Heme Oxygenase-1 Protects Gastric Mucosal Cells against Non-steroidal Anti-inflammatory Drugs
2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês
10.1074/jbc.m602074200
ISSN1083-351X
AutoresMayuko Aburaya, Ken‐ichiro Tanaka, Tatsuya Hoshino, Shinji Tsutsumi, Keitarou Suzuki, Masaki Makise, Reiko Akagi, Tohru Mizushima,
Tópico(s)Thermal Regulation in Medicine
ResumoGastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAID11113) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions. Gastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAID11113) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions. Non-steroidal anti-inflammatory drugs (NSAIDs) 2The abbreviations used are: NSAIDs, non-steroidal anti-inflammatory drugs; AMC, aminomethylcoumarin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid; β-NA, β-nicotinamide adenine dinucleotide phosphate; CHOP, C/EBP homologous transcription factor; CO, carbon monoxide; COX, cyclooxygenase; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GRP, glucose-regulated protein; HE, hematoxylin and eosin; HO-1, heme oxygenase-1; HSP, heat shock protein; IL, interleukin; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Nrf2, NF-E2-related factor 2; PG, prostaglandin; PI3K, phosphatidylinositol 3-kinase; SnMP, Sn(IV) Mesoporphyrin; TUNEL, TdT-mediated dUTP-biotin end-labeling; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid. are a useful family of therapeutics, accounting for nearly 5% of all prescribed medications (1Smalley W.E. Ray W.A. Daugherty J.R. Griffin M.R. Am. J. Epidemiol. 1995; 141: 539-545Crossref PubMed Scopus (277) Google Scholar). The anti-inflammatory actions of NSAIDs are mediated through their inhibitory effects on cyclooxygenase (COX) activity. COX is an enzyme essential for the synthesis of prostaglandins (PGs), which have a strong capacity to induce inflammation. On the other hand, NSAID use is associated with gastrointestinal complications (2Hawkey C.J. Gastroenterology. 2000; 119: 521-535Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar), with about 15-30% of chronic users of NSAIDs suffering from gastrointestinal ulcers and bleeding (3Barrier C.H. Hirschowitz B.I. Arthritis Rheum. 1989; 32: 926-932PubMed Google Scholar, 4Fries J.F. Miller S.R. Spitz P.W. Williams C.A. Hubert H.B. Bloch D.A. Gastroenterology. 1989; 96: 647-655Abstract Full Text PDF PubMed Scopus (379) Google Scholar). 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Furthermore, we suggested that both COX-inhibition and NSAID-induced cell death (such as apoptosis) in gastric mucosa are required for production of NSAID-induced gastric lesions in vivo (10Tomisato W. Tsutsumi S. Hoshino T. Hwang H.J. Mio M. Tsuchiya T. Mizushima T. Biochem. Pharmacol. 2004; 67: 575-585Crossref PubMed Scopus (104) Google Scholar). Cellular factors that suppress NSAID-induced apoptosis are therefore important for protection of gastric mucosa from NSAID-induced gastric lesions. When cells are exposed to various stressors, including NSAIDs, they induce a number of proteins, so-called stress proteins, in order to protect themselves against such stressors. Molecular chaperons are representative stress proteins. Their up-regulation in cells confers resistance to various stressors as the chaperons re-fold or degrade denatured proteins produced by stressors (11Mathew A. Morimoto R.I. Ann. N. Y. Acad. Sci. 1998; 851: 99-111Crossref PubMed Scopus (135) Google Scholar). It has been shown that cytosolic molecular chaperones (such as heat shock proteins (HSPs)) and endoplasmic reticulum (ER) molecular chaperons (such as glucose-regulated proteins (GRPs)) are up-regulated by NSAIDs and make cells resistant to NSAIDs (12Gehrmann M. Brunner M. Pfister K. Reichle A. Kremmer E. Multhoff G. Clin. Cancer Res. 2004; 10: 3354-3364Crossref PubMed Scopus (54) Google Scholar, 13Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar). Furthermore, geranylgeranylacetone (GGA), the leading anti-ulcer drug on the Japanese market, has been reported to induce HSPs at gastric mucosa that protect gastric mucosal cells against NSAIDs and other gastric irritants (14Tomisato W. Takahashi N. Komoto C. Rokutan K. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2000; 45: 1674-1679Crossref PubMed Scopus (42) Google Scholar, 15Takano T. Tsutsumi S. Tomisato W. Hoshino T. 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Flurbiprofen was from Cayman Chemicals and Loxoprofen was kindly provided by Sankyo Co. Fetal bovine serum (FBS), heme, β-nicotinamide adenine dinucleotide phosphate (β-NADP), glucose-6-phosphate dehydrogenase, glucose 6-phosphate, diclofenac, anysomycin, ibuprofen, paraformaldehyde, probenecid, proteinase K, and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma. SP600125, indomethacin and aspirin were obtained from Wako Co. Mayer's hematoxylin, 1% eosin alcohol solution and Malinol were from MUTO pure chemical Co. Terminal deoxynucleotidyl transferase (TdTase) was from TOYOBO Co. Biotin 14-ATP, Alexa Fluor 488 goat anti-rabbit immunoglobulin G, Alexa Fluor 488 conjugated with streptavidin and Lipofectamine (TM2000) were from Invitrogen. VECTASHIELD was from Vector Laboratory. SnMP was from Frontier Scientific Inc. Celecoxib was from LKT Laboratories Inc. Antibodies against HSP72, Nrf2, lamin B, GRP78 and actin were purchased from Santa Cruz Biotechnology Inc. Antibodies against HO-1 and p38 MAPK were from Stressgen and Cell Signaling Technology Inc., respectively. Acetyl-DEVD-methylcoumarin amide was from Peptide Institute Inc. O.C.T. compound was from Sakura Fintechnical. PD98059, SB203580, LY294002, and the Dual Luciferase Assay System, including a control plasmid harboring the Renilla reniformis luciferase gene (pRL-SV40), were from Promega. A plasmid containing the Photinus pyralis luciferase gene under control of the HO-1 gene promoter (pHO15luc) (34Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar) was a gift kindly donated by J. Alam (Alton Ochsner Medical Foundation). This plasmid contain 15 kbp of mouse HO-1 5′-flanking region. A plasmid expressing enhanced green fluorescent protein (EGFP) (pEGFP-C1) was obtained from Clontech Laboratories Inc. Male guinea pigs weighing 200-300 g and male Wistar rats weighing 160-200 g were purchased from Kyudo Co. The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institute of Health, and were approved by the Animal Care Committee of Kumamoto University. Gastric Damage Assay—Gastric damage assays were performed as described previously (10Tomisato W. Tsutsumi S. Hoshino T. Hwang H.J. Mio M. Tsuchiya T. Mizushima T. Biochem. Pharmacol. 2004; 67: 575-585Crossref PubMed Scopus (104) Google Scholar). Rats, which had been fasted for 24 h, were intraperitoneally injected with SnMP (dissolved in 0.1 n NaOH, adjusted to pH 7.6 with HCl). One hour later, indomethacin in 1% methylcellulose was orally administered. Three hours after the oral administration, the rats were sacrificed by decapitation under light anesthesia with ethyl ether, and the stomachs were removed and scored for hemorrhagic damage by an observer unaware of the treatment the rats had received. Calculation of the scores involved measuring the area of all lesions in millimeters squared and summing the values to give an overall gastric lesion index. Cell Culture, Transfection, and Cell Viability Assay—Gastric mucosal cells were isolated from guinea pig fundic glands, as described previously (17Hirakawa T. Rokutan K. Nikawa T. Kishi K. Gastroenterology. 1996; 111: 345-357Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 35Tomisato W. Hoshino T. Tsutsumi S. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2125-2133Crossref PubMed Scopus (13) Google Scholar). Isolated gastric mucosal cells were cultured for 12 h in RPMI 1640 containing 0.3% v/v FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin in type-I collagen-coated plastic culture plates in 5% CO2, 95% air at 37 °C. After removing non-adherent cells by washing with RPMI 1640, cells that were attached to the plate at ∼50% confluence were used. Guinea pig gastric mucosal cells prepared under these conditions have been previously characterized, with the majority (about 90%) of such cells being identified as pit cells (17Hirakawa T. Rokutan K. Nikawa T. Kishi K. Gastroenterology. 1996; 111: 345-357Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 35Tomisato W. Hoshino T. Tsutsumi S. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2125-2133Crossref PubMed Scopus (13) Google Scholar). Human gastric carcinoma cells (AGS) were cultured in RPMI1640 medium supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin in 5% CO2, 95% air at 37 °C. Unless otherwise noted, cells (0.8 × 104 cells per well in 24-well plates, 4 × 104 cells per well in 6-well plates, 6 × 105 cells in 100-mm plates) were cultured for 24 h and then used in the experiments. Transfection of cells with plasmid was carried out using Lipofectamine (TM2000) according to the manufacturer's instructions. Transfected cells were used for experiments after a 24-h recovery period. Transfection efficiency was determined in parallel plates by transfection of cells with the pEGFP-C1 control vector. Transfection efficiency was more than 80% in all experiments. NSAIDs were dissolved in Me2SO or Na2CO3 (for indomethacin only) and control experiments (without NSAIDs) were performed in the presence of the same concentrations of Me2SO or Na2CO3. Cells were exposed to NSAIDs by changing the medium. Cell viability was determined by the MTT method. Immunoblotting Analysis—Whole cell extracts and nuclear extracts were prepared as described previously (36Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3955) Google Scholar, 37Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar). The protein concentration of samples was determined by the Bradford method. Samples were applied to 8% (HSP72 and GRP78), 10% (lamin B, Nrf2, p38 MAPK, and actin) or 12% (HO-1) polyacrylamide SDS gels, subjected to electrophoresis, and proteins then immunoblotted with appropriate antibodies. Luciferase Assay—The luciferase assay was performed as described previously (7Tsutsumi S. Gotoh T. Tomisato W. Mima S. Hoshino T. Hwang H.J. Takenaka H. Tsuchiya T. Mori M. Mizushima T. Cell Death Differ. 2004; 11: 1009-1016Crossref PubMed Scopus (221) Google Scholar). Cells were transfected with 0.375 μgof each of the P. pyralis luciferase reporter plasmids (pHO15luc or its vector) and 0.125 μg of the internal standard plasmid bearing the R. reniformis luciferase reporter (pRL-SV40). P. pyralis luciferase activity in cell extracts was measured using the Dual Luciferase Assay System and then normalized for R. reniformis luciferase activity. Histological and Immunohistochemical Analysis—Gastric tissue samples were fixed in 4% buffered paraformaldehyde, embedded in O.C.T. compound and cryosectioned. Sections were stained first with Mayer's hematoxylin and then with 1% eosin alcohol solution for histological examination (hematoxylin and eosin (HE) staining). Samples were mounted with Malinol and inspected using microscopy (Olympus IX70). For immunohistochemical analysis, sections were blocked with 2.5% goat serum for 10 min and then incubated for 12 h with antibody against HO-1 (1:500 dilution) in the presence of 2.5% bovine serum albumin, and finally incubated for 1 h with Alexa Fluor 488 goat anti-mouse immunoglobulin G. Samples were mounted with VECTASHIELD and inspected using fluorescence microscopy (Olympus IX70). TdT-mediated dUTP-biotin End-labeling (TUNEL) Assay— Gastric tissue samples were fixed in 4% buffered paraformaldehyde, embedded in O.C.T. compound and cryosectioned. Sections were first incubated with protenase K (10 μg/ml) for 15 min at 37 °C, then with TdTase and biotin 14-ATP for 1 h at 37 °C and finally with Alexa Fluor 488 conjugated with streptavidin for 1 h. Samples were mounted with VECTASHIELD and inspected using fluorescence microscopy (Olympus IX70). Measurement of HO Activity—Enzymatic activity of HO was determined as described previously (38Tenhunen R. Marver H.S. Schmid R. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 748-755Crossref PubMed Scopus (1540) Google Scholar), with some modifications. Sample preparation from cultured cells: Cells were lysed by freeze-thawing and sonication in the 0.1 m potassium phosphate buffer (pH 7.4) and centrifuged at 1000 × g for 10 min. The supernatants were applied to the HO assay system (see below). Sample preparation from gastric mucosa: Gastric mucosal cells prepared from rats were homogenized in the 0.1 m potassium phosphate buffer (pH 7.4) containing 0.25 m sucrose, and centrifuged at 15,000 × g for 10 min. The supernatants were further centrifuged at 105,000 × g for 60 min. The precipitates were resuspended with the 0.1 m potassium phosphate buffer (pH 7.4) containing 0.15 m KCl and applied to the HO assay system (see below). HO Assay System—After determination of the protein concentration, samples were incubated for 60 min at 37 °C in the dark with the following reagents: heme (17 μm), rat liver cytosol (10 mg/ml), MgCl2 (2 mm), glucose-6-phosphate dehydrogenase (4 units), glucose 6-phosphate (0.85 mm), and β-NADP (2 mm) in 0.6 ml of 0.1 m potassium phosphate buffer (pH 7.4). The reaction was stopped by placing the tubes on ice. The amount of bilirubin generated was estimated with a scanning spectrophotometer and was defined as the difference between 452 and 530 nm. The HO activity is expressed as pmol of bilirubin per milligram of protein per hour. Caspase Activity Assay—The activity of caspase-3 was determined as described previously (39Hoshino T. Tsutsumi S. Tomisato W. Hwang H.J. Tsuchiya T. Mizushima T. J. Biol. Chem. 2003; 278: 12752-12758Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Briefly, cells were collected by centrifugation and suspended in extraction buffer (50 mm PIPES (pH 7.0), 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, and 1 mm dithiothreitol). Suspensions were sonicated and centrifuged, after which the supernatants were incubated with fluorogenic peptide substrates (acetyl-DEVD-methylcoumarin amide) in reaction buffer (100 mm HEPES-KOH (pH 7.5), 10% sucrose, 0.1% CHAPS, and 1 mg/ml bovine serum albumin) for 15 min at 37 °C. The release of aminomethylcoumarin (AMC) was determined using a fluorescence spectrophotometer. One unit of protease activity was defined as the amount of enzyme required to release 1 pmol of AMC/min. Measurement of the Intracellular Ca2+ Level—Intracellular Ca2+ levels were monitored as described (6Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem. 2005; 280: 31059-31067Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Briefly, cells were incubated with 4 μm fluo-3/AM in assay buffer supplemented with 0.1% bovine serum albumin, 0.04% Pluronic F127 and 2 mm probenecid, for 40 min at 37 °C. After washing twice with assay buffer, cells were suspended in assay buffer supplemented with 2 mm probenecid. Cells were transferred to a water-jacketed cuvette and the fluo-3 fluorescence was then measured with a HITACHI F-4500 spectrofluorophotometer. The intracellular Ca2+ level was calculated according to the equation [Ca2+]i = Kd(F - Fmin)/(Fmax - F), where Kd is the apparent dissociation constant (400 nm) of the fluorescent dye-Ca2+ complex. Statistical Analysis—All values are expressed as the mean ± S.D. One-way analysis of variance (ANOVA) followed by Scheffe's multiple comparison test was used for evaluation of differences between groups. The Student's t test for unpaired results was used for the evaluation of differences between two groups. Differences were considered to be significant for values of p < 0.05. NSAIDs Up-regulate HO-1—Up-regulation of HO-1 production by NSAIDs was examined in primary cultures of guinea pig gastric mucosal cells. This type of cell has been used as an in vitro model for physiological and pathological studies of gastric mucosa, because various characteristic features of gastric mucosal cells in vivo (such as vigorous secretion of mucin) are reproduced in this system (17Hirakawa T. Rokutan K. Nikawa T. Kishi K. Gastroenterology. 1996; 111: 345-357Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). As shown in Fig. 1A, treatment of cells with indomethacin up-regulated HO-1 very rapidly (within 3 h of the addition of indomethacin) and transiently (HO-1 levels returned to pre-treatment levels 24 h after the addition). Indomethacin is known to up-regulate other stress proteins (HSPs and GRPs) (12Gehrmann M. Brunner M. Pfister K. Reichle A. Kremmer E. Multhoff G. Clin. Cancer Res. 2004; 10: 3354-3364Crossref PubMed Scopus (54) Google Scholar, 13Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar). The results in Fig. 1A show that up-regulation of HO-1 by indomethacin occurs prior to that of HSP72 and GRP78. Fig. 1B shows the effects of different doses of indomethacin on HO-1 up-regulation. Up-regulation of HO-1 was just apparent at 25-50 μm indomethacin and was distinct at 200-400 μm indomethacin. These concentrations of indomethacin did not affect cell viability (Fig. 1C), showing that up-regulation of HO-1 by indomethacin is not the result of indomethacin-induced cell damage. On the other hand, up-regulation of HSP72 and GRP78 required much higher concentrations of indomethacin (Fig. 1B); in other words, up-regulation of these proteins occurs simultaneously with cell damage (Fig. 1C). We also examined up-regulation of HO-1 by other NSAIDs (diclofenac, ibuprofen, aspirin, flurbiprofen, celecoxib, and loxoprofen). All of the NSAIDs tested up-regulated HO-1 (Fig. 2) at concentrations that did not affect cell viability (data not shown). As was the case for indomethacin, some NSAIDs (diclofenac and flurbiprofen) showed two peaks in their dose response profile of HO-1 up-regulation (Fig. 2). COX exists as two subtypes, COX-1 and COX-2, for which celecoxib and flurbiprofen are COX-2-selective in their action. Results in Fig. 2 show that all NSAIDs tested increased cellular HO-1, irrespective of their COX-2 specificity. IC50 values for COX inhibition of each NSAID (40Schroeder C.P. Yang P. Newman R.A. Lotan R. Cancer Biol. Ther. 2004; 3: 847-852Crossref PubMed Scopus (31) Google Scholar, 41Ben-Chetrit E. Fischel R. Hinz B. Levy M. Rheumatol. Int. 2005; 25: 332-335Crossref PubMed Scopus (23) Google Scholar, 42Kawai S. Nishida S. Kato M. Furumaya Y. Okamoto R. Koshino T. Mizushima Y. Eur. J. Pharmacol. 1998; 347: 87-94Crossref PubMed Scopus (106) Google Scholar) are not related to the concentration required for HO-1 up-regulation (Figs. 1 and 2). Furthermore, loxoprofen is a pro-drug, meaning that its active metabolite but not itself has COX inhibitory activity (43Tanaka Y. Nishikawa Y. Hayashi R. Chem. Pharm. Bull. (Tokyo). 1983; 31: 3656-3664Crossref PubMed Scopus (28) Google Scholar). Therefore, it seems that NSAIDs up-regulate HO-1 independently of COX inhi
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