ASA and celecoxib causes
a simultaneous inhibition of PGE2 and ATL synthesis, our results
strongly support the notion that inhibition of acetylated COX-2, in the
context of COX-1 suppression, is the main mechanism involved in exacerbation
of gastric injury caused by celecoxib and, by extension, by nonselective COX-2
inhibitors. Supporting this view, we have previously shown that
LXA4 analog rescues rats from injury caused by ASA, whereas
administering rats with Boc-1, a LXA4 receptor antagonist,
exacerbates the injury (Fiorucci et al.,
2002).
An interesting observation made in this study was the demonstration that
administration of a COX inhibitor to arthritic rats potentiates the
chemoattractive effect of ASA on neutrophils
(McCafferty et al., 1995).
Indeed, we observed a 4-fold increase of MPO activity in the gastric
mucosa of arthritic rats treated with ASA plus a COX inhibitor, but not
HCT-3012, in comparison with rats treated with ASA alone. This synergistic
effect on neutrophil attraction is most likely the consequence of inhibition
of ATL formation. Previous studies have demonstrated that ATL counteracts
vascular leakage and neutrophil trafficking as well as neutrophil recruitment
induced by proinflammatory stimuli such as leukotriene B4, tumor necrosis
factor-, and IL-6 (Claria and
Serhan, 1995; Fiore and
Serhan, 1995; Takano et al.,
1997,
1998;
Clish et al., 1999;
Filep et al., 1999;
Serhan and Oliw, 2001;
Fiorucci et al., 2003a).
HCT-3012 is a prototype of a new class of anti-inflammatory agents coupled
with NO. NO-NSAIDs retain the classic therapeutic profile of native compounds
such as the ability to inhibit inflammatory response, nociception, and fever,
but spare the gastrointestinal tract
(Wallace and Cirino, 1994;
Davies et al., 1997;
Wallace et al., 1997). In the
present study, we have shown that similar to naproxen, HCT-3012 administered
in a therapeutic manner reduces joint edema formation and systemic
inflammation in arthritic rats. Furthermore, confirming previous animal data
(Cicala et al., 2000), our data
demonstrated that in contrast to naproxen, HCT-3012 was effective in reducing
circulating levels of IL-6, a well recognized marker of systemic inflammation
in this model. Animal studies have shown that NO-NSAIDs spare the stomach at
doses that completely inhibit gastric mucosal COX-1 activity
(Wallace et al., 1994),
suggesting that mechanisms other than gastric mucosal prostaglandin
preservation are involved in gastrointestinal protection afforded by these
drugs. Topical application of NSAIDs decrease gastric mucosal blood flow, an
event that leads to neutrophil recruitment in the gastric microcirculation and
plays a mechanistic role in the pathogenesis of NSAID gastropathy.
NO-releasing NSAIDs, including HCT-3012, maintain gastric mucosal blood flow
(Wallace et al., 1994) and
inhibit neutrophil function and down-regulates the expression of adhesion
molecules required for leukocyte adherence to the endothelium, an important
step involved in the process of targeting neutrophils to the gastric
microcirculation (De Caterina et al.,
1995; Khan et al.,
1996). Here, we have provided evidence that although HCT-3012
exerts a potent anti-inflammatory activity and inhibits gastric
PGE2 synthesis, it did not injure the gastric mucosa of arthritic
rats. Furthermore, in contrast to celecoxib and naproxen, HCT-3032 did not
exacerbate gastric mucosal injury caused by cotreatment of arthritic rats with
low doses of ASA. In contrast with celecoxib and naproxen, HCT-3012 did not
increase neutrophil margination into the stomach when given alone nor did it
potentiate gastric neutrophil accumulation when administered in combination
with ASA, suggesting that this compound exerts COX-independent, NO-mediated
effects.
Consistent with the fact that HCT-3012 inhibits COX-1 and COX-2, it also suppress ATL formation when coadministered in combination with ASA to arthritic rats. Although these data indicate that HCT-3012 inhibits the acetylated and nonacethylated form of COX-2, it appears that NO released by its NO-donating moiety has the potential to compensate for ATL and PGE2 deficiency in this experimental setting.
In summary, by demonstrating that ASA increases ATL formation in arthritic rats, we have provided evidence that acetylated COX-2 might play a role in an animal model relevant to a human disease. We have also shown that simultaneous administration of a low dose of ASA in combination with celecoxib increases the tendency of ASA to cause gastric injury in the rodent model of RA. Finally, our results support the notion that NO released from the NO-donating moiety of HCT-3012 attenuates gastric injury even in the absence of PGE2 and ATL.
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Footnotes |
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ABBREVIATIONS: ASA, acetylsalicylic acid; TXA2,
thromboxane A2; LX, lipoxin; COX, cyclooxygenase;
15R-HETE, 15R-hydroxyeicosatetraenoic acid; ATL,
aspirin-triggered lipoxin or 15-epi-LXA4; NO, nitric oxide; NSAID,
nonsteroidal anti-inflammatory drug; OA, osteoarthritis; RA, rheumatoid
arthritis; AA, arachidonic acid; HCT-3012,
(S)-6-methoxy--methyl-2-naphthalene-acetic acid
4-(nitrooxy)butyl ester; FCA, Freund's complete adjuvant; IL-6, interleukin-6;
ELISA, enzyme-linked immunosorbent assay; MPO, myeloperoxidase;
PGE2, prostaglandin E2; RT-PCR, reverse
transcription-polymerase chain reaction; bp, base pair(s); TBS, Tris-buffered
saline; TBST, Tris-buffered saline/Tween 20.
Address correspondence to: Dr. Stefano Fiorucci, Clinica di Gastroenterologia ed Endoscopia Digestiva, Policlinico Monteluce, 06100 Perugia, Italy. E-mail: fiorucci{at}unipg.it
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, P <0.05 versus
arthritic rats.
