Nonsteroidal anti-inflammatory drugs (NSAIDs) constitute one of the most widely used classes of drugs, with more than 70 million prescriptions and more than 30 billion over-the-counter tablets sold annually in the United States (1). Although NSAIDs are effective against pain, fever, and inflammation, their adverse effects on the gastrointestinal (GI) tract limit their usage. Long-term use of these drugs is associated with significant adverse effects, most notably gastric ulceration, bleeding, and perforation, and an increased risk of bleeding from preexisting peptic ulcers (2,3). Severe GI complications (perforated and/or bleeding peptic ulcers) occur in a significant number of patients, resulting in substantial morbidity and mortality (2).
Although there is an association between the inhibition of prostaglandin synthesis and NSAID-induced gastric damage (4,5), the gastric injury cannot be explained solely by cyclooxygenase (COX) inhibition. It has long been recognized that damage to the vascular endothelium is an extremely early event following NSAID administration (6,7). This damage appears to be caused by neutrophils that adhere to the vascular endothelium (4,6). NSAID-induced neutrophil adherence contributes to the pathogenesis of gastric mucosal damage by 2 principal mechanisms: occlusion of gastric microvessels by microthrombi leading to reduced gastric blood flow and ischemic damage (8) and release of cytotoxic reactive oxygen species (ROS) (9,10).
Heme oxygenase-1 (HO-1) breaks down the prooxidant, iron-containing heme to carbon monoxide (CO) and biliverdin, which is then converted into bilirubin (11–13). Induction of HO-1 in several animal models of disease has been shown to protect tissues and cells against ischemia/reperfusion injury, oxidative stress, inflammation, transplant rejection, and apoptosis (14,15). Conversely, humans and mice deficient in HO-1 are prone to oxidant-mediated injury (16,17). Antioxidant effects of HO-1 arise from its capacity to degrade heme from destabilized heme proteins (18) and generate biliverdin and bilirubin, both with strong antioxidant properties (19). CO is not an antioxidant (20), but it has potent antiapoptotic, anti-inflammatory, and vasodilatory effects (21).
HO-1 and its metabolites have the potential to counteract the NSAID-induced gastric injury by their inhibitory effects on leukocyte adhesion (22), their antioxidant properties (19), and their ability to restore mucosal blood flow (21); however, there is limited information as to the protective role of HO-1 on gastric injury. HO-1 is expressed in gastric epithelium and lamina propria inflammatory cells (23). Its expression is increased in inflamed stomach (23) and during the healing phase of gastric ulcers (24). A specific inhibitor of HO, tin mesoporphyrin (SnMP), was reported to exacerbate indomethacin-induced gastric lesions in rats and makes cells more prone to apoptosis (25). These results suggest that the induction of HO-1 may play an important role protecting gastric tissue from NSAID-induced gastric mucosal lesions.
In the present study, we show that the upregulation of HO-1 decreases the severity of NSAID-induced gastric ulcers, possibly by reducing neutrophil infiltration and inhibiting proinflammatory cytokine expression. Our results suggest that the induction of an anti-inflammatory and cytoprotective enzyme, HO-1, can offer a strategy to overcome the GI adverse effects that limit the use of NSAIDs.
Mice and Reagents
Healthy 8-week-old mice on a 129/EvSv background were used for the present study. Mice were maintained in microisolator cages under specific pathogen-free conditions at the animal care facility at the University of Iowa. All of the mice were maintained and treated in accordance with guidelines of the University of Iowa animal care and use committee. All materials were obtained from Sigma-Aldrich (St Louis, MO) unless otherwise specified. Tin protoporphyrin (SnPP, inhibitor of HO activity) and cobalt protoporphyrin (CoPP, inducer of HO-1) were obtained from Frontier Scientific (Logan, UT).
Mice were intraperitoneally (ip) injected with indomethacin (10 mg/kg). A specific inducer of HO-1, CoPP (26) (5 mg/kg ip, prepared in 0.1 mol/L NaOH and phosphate-buffered saline, pH 7.5) was given 24 hours before indomethacin to allow for the expression of HO-1. An inhibitor of HO activity, SnPP (40 μmol/kg ip, prepared in 0.1 mol/L NaOH and phosphate-buffered saline, pH 7.5) was given at the time of indomethacin injection. Control animals received sham injections only. Animals had free access to food and water before and after the procedure.
Assessment of Gastric Ulcers
Twenty-four hours after indomethacin injection, animals were sacrificed and stomachs were dissected open along the lesser curve. Because indomethacin-induced gastric ulcers cannot be easily seen macroscopically in mice as they are in rats (27) and the histological estimates of gastric ulcer length are not always reliable, we measured the gastric damage in our studies by counting the number of gastric ulcers per step sections, as described by Tan et al (28). After fixing in 10% neutral-buffered formalin overnight and embedding in paraffin, step sections (4-μm thick) were made vertically from the entire stomach at 1-mm intervals and stained with hematoxylin and eosin (28). Histology slides were assessed for ulcers, ranging from epithelial disruption to deep ulceration to the lamina propria in association with the inflammatory infiltrates. Number of ulcers was determined per vertical stomach section. Results were expressed as mean ± standard error of the mean.
Snap-frozen tissues from the stomach were homogenized in lysis buffer (0.15 mol/L NaCl, 5 mmol/L ethylenediaminetetraacetic acid, 1% Triton X, 10 mmol/L Tris-Cl, 100 μmol/L phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride) and protein was quantified with Micro-BCA protein assay reagent kit (Pierce Biotechnology, Rockford, IL). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 12% acrylamide resolving gels. After electrophoretic transfer to a nitrocellulose membrane, blots were blocked in Tris-buffered saline (TTBS) buffer (10 mmol/L Tris, pH 7.4, 130 mmol/L NaCl, 0.8 mmol/L disodium ethylenediaminetetraacetic acid, 1% Tween 20) with 5% nonfat dairy milk for at least 1 hour, and subsequently incubated for 1 hour with polyclonal HO-1 antibody (1:2000) (Assay Designs, Ann Arbor, MI) diluted in TTBS buffer. The specific protein was detected by using goat-anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (1:10,000) (Upstate Biotechnology, Lake Placid, NY). Blots were washed several times with TTBS buffer. Antibody-labeled bands were visualized by incubating the blots for 1 minute with enhanced chemiluminescent substrate (ECL, Amersham, Arlington Heights, IL), and exposing Kodak XAR film (Eastman Kodak, Rochester, NY) for 1 to 5 minutes.
Quantification of Neutrophils in the Stomach
The myeloperoxidase (MPO) assay was used to quantify the degree of neutrophil infiltration in the stomachs of mice, as previously described (29). The glandular stomach was weighed and subsequently homogenized in 50 mmol/L phosphate buffer containing 0.5% (wt/vol) hexadecyltrimethyl ammonium bromide, and then sonicated for 10 seconds. The samples were freeze-thawed 3 times and centrifuged at 15,000g for 20 minutes at 4 °C. The supernatants were diluted in 50 mmol/L phosphate buffer and samples were added to phosphate buffer containing 0.5 mmol/L 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate and 0.00001% H2O2. The change in absorbance at 630 nm was measured with a spectrophotometer. Data were presented as MPO activity units per gram of protein.
We measured proinflammatory cytokine expression in mice using a multiplex enzyme-linked immunosorbent assay (ELISA) assay (Aushon Systems, Billerica, MA). This assay allows simultaneous measurement of murine interleukin-1β (IL-1β), IL-17, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) proteins.
Measurement of Labile Iron Pool
Using electron paramagnetic resonance (EPR) (30–32) and desferrioxamine (DFO) (30), we measured the labile iron pool in samples of mouse stomach as a marker of oxidative stress. Briefly, stomachs were isolated, incubated with DFO, frozen in liquid nitrogen, and EPR spectra were obtained (signal height in arbitrary units; [G], magnetic field in Gauss). These spectra are a result of the EPR detectable “ferrioxamine” complex, a measure of the labile iron pool. Gastric homogenates were incubated on ice with 1 mmol/L DFO to chelate labile iron before freezing in a quartz EPR tube with liquid nitrogen. DFO forces the oxidation of Fe2+ to Fe3+ and then binds with high affinity to Fe3+, forming the EPR-detectable ferrioxamine complex. EPR spectra were obtained with a Bruker EMX spectrometer (Bruker BioSpin, Milton, OH) at 9.43 GHz and 105 K. The labile iron concentration was estimated by measuring the ferrioxamine signal (g = 4.3–1550 G) and comparing with standards.
Data were expressed as mean ± standard error of the mean. Differences between groups were analyzed using Mann-Whitney rank sum U test; P < 0.05 was considered significant.
Gastric HO-1 Expression Increases With CoPP
We first assessed whether HO-1 was expressed in the mouse stomach and its expression could be further induced. HO-1 was present in the control mouse stomach and it was unchanged after indomethacin treatment (Fig. 1). SnPP, an inhibitor of HO activity, had no effect on HO-1 expression. There was a slight induction of HO-1 if animals were treated with indomethacin plus SnPP. A well-known inducer of HO-1, CoPP increased gastric HO-1 expression; CoPP in combination with indomethacin-induced HO-1 more than CoPP alone.
CoPP Ameliorates Gastric Ulcers Caused by Indomethacin
Because the upregulation of HO-1 protects tissues against various stressors, we hypothesized that the induction of HO-1 in the stomach would have protective effects against NSAID-induced damage. To test this hypothesis, we assessed whether the use of an HO-1inducer, CoPP, would decrease NSAID-induced inflammation compared with indomethacin alone. CoPP alone did not cause morphological changes in the stomach (Fig. 2B), compared with controls, suggesting that the upregulation of HO-1 alone does not have any adverse effects on the stomach. Indomethacin induced gastric ulcers and large number of inflammatory cell infiltrates (predominantly neutrophils) in all animals (Fig. 2C). Gastric damage caused by indomethacin occurred only in the glandular portion of the stomach and mainly the antrum; the gastric fundus and the squamous portion of the stomach were spared. Response to indomethacin and CoPP was variable but consistent. In some mice that were treated with CoPP before indomethacin, ulcers were completely ameliorated (Fig. 2E). In other mice, ulcers were present, but they looked smaller (Fig. 2D). Overall, the gastric damage, estimated by the number of gastric ulcers per section, was significantly decreased in CoPP and indomethacin-treated mice compared with mice treated with indomethacin only (Fig. 2F). These results suggest that the upregulation of HO-1 by CoPP may protect the gastric mucosa from NSAID-induced ulcers.
CoPP Decreases the Expression of Proinflammatory Cytokines in Mouse Stomach in Response to Indomethacin
To investigate whether the protective effects of HO-1 against indomethacin were the result of changes in the proinflammatory cytokine expression or reflective of the severity of the inflammation, we measured cytokine levels (IFN-γ, TNF-α, IL-17, and IL-6) in the glandular stomach samples using the ELISA technique. CoPP alone had no effects on cytokine expression (Fig. 3). Stomachs from indomethacin-treated animals had increased expression of IL-6 and TNF-α (P < 0.01) but not IFN-γ and IL-17. CoPP treatment significantly decreased the expression of IL-6 and TNF-α induced by indomethacin (P < 0.01). CoPP treatment reduced baseline and indomethacin-induced IFN-γ expression (P < 0.05). These results show that HO-1 overexpression reduces gastric cytokine release induced by NSAIDs. Thus, anti-inflammatory effects of HO-1 may play an important role against gastric injury caused by NSAIDs.
CoPP Decreases Gastric Neutrophil Infiltration in the Mouse Stomach in Response to Indomethacin
To determine whether neutrophil infiltration is increased in the mouse stomach 24 hours after indomethacin, we performed MPO assays (29). MPO activity increased approximately 10-fold after indomethacin treatment (Fig. 4). MPO activity induced by indomethacin was significantly less if mice were pretreated with CoPP. These results suggest that HO-1 overexpression decreases neutrophil infiltration induced by indomethacin.
Indomethacin Does Not Induce Oxidative Stress in Mice Stomach
Because HO-1 has been shown to protect tissues and cells against oxidative stress (14,15) and HO-1 deficiency makes humans and mice prone to oxidant-mediated injury (16,17), we wondered whether the effects of CoPP were caused by the antioxidant properties of HO-1 and its metabolites. We first investigated whether indomethacin caused release of ROS in the gastric tissue by measuring tissue labile iron pools. Oxidative stress increases the cellular labile iron pools, which can be detected by EPR (30–32); this iron can in turn amplify oxidative damage (32–34). Using EPR and DFO (30), we measured the labile iron pool in samples of mouse stomach. This allowed us a unique approach to determine the presence of oxidative stress in the glandular stomach of mice. The labile iron concentration was not different in mice treated with indomethacin compared with untreated mice (Fig. 5). These data indicate that indomethacin does not generate oxidative stress in mouse stomach.
Gastric ulceration induced by NSAIDs is a major limitation to their use as anti-inflammatory drugs. The mechanisms responsible for these deleterious effects are linked to their ability to inhibit gastric prostaglandin synthesis (4,5) and vascular endothelial damage mediated by neutrophils (6,7). Neutrophils play an important role in the pathogenesis of NSAID-induced gastric mucosal injury. The severity of gastric mucosal lesions induced by indomethacin is significantly reduced by depletion of circulating neutrophils in rats or reducing leukocyte adhesion in rabbits (6). Our study supports the role of neutrophils as important players in NSAID-related gastric injury: MPO activity in the mouse stomach increased 24 hours after indomethacin, coinciding with the formation of gastric ulcers.
There are 2 likely mechanisms through which neutrophils may contribute to the indomethacin-induced gastric ulceration. Neutrophil-derived ROS have been shown to contribute significantly to the pathogenesis of gastric erosions induced by indomethacin (35). Neutrophils may also cause injury by adhering to the vascular endothelium and reducing the blood flow to the mucosa (4,6). In our study, there was no increase in the free radicals following indomethacin; thus, the deleterious effects of NSAIDs did not seem to be related to the oxidative stress. We did not assess the gastric blood flow in our study and its changes in response to indomethacin.
We observed a >4-fold increase in TNF-α expression in mouse stomach after indomethacin and the levels were much lower if mice were pretreated with CoPP. A dose-dependent increase in plasma TNF-α level has been reported in rats following NSAID administration, and its release correlates well with the gastric injury and neutrophil activation (36). Moreover, pretreating rats with specific TNF-α synthesis inhibitors or selective anti-TNF-α receptor monoclonal antibodies prevents gastric mucosal damage caused by NSAIDs (37). TNF-α may play a role in NSAID-induced neutrophil infiltration and gastric ulcers, an effect that could be antagonized by inducing HO-1.
Overexpression of HO-1 can protect organs/tissues from immune-mediated injury through either prevention of oxidative damage or its influence on infiltrating inflammatory cells. In contrast, chronic inflammatory changes appear to be the hallmark of HO-1 deficiency as reported in HO-1 knockout mice (38) and a human being with HO-1 deficiency (17). HO-1 expression is upregulated in inflammatory bowel diseases (IBDs) (39,40), and further induction of HO-1 or delivery of its degradation products can ameliorate colitis in animal models of IBD (41,42). Although the protective effects of HO-1 in the intestine are well described, there is little known about its actions in the stomach. Aburaya et al (25) explored the effects of an HO inhibitor, SnMP, on indomethacin-induced gastric lesions in rats and reported an exacerbation of lesions and increased apoptosis. Our study further demonstrates that HO-1 can be induced in the stomach and exert protection against the NSAID-induced gastric ulcers. The beneficial effects of HO-1 induction in the present study can be attributed to several factors including the degradation of prooxidant heme, formation of biliverdin and bilirubin with their antioxidant properties (19), and release of CO, which has anti-inflammatory properties (43). It is also possible that the protective effects exerted by CoPP in our study may be a direct anti-inflammatory effect of this compound, rather than its action via HO-1.
We observed that the induction of HO-1 significantly reduced indomethacin-induced MPO activity and TNF-α and IL-6 levels in the stomach. HO-1 expression and endogenously generated CO are known to inhibit leukocyte adhesion (22) and production of proinflammatory cytokines (eg, TNF-α, IL-6) (43,44). Therefore, it is likely that HO-1 and its by-products exerted their protective effects via these pathways.
In our study, indomethacin and/or induction of HO-1 had no effect on the gastric IL-17 and IFN-γ expression. This could be because they are T-cell products and our time point is early to detect T-lymphocyte activation.
Although we have not measured gastric mucosal blood flow in the present study, it is possible that CO, a by-product of HO-1, improved the blood flow to the mucosa following indomethacin administration (30,31). Properties of CO are similar to those of another gas, nitric oxide (NO), except that CO is chemically more stable (45). Interestingly, NO-releasing NSAIDs are being actively investigated in humans for their protective effects against gastric ulcers (46). NO protects gastric mucosa against NSAIDs most likely via its ability to promote blood flow (47) and inhibit leukocyte adhesion to the endothelium (48). Our study shows that the HO-1 pathway has the potential to offer a protective effect similar to that of NO.
In summary, our findings support an important role for HO-1 in the protection of gastric mucosa against the NSAID-induced damage. The pharmacological induction of HO-1 could prove to be useful to overcome the GI adverse effects limiting the use of NSAIDs.
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