Albino male Wistar rats, with 200-220 g body weight, were used throughout the study. They were fed standard laboratory chow and tap water ad libitum and were not used for at least one week after their delivery to the laboratory. The animals were housed, four in a cage, in temperature-controlled rooms on a 12-h light cycle at 22-24°C and 50-60% relative humidity. They were deprived of food 24 h before the experiment. However, free access to water ad libitum was allowed until 1 h before the beginning of experiments. Experiments were performed in accordance with the provisions of the European Community (EC) Council Directive 86-609, recognized and adopted by the Italian Government.

In experiments on gastroprotection, lansoprazole (18 or 90 µmol/kg) or its vehicle was administered by intragastric gavage. The following NSAIDs, which are associated with moderate to high levels of risk for gastric injury in clinical settings[4], were then administered by intragastric gavage at doses known to induce gastric lesions in rats: indomethacin (100 µmol/kg[12]), diclofenac (60 µmol/kg[13]), piroxicam (150 µmol/kg[14]), and ketoprofen (150 µmol/kg[15]). In all cases, a 30 min interval was allowed between lansoprazole and NSAID administration. Four hours after treatment with NSAIDs, the animals were sacrificed by cervical dislocation, their stomachs were rapidly removed and processed for the evaluation of the following parameters: (1) histomorphometric analysis of gastric mucosal damage; (2) quantitative estimation of mucosal inflammatory activity; (3) assay of malondialdehyde (MDA), non-proteic sulfhydryl compounds and prostaglandin E2 (PGE2) in the mucosal layer; (4) assessment of mucosal expression of COX isoforms (COX-1, COX-2).

In an additional set of experiments, the effects of lansoprazole (18 or 90 µmol/kg) or its vehicle were assayed on gastric acid secretion in rats subjected to pylorus ligation. Dose levels of lansoprazole were selected on the basis of previous studies in rat models of gastric injury, showing that this PPI exerted maximal protective actions at 90 µmol/kg[9].

Histomorphometric evaluation of gastric mucosal damage The histomorphometric estimation of gastric mucosal damage was carried out as previously described[16]. Briefly, the stomach was opened along the greater curvature, gently washed with saline (154 mmol/L NaCl), pinned upon a cork plate with the mucosal surface turned upwards, and fixed in 10% formalin buffered with phosphate for 24 h at 4°C. Each stomach was dissected in parallel strips perpendicular to the lesser curvature at a distance of 2 mm. The strips from each stomach were sequentially placed on a glass slide and oriented with the side of each strip distal to the pylorus upwards. A solution of melted 3% agar was gently poured on the strips and quickly cooled at 4°C to induce solidification. The agar block was then removed from the glass slide, dehydrated, and embedded in paraffin wax. Three micrometers thick paraffin sections were cut using a microtome and stained with hematoxylin and eosin. Sections were examined by light microscopy and the length of both total and damaged mucosa was evaluated by means of a micrometric scale. The lesion index was estimated as the length fraction of damaged mucosa over the total length of mucosa, and expressed in percentage values.

Determination of mucosal myeloperoxidase content Myeloperoxidase (MPO) was assayed as previously reported by Pacheco et al[17] and assumed as a quantitative index to estimate the degree of mucosal infiltration by polymorp-honuclear cells elicited by treatment with test NSAIDs. Briefly, specimens of mucosa were rapidly scraped from the underlying tissue layers of gastric wall using two glass slides kept cold on ice. Mucosal samples (300 mg) were homogenized 3 times (30 s each) at 4°C with a polytron homogenizer (Cole Parmer Homogenizer, Vernon Hills, IL, USA) in 1 mL of ice-cold 50 mmol/L phosphate buffer (pH 6.0) containing 0.5% of hexadecyltrime-thylammonium bromide to prevent the pseudoperoxidase activity of hemoglobin as well as to solubilize membrane-bound MPO. The homogenate was sonicated for 10 s, frozen-thawed 3 times and spun by centrifugation for 20 min at 18 000 g. The supernatant was then recovered and used for determination of MPO concentration by means of enzyme-linked immunosorbent assay (ELISA) (Bioxytech, Oxis International Inc., Portland, OR, USA). All samples were assayed within 2 d after collection. The results were expressed as ng of MPO per 100 mg of gastric mucosa.

Quantitative evaluation of mucosal oxidative damage MDA concentrations in samples of gastric mucosa were determined to obtain quantitative estimations of membrane lipid peroxidation evoked by test NSAIDs. For this purpose, specimens of mucosa were scraped from the underlying tissue layers of gastric wall using two glass slides kept cold on ice. The mucosa was weighed, minced by forceps, homogenized in 2 mL of cold buffer (Tris-HCl 20 mmol/L, pH 7.4) using a polytron homogenizer (Cole Parmer Homogenizer), and spun by centrifugation at 1 500 g for 10 min at 4°C. Aliquots of supernatants were then used for subsequent assay procedures. Mucosal MDA concentrations were estimated by colorimetric assay (Calbiochem-Novabiochem Corporation, San Diego, CA, USA), and the results were expressed as nmol of MDA per milligram of gastric mucosa.

Assay of sulfhydryl compounds in gastric mucosa The concentrations of reduced glutathione (GSH) in specimens of gastric mucosa were determined to estimate the tissue content of non-proteic sulfhydryl compounds[18]. For this purpose, samples of mucosa were scraped from the underlying layers of gastric wall using two glass slides kept cold on ice. The scraped mucosa was weighed, minced by forceps, homogenized in 2 mL of cold buffer [0.4 mol/L 2-(N-morpholino)-ethanesulfonic acid, 0.1 mol/L phosphate, and 2 mmol/L ethylendiaminotetracetic acid (EDTA), pH 6] using a polytron homogenizer (Cole Parmer Homogenizer), and spun by centrifugation at 10 000 r/min for 15 min at 4°C. Aliquots of supernatants were deproteinated by a solution containing 1.25 mol/L metaphosphoric acid and 4 mol/L triethanolamine, to avoid interferences due to particulate components or proteic sulfhydryl groups, and then used for the subsequent assay procedures. Gastric mucosal levels of GSH were determined by enzyme colorimetric assay (Cayman Chemicals, Ann Arbor, MI, USA), and the results were expressed as nmol of GSH per milligram of gastric mucosa.

Enzyme immunoassay of PGE2 in gastric mucosa was performed by means of a commercial kit (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, specimens of mucosa were rapidly scraped from the underlying tissue layers of gastric wall using two glass slides kept cold on ice. The mucosa was weighed, minced by forceps, and homogenized in 1 mL of cold phosphate buffer (PBS 0.1 mol/L, pH 7.4, containing 1 mmol/L EDTA and 10 µmol/L indomethacin) per gram of tissue using a polytron homogenizer (Cole Parmer Homogenizer, Vernon Hills, IL, USA). The resulting homogenate was added with an equal volume of absolute ethanol, and stirred with vortex. After 5-min incubation at room temperature, the homogenate was spun by centrifugation at 1 500 r/min for 10 min at 4°C. The supernatant was added with 1 mol/L HCl until pH 4. Before the assay, samples were subjected to purification by means of superclean LC-18 SPE columns (Sigma Co., St. Louis, MO, USA). For this purpose, 0.5 mL of sample was added to 2 mL of ethanol and vortexed. After incubation at room temperature for 5 min, the sample was spun by centrifugation at 3 000 r/min for 10 min. The supernatant was then removed and applied to the LC-18 SPE column which was previously activated with 5 mL of methanol and then with 5 mL of ultrapure water. The column was subsequently washed with 5 mL of ultrapure water and 5 mL of hexane. PGE2 was then eluted with 5 mL of ethyl acetate containing 1% methanol. The eluted ethyl acetate fractions were collected and evaporated to dryness with nitrogen. Aliquots were then used for subsequent enzyme immunoassay. PGE2 concentration was expressed as nanogram per gram of mucosal tissue.

Specimens of mucosa were scraped from the underlying tissue layers of gastric wall using two glass slides kept cold on ice. Mucosal samples were weighed and homogenized in lysis buffer containing: HEPES 10 mmol/L, NaCl 30 mmol/L, EDTA 0.2 mmol/L, phenylmethylsulphonyl fluoride 2 mmol/L, leupeptin 10 µg/mL, aprotinin 10 µg/mL, sodium fluoride 1 mmol/L, sodium orthovanadate 1 mmol/L, glycerol 2%, MgCl₂ 0.3 mmol/L, and Triton-X 100 1%, using the polytron homogenizer. Mucosal homogenates were spun by centrifugation at 20 000 g for 15 min at 4°C, and the resulting supernatants were then separated from pellets and stored at -80°C. Protein concentration was determined in each sample by the Bradford method (Protein Assay kit, Bio-Rad, Hercules, CA, USA). To perform Western blot analysis of COX-1 and COX-2, equivalent amounts of protein lysates (50 µg) were separated by electrophoresis on sodium dodecylsulfate polyacrylamide gel (8%) and transferred onto a nitrocellulose membrane. The blots were then blocked overnight with 5% non-fat dried milk in phosphate buffered saline, and incubated overnight at room temperature with goat polyclonal antiserum raised against rat COX-1 or COX-2 (dilution 1:1000). After repeated washings with 0.1% Tween-20 in Tris-buffered saline, a peroxidase-conjugated rabbit anti-goat antibody (dilution 1:10000) was added for 1 h at room temperature. After repeated washings with 0.1% Tween-20 in Tris-buffered saline, the immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences Europe GmbH, Cologno Monzese, Italy). The relative expression of COX-1 or COX-2 was quantified by densitometric analysis with NIH Image computer program (Scion Corporation, USA).

Pylorus ligation was carried out as previously described[19]. Briefly, during a short anesthesia with diethyl ether, the abdomen was opened by a midline laparotomy and the duodenum was exteriorized. Then, lansoprazole (18 or 90 µmol/kg) or its vehicle was directly injected into the distal portion of duodenum by means of a 25-gauge needle, the pylorus was ligated, the abdominal incision was closed with clips, and the animals were allowed to recover from anesthesia for 10 min. Two hours after pylorus ligation, the esophageal-gastric junction was ligated, and the whole stomach was excised. The gastric content was emptied, carefully collected in graduated centrifuge tubes, and spun by centrifugation at 3 000 r/min for 10 min. Samples with more than 0.5 mL of sediment were discarded. The level of acidity was measured by automatic potentiometric titration to pH 7.0 with 0.01 mol/L NaOH, using an Autotitrator pH Meter PHM82 (Radiometer, Copenhagen, Denmark), and evaluated as H+ output. The effect of lansoprazole was expressed as µEqH+/2 h.

To evaluate whether lansoprazole is endowed with direct antioxidant properties, the drug was incubated in a reaction mixture where human native low density lipoproteins (LDLs) were subjected to oxidation upon exposure to CuSO₄ at 37°C for 120 min, in accordance with a standardized method[20]. Lansoprazole was assayed at concentrations ranging from 1 to 300 µmol/L. The reaction mixture consisted of 2 mL of a phosphate buffer (10 mmol/L KH₂PO₄/K₂HPO₄, pH 5.3) containing LDLs 150 µg/mL and CuSO₄ 1 µmol/L. At the end of incubation, the oxidative reaction was stopped by addition of buthyldihy-droxytoluene 0.5 mol/L in acetonitrile, and the extent of LDL oxidation was estimated by measurement of 8-iso-prostaglandin F2α (8-iso-PGF2α) concentrations. For this purpose, 100-µL aliquots of the reaction mixture were used to assay 8-iso-PGF2α by means of a commercial kit for competitive enzyme-linked immunoassay (Cayman Chemicals, Ann Arbor, MI, USA). The results were expressed as pg of 8-iso-PGF2α per mL.

The results are given as mean±SE. The statistical significance of data was evaluated by one-way analysis of variance (ANOVA) followed by post hoc analysis by Student-Newman-Keuls test, and P values lower than 0.05 were considered significant; “n” indicates the number of experiments. All statistical procedures were performed by personal computer programs. Drugs The following drugs and reagents were used: indomethacin, ketoprofen, diclofenac, piroxicam, human native low density lipoproteins, butyldihy-droxytoluene, HEPES, EDTA, phenylmethylsulphonyl fluoride, leupeptin, aprotinin, sodium orthovanadate, glycerol, Triton-X, Tween-20, sodium dodecylsulfate, hexadecyltrimethylammonium bromide (Sigma Chemicals Co., St. Louis, MO, USA); lansoprazole (kindly provided by Takeda Italia Farmaceutici, Rome, Italy); diethyl ether (Mallinckrodt Baker BV, Deventer, The Netherlands); polyacrylamide (Bio-Rad, Hercules, CA, USA); goat anti-rat COX-1 and COX-2 antibodies, peroxidase-conjugated rabbit anti-goat antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Other reagents were of analytical grade. Lansoprazole and test NSAIDs were suspended in 1% methocel and administered in a volume of 0.5 mL.

Effects of lansoprazole on gastric mucosal damage induced by NSAIDs Intragastric administration of test NSAIDs caused various degrees of macroscopically evident mucosal damage (not shown). Histological examination of gastric sections showed that treatment with test NSAIDs elicited necrotic mucosal lesions associated with widespread areas of infiltration by polymorphonuclear cells in the upper part of the submucosal layer and the lower part of mucosa (Figure 1). Histomorphometric analysis of gastric tissue revealed negligible lesions of surface epithelium in control animals, as well as those receiving lansoprazole alone, with a total damage accounting for (0.27 ± 0.12)% (control), (0.18 ± 0.11)% (lansoprazole 18 µmol/kg) and (0.09 ± 0.03)% (lansoprazole 90 µmol/kg). By contrast, in animals treated with indomethacin, diclofenac, piroxicam or ketoprofen, histomorphometry displayed a necrotic damage affecting (6.38 ± 0.32)%, (4.48 ± 0.26)%, (6.43 ± 0.54)%, and (5.62 ± 0.39)% of gastric mucosa, respectively (Figure 2). Under these conditions, pretreatment with lansoprazole (90 µmol/kg) caused marked reductions of NSAID-induced gastric mucosal injury (Figure 2), which was associated with a concomitant decrease in the degree of infiltration by polymorphonuclear cells (Figure 1). When assayed at the dose of 18 µmol/kg, lansoprazole exerted a slight influence on NSAID-induced mucosal damage, achieving a level of statistical significance only in animals exposed to diclofenac (Figure 2).

Open in New Tab   Full Size Figure   Download Figure

Figure 1 H&E-stained sections of rat gastric mucosa. A, treatment with indomethacin 100 µmol/kg, A: severe lesion with necrosis of gastric mucosa; B: several polymorphonuclear cell scan be detected (arrows); C and D: Rats treated with lansoprazole 90 mmol/kg plus indomethacin 100 µmol/kg, C: less severe lesion with lysis of mucosal cells; D: few polymorphonuclear cells can be evidenced (arrows). A and C: original magnification 100; bar = 150 µm. B and D: original magnification 170 x; bar = 60 µm.

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Histomorphometric evaluation of gastric mucosal damage in rats subjected to intragastric administration of indomethacin (IND, 100 µmol/kg), diclofenac (DIC, 60 µmol/kg), piroxicam (PIR, 150µmol/kg) or ketoprofen (KET, 150 µmol/kg) either alone or in the presence of lansoprazole (LAN, 18 and 90 µmol/kg). Each column indicates the mean value obtained from 6-8 animals ± SE (vertical lines). ^aP < 0.05 vs control values; ^cP < 0.05 between the respective values obtained in animals treated with a test NSAID alone.

MPO levels in the gastric mucosa of control animals were 3.57 ± 0.78 ng/100 mg, and this value was not significantly affected by treatment with lansoprazole alone (90 mmol/kg). In this set of experiments, indomethacin, and piroxicam induced marked increments of mucosal MPO concentrations (+116.8% and +131.1%, respectively), which no longer occurred after pretreatment of animals with lansoprazole (Figure 3A).

Open in New Tab   Full Size Figure   Download Figure

Figure 3 MPO (A), MDA (B) and GSH (C) levels in the gastric mucosa of rats treated with indomethacin (IND, 100 µmol/kg) or piroxicam (PIR, 150 µmol/kg), either alone or in the presence of lansoprazole (LAN, 90 µmol/kg). Each column represents the mean value obtained from 7 to 10 animals ± SE. ^aP < 0.05 vs control values; ^cP < 0.05 between the respective values obtained in animals treated with indomethacin or piroxicam alone.

MDA levels in the gastric mucosa of control rats were 5.6 ± 1.4 nmol/mg tissue. Lansoprazole alone (90 µmol/kg) did not influence basal MDA concentrations. Treatment with indomethacin or piroxicam caused a significant increase in mucosal MDA concentrations (+148.2% and +112.5%, respectively), and pretreatment with lansoprazole significantly prevented the tissue accumulation of MDA promoted by test NSAIDs (Figure 3B).

In control animals, mucosal GSH levels reached 0.064 ± 0.006 nmol/mg. Under these conditions, lansoprazole alone (90 µmol/kg) did not significantly affect the mucosal concentration of endogenous sulfhydryl compounds. Treatment with indomethacin or piroxicam elicited a significant reduction of mucosal GSH levels (-70.3% and -79.7%, respectively). However, the decrease in gastric GSH concentration caused by test NSAIDs no longer occurred upon pretreatment of animals with lansoprazole (Figure 3C).

Basal PGE2 levels in the gastric mucosa of control animals accounted for 151.9 ± 13.3 ng/g, this value being not significantly affected by administration of lansoprazole alone (90 µmol/kg). All test NSAIDs caused a significant reduction in mucosal PGE2 levels. Marked inhibitory effects were observed in animals treated with indomethacin, piroxicam and ketoprofen (-74.8%, -79% and -86.6%, respectively), whereas a moderate inhibitory action could be detected in diclofenac-treated rats (-48.4%). Pretreatment with lansoprazole did not significantly interfere with the inhibitory actions of test NSAIDs on mucosal PGE2 production (Figure 4).

Open in New Tab   Full Size Figure   Download Figure

Figure 4 PGE2 levels in the gastric mucosa of rats treated with indomethacin (IND, 100 µmol/kg), diclofenac (DIC, 60 µmol/kg), piroxicam (PIR, 150 µmol/kg) or ketoprofen (KET, 150 µmol/kg), either alone or in the presence of lansoprazole (LAN, 90 µmol/kg). Each column represents the mean value obtained from 6 to 8 animals ± SE (vertical lines). ^aP < 0.05 vs control values; ^cP < 0.05 between the respective values obtained in animals treated with a test NSAID alone.

The protein expression of COX isoforms was evaluated by Western blot analysis of gastric mucosal samples obtained from animals subjected to vehicle, indomethacin, lansoprazole or lansoprazole plus indomethacin administration. Western blot assay revealed the expression of both COX-1 and COX-2 in the gastric mucosa of control animals as well as in rats treated with lansoprazole alone (90 µmol/kg) (Figure 5). The densitometric analysis of immunoreactive bands showed that the relative expression of COX-1 or COX-2, in the presence of lansoprazole, did not differ significantly from that estimated in control animals. Treatment with indomethacin was followed by a marked enhancement of COX-2, but not COX-1, expression in the gastric mucosa. A similar effect was exerted by indomethacin upon pre-treatment of animals with lansoprazole (Figure 5).

Open in New Tab   Full Size Figure   Download Figure

Figure 5 Western blot analysis of COX-1 and COX-2protein expression in gastric mucosal lysates obtained from animals treated with indomethacin (IND), lansoprazole (LAN, 90 µmol/kg) or lansoprazole plus indomethacin (LAN+IND). ^aP < 0.05 vs the control values (CON).

In control animals subjected to pylorus ligation for 2 h and pretreated with lansoprazole vehicle the gastric acid output accounted for 153.7 ± 5.8 µEqH+/2 h (n = 6). The administration of lansoprazole (18 or 90 µmol/kg) at the time of pylorus ligation caused a significant decrease in acidity (-81.4% and -93.1%, respectively; Figure 6).

Open in New Tab   Full Size Figure   Download Figure

Figure 6 Rats subjected to pylorus ligation. Effect of lansoprazole (LAN, 18 and 90 µmol/kg) on gastric acid secretion. Each column represents the mean value obtained from six experiments ±SE(vertical lines). ^aP < 0.05 vs the control values.

In control experiments, 8-iso-PGF2α generation promoted by CuSO4-induced oxidation of LDLs accounted for 1 261.3 ± 113.7 pg/mL. When the oxidative reaction was carried out in the presence of increasing concentrations of lansoprazole (1-300 µmol/L), the production of 8-iso-PGF2α underwent a progressive decrease. Under these conditions, the antioxidant activity of lansoprazole achieved a significant level at 3 µmol/L (-17%), and a maximal effect could be detected at the concentration of 100 µmol/L (-85.9%, Figure 7).

Open in New Tab   Full Size Figure   Download Figure

Figure 7 8-Iso-PGF2α levels generated in vitro upon oxidation of native LDLs by CuSO4, either in the absence (C, control) or in the presence of increasing concentrations of lansoprazole (LAN). Each column represents the mean value obtained from 5-6 experiments±SE (vertical lines). ^aP < 0.05 vs the control values.