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• W2132790109 abstract "Background & Aims: Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal erosions and ulcers. Apoptosis is one of the mechanisms. The role of survivin, an antiapoptosis protein, in NSAID-induced gastric injury is unknown. We examined the role of survivin in NSAID-induced gastric mucosal and gastric cell injury. Methods: We examined: (1) the effects of indomethacin (nonselective NSAID), celecoxib and NS-398 (cyclooxygenase [COX]-2-selective NSAIDs), SC-560 (a COX-1-selective NSAID), and SC-560 plus celecoxib on survivin expression and extent of injury in rat gastric mucosa; (2) the effects of indomethacin, NS-398, SC-560, and SC-560 plus NS-398 on survivin expression and injury in gastric epithelial (RGM-1) cells; and (3) the effects of survivin suppression with small interfering RNA (siRNA) on RGM-1 cell integrity at baseline and following indomethacin injury. Results: Indomethacin treatment dose-dependently reduced survivin protein levels and caused severe injury of gastric mucosa and RGM-1 cells. Suppression of survivin expression with siRNA in RGM-1 cells caused cell damage and increased susceptibility to injury by indomethacin. Celecoxib treatment caused exfoliation of the mucosal surface epithelium, but neither caused deep erosions or altered survivin expression. Neither NS-398 nor SC-560 treatment altered survivin levels or produced injury in vivo or in vitro. COX-1 and COX-2 inhibitor combination caused injury in vivo and in vitro but did not decrease survivin expression. Conclusions: (1) Indomethacin, but not selective COX-1 or COX-2 inhibitors alone or in combination, reduces survivin expression in gastric mucosal cells and (2) significant reduction of survivin precedes greater severity of gastric injury. Background & Aims: Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal erosions and ulcers. Apoptosis is one of the mechanisms. The role of survivin, an antiapoptosis protein, in NSAID-induced gastric injury is unknown. We examined the role of survivin in NSAID-induced gastric mucosal and gastric cell injury. Methods: We examined: (1) the effects of indomethacin (nonselective NSAID), celecoxib and NS-398 (cyclooxygenase [COX]-2-selective NSAIDs), SC-560 (a COX-1-selective NSAID), and SC-560 plus celecoxib on survivin expression and extent of injury in rat gastric mucosa; (2) the effects of indomethacin, NS-398, SC-560, and SC-560 plus NS-398 on survivin expression and injury in gastric epithelial (RGM-1) cells; and (3) the effects of survivin suppression with small interfering RNA (siRNA) on RGM-1 cell integrity at baseline and following indomethacin injury. Results: Indomethacin treatment dose-dependently reduced survivin protein levels and caused severe injury of gastric mucosa and RGM-1 cells. Suppression of survivin expression with siRNA in RGM-1 cells caused cell damage and increased susceptibility to injury by indomethacin. Celecoxib treatment caused exfoliation of the mucosal surface epithelium, but neither caused deep erosions or altered survivin expression. Neither NS-398 nor SC-560 treatment altered survivin levels or produced injury in vivo or in vitro. COX-1 and COX-2 inhibitor combination caused injury in vivo and in vitro but did not decrease survivin expression. Conclusions: (1) Indomethacin, but not selective COX-1 or COX-2 inhibitors alone or in combination, reduces survivin expression in gastric mucosal cells and (2) significant reduction of survivin precedes greater severity of gastric injury. Nonsteroidal anti-inflammatory drugs (NSAIDs) cause gastric erosions and gastric and duodenal ulcers, often resulting in gastrointestinal bleeding and perforation. 1Wallace J.L. Pathogenesis of NSAID-induced gastroduodenal mucosal injury.Best Pract Res Clin Gastroenterol. 2001; 15: 691-703Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 2Laine L. The gastrointestinal effects of nonselective NSAIDs and COX-2-selective inhibitors.Semin Arthritis Rheum. 2002; 32: 25-32Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar They also interfere with gastric ulcer healing. 3Schmassmann A. Peskar B.M. Stettler C. Netzer P. Stroff T. Flogerzi B. Halter F. Effects of inhibition of prostaglandin endoperoxide synthase-2 in chronic gastro-intestinal ulcer models in rats.Br J Pharmacol. 1998; 123: 795-804Crossref PubMed Scopus (290) Google Scholar, 4Brzozowski T. Konturek P.C. Konturek S.J. Sliwowski Z. Pajdo R. Drozdowicz D. Ptak A. Hahn E.G. Classic NSAID and selective cyclooxygenase (COX)-1 and COX-2 inhibitors in healing of chronic gastric ulcers.Microsc Res Tech. 2001; 53: 343-353Crossref PubMed Scopus (124) Google Scholar The best recognized pharmacologic action of NSAIDs is inhibition of prostaglandin synthesis due to blocking cyclooxygenase (COX) isoenzymes, COX-1 and COX-2. Clinical and experimental studies indicate that selective COX-1 inhibitors (ie, SC-560) and selective COX-2 inhibitors (ie, celecoxib and NS-398) are less injurious to the gastric mucosa than nonselective inhibitors (ie, indomethacin). 2Laine L. The gastrointestinal effects of nonselective NSAIDs and COX-2-selective inhibitors.Semin Arthritis Rheum. 2002; 32: 25-32Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 5Wallace J.L. McKnight W. Reuter B.K. Vergnolle N. NSAID-induced gastric damage in rats requirement for inhibition of both cyclooxygenase 1 and 2.Gastroenterology. 2000; 119: 706-714Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 6Tanaka A. Araki H. Komoike Y. Hase S. Takeuchi K. Inhibition of both COX-1 and COX-2 is required for development of gastric damage in response to nonsteroidal antiinflammatory drugs.J Physiol Paris. 2001; 95: 21-27Crossref PubMed Scopus (137) Google Scholar NSAIDs cause gastrointestinal damage through a variety of mechanisms, including necrotic and apoptotic cell death. 7Slomiany B.L. Piotrowski J. Slomiany A. Induction of tumor necrosis factor-alpha and apoptosis in gastric mucosal injury by indomethacin effect of omeprazole and ebrotidine.Scand J Gastroenterol. 1997; 32: 638-642Crossref PubMed Scopus (79) Google Scholar, 8Kusuhara H. Matsuyuki H. Matsuura M. Imayoshi T. Okumoto T. Matsui H. Induction of apoptotic DNA fragmentation by nonsteroidal anti-inflammatory drugs in cultured rat gastric mucosal cells.Eur J Pharmacol. 1998; 360: 273-280Crossref PubMed Scopus (56) Google Scholar, 9Szabo I. Tarnawski A.S. Apoptosis in the gastric mucosa molecular mechanisms, basic and clinical implications.J Physiol Pharmacol. 2000; 51: 3-15PubMed Google Scholar, 10Imamine S. Akbar F. Mizukami Y. Matsui H. Onji M. Apoptosis of rat gastric mucosa and of primary cultures of gastric epithelial cells by indomethacin role of inducible nitric oxide synthase and interleukin-8.Int J Exp Pathol. 2001; 82: 221-229Crossref PubMed Scopus (12) Google Scholar The molecular mechanisms of NSAID-induced cell death are not clear, but in vivo and in vitro studies indicate that NSAIDs activate proinflammatory factors such as tumor necrosis factor α and proapoptotic factors such as caspases, Bax, and p53. 7Slomiany B.L. Piotrowski J. Slomiany A. Induction of tumor necrosis factor-alpha and apoptosis in gastric mucosal injury by indomethacin effect of omeprazole and ebrotidine.Scand J Gastroenterol. 1997; 32: 638-642Crossref PubMed Scopus (79) Google Scholar, 11Piotrowski J. Slomiany A. Slomiany B.L. Activation of apoptotic caspase-3 and nitric oxide synthase-2 in gastric mucosal injury induced by indomethacin.Scand J Gastroenterol. 1999; 34: 129-134Crossref PubMed Scopus (53) Google Scholar, 12Fiorucci S. Santucci L. Cirino G. Mencarelli A. Familiari L. Soldato P.D. Morelli A. IL-1 beta converting enzyme is a target for nitric oxide-releasing aspirin new insights in the antiinflammatory mechanism of nitric oxide-releasing nonsteroidal antiinflammatory drugs.J Immunol. 2000; 165: 5245-5254PubMed Google Scholar, 13Fujii Y. Matsura T. Kai M. Matsui H. Kawasaki H. Yamada K. Mitochondrial cytochrome c release and caspase-3-like protease activation during indomethacin-induced apoptosis in rat gastric mucosal cells.Proc Soc Exp Biol Med. 2000; 224: 102-108Crossref PubMed Scopus (58) Google Scholar, 14Zhu G.H. Wong B.C. Ching C.K. Lai K.C. Lam S.K. Differential apoptosis by indomethacin in gastric epithelial cells through the constitutive expression of wild-type p53 and/or up-regulation of c-myc.Biochem Pharmacol. 1999; 58: 193-200Crossref PubMed Scopus (44) Google Scholar, 15Zhou X.M. Wong B.C. Fan X.M. Zhang H.B. Lin M.C. Kung H.F. Fan D.M. Lam S.K. Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak.Carcinogenesis. 2001; 22: 1393-1397Crossref PubMed Scopus (122) Google Scholar Because NSAIDs activate proapoptotic pathways, the possibility arises that they may also modulate antiapoptotic factors. Survivin, a 16.5-kilodalton protein, is a member of the inhibitors of apoptosis protein family and a broad-spectrum suppressor of cell death. Survivin inhibits apoptosis by binding to caspase-3 and caspase-7 and also inhibits caspase-independent cell death. 16Shin S. Sung B.J. Cho Y.S. Kim H.J. Ha N.C. Hwang J.I. Chung C.W. Jung Y.K. Oh B.H. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7.Biochemistry. 2001; 40: 1117-1123Crossref PubMed Scopus (674) Google Scholar, 17O’Connor D.S. Grossman D. Plescia J. Li F. Zhang H. Villa A. Tognin S. Marchisio P.C. Altieri D.C. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin.Proc Natl Acad Sci U S A. 2000; 97: 13103-13107Crossref PubMed Scopus (586) Google Scholar, 18Liu T. Brouha B. Grossman D. Rapid induction of mitochondrial events and caspase-independent apoptosis in survivin-targeted melanoma cells.Oncogene. 2004; 23: 39-48Crossref PubMed Scopus (159) Google Scholar, 19Shankar S.L. Mani S. O’Guin K.N. Kandimalla E.R. Agrawal S. Shafit-Zagardo B. Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways.J Neurochem. 2001; 79: 426-436Crossref PubMed Scopus (121) Google Scholar A preliminary study from our laboratory showed that in human colon cancer cells, indomethacin increases the expression of proapoptotic genes and suppresses the expression of antiapoptotic genes, including survivin. 20Huang R. Chai J. Tarnawski A.S. Do NSAIDs activate apoptosis in human colon cancer cells? Identification of specific genes and pathways involved (abstr).Gastroenterology. 2003; 124: A-239Google Scholar Survivin is expressed in both human and rat gastric mucosa, 21Chiou S.K. Moon W.S. Jones M.K. Tarnawski A.S. Survivin expression in the stomach implications for mucosal integrity and protection.Biochem Biophys Res Commun. 2003; 305: 374-379Crossref PubMed Scopus (63) Google Scholar in the mucosal surface epithelial cells, which are directly exposed to damaging agents such as NSAIDs, and in neck cells, which are progenitor cells. Survivin expression is not found or is minimal in other cells of the gastric mucosa. 21Chiou S.K. Moon W.S. Jones M.K. Tarnawski A.S. Survivin expression in the stomach implications for mucosal integrity and protection.Biochem Biophys Res Commun. 2003; 305: 374-379Crossref PubMed Scopus (63) Google Scholar This expression pattern, together with its known broad-spectrum anti-cell death function, suggests that survivin plays a role in gastric mucosal integrity and protection of gastric mucosa against injury. We hypothesized that NSAIDs can suppress survivin expression in the gastric mucosa, and that this, in turn, facilitates mucosal injury. To test this hypothesis, we administered pharmacologically effective doses of indomethacin, celecoxib, NS-398, and SC-560 alone or in combination with celecoxib to rats and analyzed survivin protein levels in the gastric mucosa and its relation to mucosal injury and apoptosis. We also examined the effects of indomethacin, NS-398, SC-560 alone, or SC-560 in combination with NS-398 on survivin protein levels and injury in cultured normal gastric mucosal epithelial (RGM-1) cells. We further examined whether suppression of survivin expression with small interfering RNA (siRNA) in RGM-1 cells would cause cell injury and/or increase susceptibility of RGM-1 cells to injury by indomethacin. To investigate the mechanisms by which NSAIDs may suppress survivin, we examined the relationship between prostaglandin E2 (PGE2) synthesis (COX activity) and survivin expression and tested the effect of exogenous PGE2 treatment on injury and survivin protein levels in the gastric mucosa and RGM-1 cells. These studies were approved by the Subcommittee for Animal Studies of the Long Beach Department of Veterans Affairs Medical Center. Male Sprague-Dawley rats (Charles River Labs, Wilmington, MA) weighing 225–250 g were fasted for 16 hours. Groups of 10 or 12 rats each were treated as follows: with either (1) 5, 20, or 45 mg/kg indomethacin (Sigma Chemical Co, St Louis, MO) or (2) vehicle (.2 mol/L Na2CO3 + NaH2PO4 in 4:1 ratio) intraperitoneally for 8 hours; (3) 10 mg/kg NS-398 (Cayman Chemical Co, Ann Arbor, MI); (4) 40 mg/kg SC-560 (Searle & Co, Chicago, IL); or (5) vehicle (dimethyl sulfoxide) intraperitoneally for 8 hours or (6) 10 mg/kg of celecoxib (Searle & Co) intragastrically for 8 hours. Additional groups of rats (n = 8) received either vehicle or a combination of 40 mg/kg SC-560 (intraperitoneally) and 10 mg/kg celecoxib (intragastrically) for 8 hours. The indomethacin concentrations administered were within the range that was previously used to induce gastric injury in rats. 22Irwin Jr, F.L. Sarfeh I.J. Tanoue K. Chaurasia O.P. Tarnawski A. The role of extracellular matrix in injury to gastric mucosa by indomethacin.J Clin Gastroenterol. 1995; 21: S18-S23PubMed Google Scholar, 23Mourad F.H. Khuri M. Shouaib F. Nassar C.F. Protective effect of the nitric oxide donor molsidomine on indomethacin and aspirin-induced gastric injury in rats.Eur J Gastroenterol Hepatol. 2000; 12: 81-84Crossref PubMed Scopus (14) Google Scholar The celecoxib, NS-398, and SC-560 concentrations used were pharmacologically effective concentrations that were previously used to inhibit COX-1 and COX-2 activity in the gastric mucosa of rats. 5Wallace J.L. McKnight W. Reuter B.K. Vergnolle N. NSAID-induced gastric damage in rats requirement for inhibition of both cyclooxygenase 1 and 2.Gastroenterology. 2000; 119: 706-714Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 24Brzozowski T. Konturek P.C. Moran A.P. Kwiecien S. Pajdo R. Konturek S.J. Drozdowicz D. Ptak A. Pawlik W. Hahn E.G. Enhanced resistance of gastric mucosa to damaging agents in the rat stomach adapted to Helicobacter pylori lipopolysaccharide.Digestion. 2003; 67: 195-208Crossref PubMed Scopus (16) Google Scholar To examine effects of exogenous prostaglandins on indomethacin-induced gastric injury and survivin reduction, groups of 8 rats each were treated as follows: (1) with vehicle only, (2) with 5 μg/kg 16,16 dimethyl PGE2 (Cayman Chemical Co) for 8 hours, (3) pretreated with 5 μg/kg PGE2 intraperitoneally for 1 hour and then treated with 20 mg/kg indomethacin for 7 hours, or (4) pretreated with vehicle for 1 hour and then with 20 mg/kg indomethacin for 7 hours. The exogenous PGE2 dose used in this study was previously shown to be protective against alcohol-induced gastric injury in rats. 25Garrick T. Kolve E. Kauffman Jr, G.L. Prostaglandin requirements are greater for protection in cold restraint-induced than alcohol-induced gastric mucosal injury.Dig Dis Sci. 1986; 31: 401-405Crossref PubMed Scopus (13) Google Scholar After treatments, the rats underwent laparotomy under ketamine (75 mg/kg)/xylazine (8 mg/kg) anesthesia. Their stomachs were excised, and the rats were killed. Gastric samples for biochemical studies were prepared by scraping the mucosa and freezing it immediately in liquid nitrogen. Gastric samples for histologic and immunohistochemical studies were fixed in 10% buffered formalin, embedded in paraffin, and sectioned. The extent of gastric injury in rats treated with indomethacin or SC-560 plus celecoxib was quantified macroscopically. The stomachs were opened along the greater curvature and photographed using a Cyber-shot digital camera (Sony Corp, Tokyo, Japan). The area of visible mucosal erosions was measured using Photoshop 5.0 (Adobe Systems Inc, San Jose, CA). Mucosal damage was expressed as percent damage of the entire gastric mucosa and calculated as follows: % Gastric Mucosal Damage=(Total Lesion Area/Total Area of the Gastric Mucosa)×100 Histologic injury of NSAID-treated gastric mucosa was evaluated as described in our previous studies. 26Tarnawski A. Hollander D. Stachura J. Krause W.J. Gergely H. Prostaglandin protection of the gastric mucosa against alcohol injury—a dynamic time-related process.Gastroenterology. 1985; 88: 334-352Abstract Full Text PDF PubMed Scopus (176) Google Scholar In brief, standardized mucosal strips (∼1 cm in length and .5 cm wide) were cut from approximately the same area in each stomach from the forestomach to the pylorus through the entire glandular mucosa. Mucosal specimens were quantified morphometrically under light microscopy at 100× magnification. Erosions >50% mucosal depth were scored. Gastric mucosal samples were lysed and caspase-3 activity was determined using a colorimetric CasPASE Apoptosis Assay Kit (Genotechnologies, Inc, St Louis, MO), following the manufacturer’s instructions. Briefly, irreversible cleavage of the synthetic substrate, DEVD, liberated a conjugated chromogenic dye, AFC, and produced an optical change that was detected at 390–400 nm using a DU640B spectrophotometer (Beckman, Fullerton, CA). OD readings were taken at the time the substrate was added (t = 0) and after 1 hour of cleavage (t = 1). The total protein concentration in each sample was quantitated, and the change in caspase activity in each sample was calculated as follows: OD (t=1)−OD (t=0)/μg Total Protein Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed on gastric wall sections using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen Co, Purchase, NY), following the manufacturer’s instructions. Eight to 10 tissue specimens were stained for each treatment. The proportion of apoptotic cells was counted in 5 randomly selected fields on each mucosal section under 200× magnification. Fold increase in caspase-3 activity and TUNEL staining from each NSAID treatment were compared with those from vehicle treatment. Rats were treated with NSAIDs for 8 hours. PGE2 production in the gastric mucosa was assessed using a modification of the method described by Whittle et al. 27Whittle B.J. Hansen D. Salmon J.A. Gastric ulcer formation and cyclo-oxygenase inhibition in cat antrum follows parenteral administration of aspirin but not salicylate.Eur J Pharmacol. 1985; 116: 153-157Crossref PubMed Scopus (23) Google Scholar Gastric mucosal samples were minced in Tris-HCl buffer (150 mmol/L, pH 8.2) for 1 minute and centrifuged at 9000 rpm for 1 minute. Supernatant was discarded, 1.5 mL of fresh Tris-HCl buffer was added, and then the specimen was vortexed for 1 minute followed by addition of 10 mmol/L indomethacin to terminate PGE2 production. The samples were centrifuged for 1 minute at 9000 rpm, and PGE2 content was determined by enzyme immunoassay using PGE2 enzyme immunoassay kits (Cayman Chemical Co) according to the manufacturer’s instructions. After measuring protein concentrations of the tissue specimens, PGE2 production per milligram protein per minute was determined for each sample. Relative gastric mucosal PGE2 production in each NSAID-treated group was compared with that in the vehicle-treated group and expressed as percent inhibition of PGE2 production. Normal rat gastric RGM-1 epithelial cells (established by Hirofumi Matsui, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan) were cultured in Dulbecco’s modified Eagle medium/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. Cells were grown in 100-mm tissue culture dishes until they were about 60% confluent, incubated for 24 hours in serum-free Dulbecco’s modified Eagle medium/F12 medium, and treated with 10 μmol/L, 50 μmol/L, .1 mmol/L, and .5 mmol/L of indomethacin, SC-560, or SC-560 and NS-398 in combination and with 10 μmol/L, 50 μmol/L, and .1 mmol/L of NS-398 for 6 and 24 hours. These concentrations of indomethacin and NS-398 were shown to inhibit re-epithelialization of gastric epithelial monolayers in culture. 28Pai R. Szabo I.L. Giap A.Q. Kawanaka H. Tarnawski A.S. Nonsteroidal anti-inflammatory drugs inhibit re-epithelialization of wounded gastric monolayers by interfering with actin, Src, FAK, and tensin signaling.Life Sci. 2001; 69: 3055-3071Crossref PubMed Scopus (48) Google Scholar Because NS-398 inhibited monolayer wound healing at lower concentrations than indomethacin, 28Pai R. Szabo I.L. Giap A.Q. Kawanaka H. Tarnawski A.S. Nonsteroidal anti-inflammatory drugs inhibit re-epithelialization of wounded gastric monolayers by interfering with actin, Src, FAK, and tensin signaling.Life Sci. 2001; 69: 3055-3071Crossref PubMed Scopus (48) Google Scholar we therefore used lower NS-398 concentrations for our current study. Celecoxib was not used in in vitro studies because it is insoluble in culture medium. To examine effects of exogenous prostaglandins on indomethacin-induced cell injury and survivin reduction, RGM-1 cells were treated with .1 μmol/L, 1 μmol/L, or 10 μmol/L of PGE2 for 6.5 hours or pretreated with the same concentrations of PGE2 for 30 minutes and then treated with .5 mmol/L indomethacin for 6 and 24 hours. At 6 hours after NSAID treatment, before onset of apparent cellular damage, cells were collected for Western blot analysis. At 6 and 24 hours, the culture supernatant was collected for cell injury assay (lactate dehydrogenase [LDH] release) as described below. Survivin siRNA was custom synthesized (Qiagen, Valencia, CA). The siRNA sequences used in our studies are as follows: sense orientation, r(UGAGCCUGAUUUGGCCCAG)d(TT); antisense orientation, r(CUGGGCCAAAUCAGGCUCA)d(TT). RGM-1 cells were seeded into 6-well plates at 105 cells per well 24 hours before transfection. Nine wells were used per treatment and condition. A total of 200 nmol/L of survivin siRNA or equimolars of control nonspecific RNA (Qiagen) were transfected into RGM-1 cells using Oligofectamine Reagent (Invitrogen) according to the manufacturer’s instructions. At 24 hours after transfection, cells were incubated in serum-free medium. Between 40 and 56 hours after transfection, cells were treated with .5 mmol/L indomethacin for 16 hours or left untreated. Then culture supernatants were collected for assessment of cell injury using LDH release assay as described below. Additional transfected cells were lysed after the same incubation times to examine survivin expression by Western blot analysis. Cellular damage was determined and quantitated using the Cytoscan LDH Colorimetric Assay Kit (Geno Technology Inc, St Louis, MO) according to the manufacturer’s instructions. Briefly, release of cytosolic LDH enzyme into the culture supernatant upon cell membrane damage triggered an enzymatic reaction resulting in conversion of a tetrazolium salt into a red color formazan. The amount of formazan is proportional to the amount of damaged cells and was measured at 490 nm using a DU640B spectrophotometer (Beckman). Percent cell injury was determined using the following method: %Cell Injury=(Experimental OD490−Spontaneous OD490)Maximum LDH Release(OD490)×100 Spontaneous OD was obtained from the culture supernatant of vehicle-treated controls, and maximum OD was read from the positive control (cells disintegrated by sonication). Rat gastric mucosal specimens were homogenized with a Polytron homogenizer in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.9), 1.5 mmol/L MgCl2, 550 mmol/L NaCl, .2 mmol/L EDTA, 2 mmol/L dithiothreitol, and 20% (vol/vol) glycerol. RGM-1 cells were lysed in the same buffer. Total protein was extracted from the supernatant after centrifugation, and 100 μg per sample was separated on a 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis for determination of survivin expression and then transferred onto nitrocellulose membranes. The membranes were blocked in skim milk, incubated with rabbit polyclonal anti-survivin antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 hour, and then incubated with peroxidase conjugated goat anti-rabbit or goat anti-mouse antibodies (Sigma Chemical Co) for 1 hour. Survivin protein signals were visualized using enhanced chemiluminescence reagent (Amersham Life Science, Piscataway, NJ) and by exposure to Kodak X-Omat film (Eastman Kodak, Pittsburgh, PA). The membranes were stripped and reincubated with monoclonal anti-β-actin antibody (Sigma Chemical Co) and then with peroxidase-conjugated anti-mouse antibody (BD Transduction Laboratories, Lexington, KY). Protein signal densities were quantified using a Metamorph Imaging System (version 3.0; Universal Imaging Corp, Downingtown, PA). Protein signal densities were subtracted from background densities and normalized to the corresponding β-actin signal densities. Rat gastric sections were deparaffinized in Histoclear (Dako, Carpinteria, CA) and hydrated in serial dilutions of ethanol. Antigen was exposed by heating the samples in Target Retrieval Solution (Dako) for 10 minutes at high-power microwave setting. Gastric sections were treated with serum-free protein block solution (Dako) to block nonspecific signal, incubated with rabbit-polyclonal anti-survivin antibody at 1:100 dilution for 16 hours at 4°C, and then stained with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Sigma Chemical Co) at 1:100 dilution for 30 minutes. Staining was visualized using a Nikon Optiphot epifluorescence microscope (Nikon, Inc, Melville, NY) with Omega filter fluorescein isothiocyanate/Texas red. Comparisons of macroscopic and histologic mucosal injury were performed with the Kruskal-Wallis and Wilcoxon tests and expressed as percent of mucosal area ± SD, as described in our previous study. 26Tarnawski A. Hollander D. Stachura J. Krause W.J. Gergely H. Prostaglandin protection of the gastric mucosa against alcohol injury—a dynamic time-related process.Gastroenterology. 1985; 88: 334-352Abstract Full Text PDF PubMed Scopus (176) Google Scholar The relative standardized protein signals from Western blots, cellular injury, and apoptosis caused by NSAIDs were expressed as mean ± SD. Student 2-tailed t test was used to compare data between 2 groups. One-way analysis of variance and Bonferroni’s correction were used to compare data among 3 or more groups. P < .05 was considered statistically significant. Vehicle-treated rats had no gastric mucosal damage. Indomethacin treatment caused visible gastric lesions at all doses used, and the severity of injury was dose dependent (Figure 1 A). Treatment with SC-560 and celecoxib in combination also caused visible gastric damage comparable to that caused by the lowest dose (5 mg/kg) of indomethacin (Figure 1 A). In contrast, celecoxib, NS-398, or SC-560 alone did not cause any visible gastric mucosal damage. The gastric mucosa of vehicle-treated rats was normal (Figure 1B). Both indomethacin and SC-560 plus celecoxib induced exfoliation of the surface epithelial cells and deep erosions (Figure 1 C and D and Table 1). The gastric mucosa of celecoxib-treated rats showed signs of superficial injury, with exfoliation of surface epithelium in ∼20% of the mucosal section length (Figure 1 E and Table 1). However, even the most superficial mucosal microvessels were intact and deep injury in the form of erosions was absent in the celecoxib-treated gastric mucosa. NS-398 or SC-560 treatment did not produce any appreciable gastric mucosal damage (Figure 1 F and G, respectively, and Table 1).Table 1Histologic Injury in Gastric Mucosa of NSAID-Treated RatsTreatmentsPercent injuryVehicle3.8 ± 7.4Indomethacin (5 mg/kg)28.1 ± 16.5aP < .01.Indomethacin (20 mg/kg)57.5 ± 16.7aP < .01.Indomethacin (45 mg/kg)62.5 ± 16.6aP < .01.SC-560 + celecoxib33.8 ± 18.5aP < .01.Celecoxib (10 mg/kg)20.6 ± 13.2aP < .01.NS-398 (10 mg/kg)5.0 ± 7.0SC-560 (40 mg/kg)7.5 ± 8.8NOTE. Values represent mean ± SD.a P < .01. Open table in a new tab NOTE. Values represent mean ± SD. Indomethacin treatment significantly and dose-dependently decreased survivin protein levels in the gastric mucosa compared with vehicle treatment (Figure 2 A and F). Immunofluorescence staining showed that survivin signal is markedly reduced in mucosal areas bordering indomethacin-induced erosions (Figure 3). In contrast, treatment with celecoxib, NS-398, or SC-560 did not alter survivin protein levels (Figure 2 B–D and F) or survivin localization (Figure 3) in the gastric mucosa. Treatment with SC-560 and celecoxib in combination significantly increased survivin protein level compared with vehicle treatment (Figure 2 E and F), and survivin signal is present in the mucosa bordering erosions (Figure 3).Figure 3Immunofluorescence staining of survivin protein in the gastric mucosa of rats treated with NSAIDs for 8 hours. Treatments are indicated above each panel. White lines mark the borders of erosions. (Original magnification 100×.)View Large Image Figure ViewerDownload (PPT) Indomethacin treatment dose-dependently increased caspase-3 activity and TUNEL staining in the gastric mucosa compared with vehicle treatment (Table 2). Celecoxib treatment alone or in combination with SC-560 increased caspase-3 activi" @default.
• W2132790109 created "2016-06-24" @default.
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• W2132790109 date "2005-01-01" @default.
• W2132790109 modified "2023-09-26" @default.
• W2132790109 title "Survivin: A novel target for indomethacin-induced gastric injury" @default.
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