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Rolipram, a specific type IV phosphodiesterase inhibitor, ameliorates indomethacin-induced gastric mucosal injury in rats
Pathophysiology 9 (2003) 195 /200 www.elsevier.com/locate/pathophys
Rolipram, a specific type IV phosphodiesterase inhibitor, ameliorates indomethacin-induced gastric mucosal injury in rats Chieko Nakamura, Michiro Otaka *, Masaru Odashima, Mario Jin, Noriaki Konishi, Youhei Horikawa, Tamotsu Matsuhashi, Sumio Watanabe First Department of Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita city, Akita 010-8543, Japan Received 18 December 2002; received in revised form 17 January 2003; accepted 22 January 2003
Abstract Inhibition of type IV phosphodiesterase activity is beneficial in various inflammation mediated by its function to suppress the production of inflammatory cytokines in inflammatory cells. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are well known to develop gastric mucosal lesion. As pathogenesis of indomethacin induced gastric mucosal lesion, activation of neutrophils and inflammatory cytokine production play critical roles. However, the effect of phosphodiesterase inhibitors on development of gastric mucosal lesion has not been reported. In the present study, we examined the effect of specific type IV phosphodiesterase inhibitor (rolipram) on NSAIDs-induced gastric mucosal lesion. Also, we examined the effect of rolipram on tissue prostaglandin E2 (PGE2) production. Indomethacin-induced gastric mucosal injury was produced by the intragastric administration of indomethacin (30 mg/kg). Rolipram was injected to the rats intraperitoneally 30 min before the indomethacin administration. Ulcer index and tissue myeloperoxidase (MPO) activity of the gastric mucosa was evaluated. The gastric concentration of PGE2 was determined by RIA. Gastric mucosal lesion induced by indomethacin was significantly inhibited with treatment of rolipram. Mucosal MPO activity was also suppressed by administration of rolipram. Gastric mucosal PGE2 concentration was not affected by intraperitoneal injection of rolipram. Based on these data, the beneficial effects of rolipram on indomethacin-induced gastric mucosal injury may be attributed to its anti-inflammatory properties. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: NSAIDs; Mucosal injury; Phosphodiesterase inhibitor; Neutrophil
1. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are widely used as anti-inflammatory, analgesic agents [1 /3]. However, gastrointestinal injury is a serious adverse effect of NSAIDs, and effective strategies to protect the gastrointestinal mucosa are required. Many studies have been reported to understand the mechanisms for the development of indomethacin-induced mucosal lesion [4 /6]. Although, as the mechanism of gastric lesion induced by NSAIDs, suppression of endogenous prostaglandin (PG) production resulted from cyclooxigeanse (COX) inhibition is thought to be crucial, the exact pathogenic mechanism
* Corresponding author. Tel.: /81-18-834-1111; fax: /81-18-8362611. E-mail address: [email protected] (M. Otaka).
remains to be elucidated [7]. Several investigators have reported that intraperitoneal injection of anti-neutrophil serum and immunoneutralization of adhesion molecules on neutrophils and endothelial cells significantly attenuate the gastric mucosal injury induced by NSAIDs [8,9]. Therefore, activation and infiltration of neutrophils are critical factors to develop gastric mucosal lesion induced by NSAIDs. On the other hand, recently, the potential usefulness of a specific type IV phosphodiesterase inhibitor, rolipram, as a novel anti-inflammatory agent has been reported [10,11]. A specific type IV phosphodiesterase inhibitor by suppression of breakdown of cAMP to 5AMP increases intracellular cAMP in neutrophils and decreases TNF-a and N -formyl-methionyl-leucyl-phenylamine (FMLP)-stimulated neutrophil adherence, production of reactive oxygen species, and degranulation [12]. In addition, rolipram has been reported to ameliorate experimentally induced colonic
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mucosal lesion and ischemia reperfusion injury of kidney in animal models [13]. Also, it has been reported that rolipram prevented NSAIDs-induced enteropathy [14]. However, the effect of rolipram or significance of type IV phosphodiesterase in the pathogenesis of gastric mucosal lesion has not been reported. In this study, we examined the effect of a specific type IV phosphodiesterase inhibitor, rolipram, on indomethacin-induced gastric mucosal lesion and myeloperoxidase (MPO) activity in the gastric mucosa to understand the pathogenesis of NSAIDs-induced gastric mucosal lesion. Also, the effect of rolipram on prostaglandin E2 (PGE2) concentration in gastric mucosa was studied.
2. Materials and methods 2.1. Animals Eight-week-old male Sprague /Dawley rats (250 /300 g) were fed on standard laboratory diet and water ad libitum, and kept in cages in temperature (239/2 8C) and humidity (559/5%) controlled room with a 12:00 h dark:12:00 h light cycle before and during the experiment. All of the experiments were performed in the animals deprived of food for 24 h but with free access to water before experiments. This experiment is approved by the Akita University Animal Care Committee. 2.2. Chemicals Specific phosphodiesterase IV inhibitor, rolipram, was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rolipram was dissolved in small volume of dimethylsulfoxide and then diluted with physiological saline just before injection. 2.3. Effect of rolipram on indomethacin-induced gastric mucosal lesion
2.4. Effect of rolipram on myeloperoxidase (MPO) concentration in the gastric mucosa An assay of gastric mucosal MPO activity was applied to quantify the degree of neutrophil infiltration. Three hundred milligrams of scraped mucosa was homogenized for 30 s with polytron homogenizer (PT 1200, KInematica AG, Littau, Switzerland) in 1.0 ml of icecold 0.5% hexadecyliltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0). Hexadecyliltrimethylammonium bromide is used to negate the pseudoperoxidase activity of the hemoglobin and to solubilize membrane-bound MPO. The homogenate was sonicated (U50 IKA Werke GrmlH and Co. KG, Staufen, Germany) for 10 s, freeze-thawed three times and centrifuged for 20 min at 18 000 /g . The supernatant was taken for determination of the enzyme activity utilizing an ELISA kit (Bioxytech, Oxis International, Inc. Portand, OR, USA). The change in absorrbance at 405 nm was measured with a spectrophotometer (Microplate reader model 3550, Bio-Rad, Hercules, CA, USA). The concentration of MPO was expressed as nanogram per mg protein using Bradford’s method [16]. 2.5. Effect of rolipram on mucosal content of PGE2 To determine the effect of rolipram on PGE2 synthesis, the animals were sacrificed 6 h after rolipram administration and the stomach was removed. A part of fundic mucosa (about 100 mg) was exised for determination of PGE2 synthesis according to the method described previously [17]. The samples were weighed, finely minced with scissors for 15 s, then suspended in 1.0 ml of 10 nM sodium phosphate buffer (pH 7.4). The samples were then incubated in a shaking bath (37 8C) for 29 min followed by centrifugation (9000 /g for 30 s). The supernatant was frozen and subsequent determination of PGE2 was performed by specific radioimmunoassays using PGE2 [125I] RIA kit (Dupont/NEN, Boston, MA, USA). 2.6. Data analysis
Indomethacin-induced gastric injury was produced by the intragastric administration of indomethacin (30 mg/ kg). Indomethacin was dissolved in 3% sodium bicarbonate solution and 1 ml of the solution was administrated. Rolipram (0.125, 0.25, 0.5 mg/kg, n /5 in each dose) or vehicle was intraperitoneally injected 30 min prior to indomethacin administration. Control rats were given only physiological saline. The animals were sacrificed by stunning and cervical dislocation 6 h after indomethacin administration and the stomach was removed. The gastric mucosal lesions were measured by two independent observers blind to the treatment. The ulcer index was calculated as the sum of the lengths of all lesions [15].
All data were expressed as mean9/S.E.M. Statistical analysis was performed using Mann /Whitney U -test. P value B/0.05 was considered to be statistically significant.
3. Results 3.1. Effect of rolipram on indomethacin-induced gastric mucosal injury Indomethacin caused multiple hemorrhagic erosions and bleeding developed in the glandular stomach 6 h
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Fig. 1. Macroscopic findings of the gastric mucosa 6 h after administration of indomethacin (30 mg/kg) with or without pretreatment with rolipram (0.5 mg/kg, i.p.). Control: treated with vehicle. Multiple hemorrhagic erosions in the glandular stomach of rats 6 h after administration of indomethacin. Pretreatment with rolipram at a dose of 0.5 mg/kg markedly reduced these erosions.
after indomethacin administration. In contrast, pretreatment of rolipram inhibited the gastric lesions (Fig. 1). The ulcer index in vehicle-treated rats was 46.59/2.5 mm. The ulcer index in rats pretreated with rolipram was significantly suppressed to 14.59/1.94 mm (0.125 mg/kg, P B/0.001), 7.49/0.93 mm (0.25 mg/kg, P B/ 0.001), or 3.69/1.57 mm (0.5 mg/kg, P B/0.001) in a dose dependent manner (Fig. 2). The protective effect of rolipram was confirmed histologically. Indomethacin administration resulted in large areas of epithelial crypt
loss, predominantly neutrophilic infiltrate throughout the mucosa and sub-mucosa, erosions, and mucosal bleeding. In contrast, pre-treatment with rolipram resulted in smaller erosions with few neutrophils (Fig. 3).
3.2. Effect of rolipram on myeloperoxidase activity in gastric mucosa Fig. 4 shows the gastric mucosal MPO concentration in rats 6 h after indomethacin administration. The MPO activity in vehicle-treated rats was 15.89/2.77 ng/mg protein. The MPO concentration was suppressed by rolipram pretreatment to 9.179/1.11 ng/mg protein (0.125 mg/kg, P B/0.05), 7.349/2.35 ng/mg protein (0.25 mg/kg, P B/0.05), or 0.629/0.61 ng/mg protein (0.5 mg/kg, P B/0.05) compared with vehicle-treated rats.
3.3. Effect of rolipram on mucosal content of PGE2
Fig. 2. Ulcer index of rats 6 h after administration of indomethacin with or without pretreatment with rolipram (0.125, 0.25, 0.5 mg/kg, i.p.), (n /5 in each group) * P B/0.001, significant difference compared with vehicle treated group.
The concentration of PGE2 was 37.29/14.5 pg/mg in vehicle-treatment rats. Rolipram administration did not interfere with reduction of gastric PGE2 concentration (Fig. 5).
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Fig. 3. Microscopic findings of gastric mucosa 6 h after administration of indomethacin with or without pretreatment with rolipram (0.5 mg/kg, i.p.). Original magnification 100 and 200 / (H and E staining). Hemorrhagic mucosal erosions and inflammatory cell infiltration developed in the glandular stomach of rats 6 h after the administration of indomethacin. Pretreatment of rolipram at a dose of 0.5 mg/kg markedly inhibited these changes.
Fig. 4. Effect of rolipram on MPO concentration in the gastric mucosa (n /5 in each group). * P B/0.05, significant difference compared with vehicle treated group.
Fig. 5. Effect of rolipram on PGE2 concentration in the gastric mucosa. NS, statistically no significant difference.
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4. Discussion The present study demonstrated for the first time that rolipram prevents indomethacin-induced gastric mucosal injury. Rolipram significantly inhibited the increase in MPO concentration and neutrophil accumulation index in the gastric mucosa 6 h after indomethacin administration. Various mechanisms and factors, such as stimulation of gastric acid, inflammatory cell infiltration, cytokines, mucosal blood flow, and free radicals have been known to contribute to the development of NSAIDs-induced gastric mucosal damage [18]. Recently, it has been reported that neutrophil depletion by intraperitoneal injection of anti-neutrophil serum significantly attenuates the gastric mucosal injury induced by indomethacin [19]. Also, it has been proved that immunoneutralization of the CD11b/CD18 adherence complex on neutrophils inhibited indomethacininduced gastric injury [20]. Therefore, neutrophilsmediated inflammation is one of the critical factors in the development of indomethacin-induced gastric mucosal injury. Rolipram has initially been developed and studied in clinical trials as an antidepressant [21]. Recently, the potential usefulness of specific type IV phosphodiesterase inhibitor, rolipram, as a novel antiinflammatory agent has been reported [22,23]. Agent that elevate the concentration of intracellular cAMP can inhibit inflammatory cell activities such as cytokine production, chemotaxis, cytotoxicity and cell aggregation [24 /26]. Rolipram inhibits neutrophil activation and TNF-a mediated neutrophil adhesion to the vascular endothelium. And it reduces the synthesis and release of reactive oxygen species. In addition, rolipram has been reported to inhibit experimentally-induced colonic lesion in animal model [27]. These findings led us to examine the effect of rolipram on indomethacininduced gastric mucosal lesion. In this study, we clearly demonstrated that the rolipram ameliorated the development of gastric mucosal injury induced by indomethacin administration and highest dose tested caused an approximately 90% reduction in gastric mucosal lesion. Histopathological findings showed the infiltration of inflammatory cells in submucosal layer was dramatically suppressed by rolipram. Further, MPO activity increases in the gastric mucosa 6 h after indomethacin administration in rats treated with vehicle solution, and the increase in the MPO concentration is significantly inhibited by treatment with rolipram. These results indicate that the inhibition of neutrophil infiltration by rolipram may be one of the protective factors decreasing indomethacin-induced gastric mucosal injury. It has been reported that PGE2 prevented gastric mucosal damage by indomethacin in human and animals [28]. However, the protective effect of rolipram did not depend on gastric mucosal prostaglandin synthesis, since pretreatment with rolipram, which reduced gastric
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damage had no effect on the gastric mucosal prostaglandin concentration. On the other hand, phosphodiesterase inhibitor is known to inhibit TNF-a production in vitro and in vivo [29]. The predominant phosphodiesterase isoenzyme family in monocytes, a main source of TNF-a production, is the type IV phosphodiesterase. The specific type IV phosphodiesterase inhibitor, rolipram, is a 500-fold more potent inhibitor of TNF synthesis compared with nonspecific phoshodiesterase inhibitor, pentoxifylline, in human mononuclear cells [30]. TNF-a is a proinflammatory cytokine and has recently been shown to be a crucial mediator of NSAIDs induced gastric mucosal injury [31]. Also, TNF-a is a cytokine that strongly stimulates neutrophil adherence by inducing the synthesis and expression of adhesion molecules on endothelial cells and neutrophils [32,33]. Furthermore, studies on experimental models have shown that intravenous administration of TNF-a produces extensive neutrophil infiltration within the micorvasculature of the digestive tract. In addition, portal infusion of TNF-a caused gastric and small intestinal damage in rats [34]. It has been also shown that NSAIDs administration increases TNF-a concentrations in both humans and experimental animals [35,36]. These evidences indicate that the anti-ulcer effect of rolipram might be caused by inhibition of TNF-a production.
5. Conclusion Rolipram prevented indomethacin-induced gastric damage in rats. The effect of rolipram is not related to the synthesis of prostaglandins but depended on inhibition of neutrophil infiltration to gastric mucosa. These results indicate that agents which modulates cAMP could be clinically useful for the therapy of NSAIDsinduced gastropathy.
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[21] L.L. Jeffrey, V.P. Michael, E.G. Lee, R.S. Christopher, The toxicity of repeated exposures to rolipram, a type IV phosphodiesterase inhibitor in rats, Pharmacol. Toxicol. 78 (1996) 44 /49. [22] J.T. Theudore, Phosphodiesterase isozymes, Am. J. Respir. Crit. Care Med. 157 (1998) 351 /370. [23] A.B. Joseph, Cyclic nucleotide phosphodiesterases: functional implication of multiple isoforms, Physiol. Rev. 75 (1995) 725 / 748. [24] T.J. Torphy, Phosphodiesterase isozymes: molecular targets for novel antiasthma agents, Am. J. Respir. Crit. Care Med. 157 (1998) 351 /370. [25] A.R. Moore, D.A. Willoughby, The role of cAMP regulation in controlling inflammation, Clin. Exp. Immunol. 101 (1995) 387 / 389. [26] H.R. Bourne, L.M. Lichtenstein, K.L. Melmon, C.S. Henney, Y. Weinstein, G.M. Shearer, Modulation of inflammation and immunity by cyclic AMP, Science 184 (1974) 19 /28. [27] H. Gunther, B. Christoph, S. Britta, A. Stefan, S. Johannes, T. Katharina, E. Andreas, A.L. Hans, E. Stefan, Specific type IV phophodiesterase inhibitor rolipram mitigates experimental colitis in mice, J. Pharmacol. Exp. Ther. 292 (2000) 22 /30. [28] B.A. Caroline, D. McCafferty, W.T. Allan, G.S. Mark, L.W. John, Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence, Am. J. Physiol. 270 (1996) G42 /G48. [29] L. Sekut, D. Yarnall, S.A. Stimpson, L.S. Noel, R. Bateman-Fite, R.L. Clark, M.F. Brackeen, J.A. Menius, Jr, K.M. Connolly, Anti-inflammatory activity of phosphodiesterase(PDE)-IV inhibitors in acute and chronic models of inflammation, Clin. Exp. Immunol. 100 (1995) 126 /132. [30] J. Semmler, H. Wachtel, S. Endres, The specific type IV phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-alpha production by human mononuclear cells, Int. J. Immunopharmacol. 15 (1993) 409 /413. [31] L. Santucci, M. Fiorucci, M. Giansanti, P.M. Brunori, F.M. Di Matteo, A. Morelli, Pentoxifylline prevents indomethacin induced acute gastric mucosal damage in rats: role of tumor necrosis factor alpha, Gut 35 (1994) 909 /915. [32] T. Yoshikawa, H. Takano, Y. Naito, H. Oyamada, S. Ueda, M. Kondo, Augmentative effects of tumor necrosis factor-alpha (human, natural type) on polymorphonuclear leukocyte-derived superoxide generation induced by various stimulants, Int. J. Immunopharmacol. 14 (1992) (1992) 1391 /1398. [33] S. Kokura, R.E. Wolf, T. Yoshikawa, D.N. Granger, T.Y. Aw, Tlymphocyte-derived tumor necrosis factor exacerbates anoxiareoxygenation-induced neutrophil-endothelial cell adhesion, Circ. Res. 86 (2000) 205 /213. [34] M.P. Kahky, C.O. Daniel, A.B. Cruz, H.V. Gaskill, III, Portal infusion of tumor necrosis factor increases mortality in rats, J. Surg. Res. 49 (1990) 138 /145. [35] M. Spatafora, G. Chiappara, D. D’Amico, D. Volpes, M. Melis, E. Pace, A. Merendino, Effect of indomethacin on the kinetics of tumour necrosis factor alpha release and tumour necrosis factor alpha gene expression by human blood monocytes, Pharmacol. Res. 23 (1991) 247 /257. [36] G.D. Martich, M.M. Parker, R.E. Cunnion, A.F. Suffredini, Effects of ibuprofen and pentoxifylline on the cardiovascular response of normal humans to endotoxin, J. Appl. Physiol. 73 (1992) 925 /931.
Rolipram, a specific type IV phosphodiesterase inhibitor, ameliorates indomethacin-induced gastric mucosal injury in rats Chieko Nakamura, Michiro Otaka *, Masaru Odashima, Mario Jin, Noriaki Konishi, Youhei Horikawa, Tamotsu Matsuhashi, Sumio Watanabe First Department of Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita city, Akita 010-8543, Japan Received 18 December 2002; received in revised form 17 January 2003; accepted 22 January 2003
Abstract Inhibition of type IV phosphodiesterase activity is beneficial in various inflammation mediated by its function to suppress the production of inflammatory cytokines in inflammatory cells. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are well known to develop gastric mucosal lesion. As pathogenesis of indomethacin induced gastric mucosal lesion, activation of neutrophils and inflammatory cytokine production play critical roles. However, the effect of phosphodiesterase inhibitors on development of gastric mucosal lesion has not been reported. In the present study, we examined the effect of specific type IV phosphodiesterase inhibitor (rolipram) on NSAIDs-induced gastric mucosal lesion. Also, we examined the effect of rolipram on tissue prostaglandin E2 (PGE2) production. Indomethacin-induced gastric mucosal injury was produced by the intragastric administration of indomethacin (30 mg/kg). Rolipram was injected to the rats intraperitoneally 30 min before the indomethacin administration. Ulcer index and tissue myeloperoxidase (MPO) activity of the gastric mucosa was evaluated. The gastric concentration of PGE2 was determined by RIA. Gastric mucosal lesion induced by indomethacin was significantly inhibited with treatment of rolipram. Mucosal MPO activity was also suppressed by administration of rolipram. Gastric mucosal PGE2 concentration was not affected by intraperitoneal injection of rolipram. Based on these data, the beneficial effects of rolipram on indomethacin-induced gastric mucosal injury may be attributed to its anti-inflammatory properties. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: NSAIDs; Mucosal injury; Phosphodiesterase inhibitor; Neutrophil
1. Introduction Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are widely used as anti-inflammatory, analgesic agents [1 /3]. However, gastrointestinal injury is a serious adverse effect of NSAIDs, and effective strategies to protect the gastrointestinal mucosa are required. Many studies have been reported to understand the mechanisms for the development of indomethacin-induced mucosal lesion [4 /6]. Although, as the mechanism of gastric lesion induced by NSAIDs, suppression of endogenous prostaglandin (PG) production resulted from cyclooxigeanse (COX) inhibition is thought to be crucial, the exact pathogenic mechanism
* Corresponding author. Tel.: /81-18-834-1111; fax: /81-18-8362611. E-mail address: [email protected] (M. Otaka).
remains to be elucidated [7]. Several investigators have reported that intraperitoneal injection of anti-neutrophil serum and immunoneutralization of adhesion molecules on neutrophils and endothelial cells significantly attenuate the gastric mucosal injury induced by NSAIDs [8,9]. Therefore, activation and infiltration of neutrophils are critical factors to develop gastric mucosal lesion induced by NSAIDs. On the other hand, recently, the potential usefulness of a specific type IV phosphodiesterase inhibitor, rolipram, as a novel anti-inflammatory agent has been reported [10,11]. A specific type IV phosphodiesterase inhibitor by suppression of breakdown of cAMP to 5AMP increases intracellular cAMP in neutrophils and decreases TNF-a and N -formyl-methionyl-leucyl-phenylamine (FMLP)-stimulated neutrophil adherence, production of reactive oxygen species, and degranulation [12]. In addition, rolipram has been reported to ameliorate experimentally induced colonic
0928-4680/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0928-4680(03)00005-1
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mucosal lesion and ischemia reperfusion injury of kidney in animal models [13]. Also, it has been reported that rolipram prevented NSAIDs-induced enteropathy [14]. However, the effect of rolipram or significance of type IV phosphodiesterase in the pathogenesis of gastric mucosal lesion has not been reported. In this study, we examined the effect of a specific type IV phosphodiesterase inhibitor, rolipram, on indomethacin-induced gastric mucosal lesion and myeloperoxidase (MPO) activity in the gastric mucosa to understand the pathogenesis of NSAIDs-induced gastric mucosal lesion. Also, the effect of rolipram on prostaglandin E2 (PGE2) concentration in gastric mucosa was studied.
2. Materials and methods 2.1. Animals Eight-week-old male Sprague /Dawley rats (250 /300 g) were fed on standard laboratory diet and water ad libitum, and kept in cages in temperature (239/2 8C) and humidity (559/5%) controlled room with a 12:00 h dark:12:00 h light cycle before and during the experiment. All of the experiments were performed in the animals deprived of food for 24 h but with free access to water before experiments. This experiment is approved by the Akita University Animal Care Committee. 2.2. Chemicals Specific phosphodiesterase IV inhibitor, rolipram, was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rolipram was dissolved in small volume of dimethylsulfoxide and then diluted with physiological saline just before injection. 2.3. Effect of rolipram on indomethacin-induced gastric mucosal lesion
2.4. Effect of rolipram on myeloperoxidase (MPO) concentration in the gastric mucosa An assay of gastric mucosal MPO activity was applied to quantify the degree of neutrophil infiltration. Three hundred milligrams of scraped mucosa was homogenized for 30 s with polytron homogenizer (PT 1200, KInematica AG, Littau, Switzerland) in 1.0 ml of icecold 0.5% hexadecyliltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0). Hexadecyliltrimethylammonium bromide is used to negate the pseudoperoxidase activity of the hemoglobin and to solubilize membrane-bound MPO. The homogenate was sonicated (U50 IKA Werke GrmlH and Co. KG, Staufen, Germany) for 10 s, freeze-thawed three times and centrifuged for 20 min at 18 000 /g . The supernatant was taken for determination of the enzyme activity utilizing an ELISA kit (Bioxytech, Oxis International, Inc. Portand, OR, USA). The change in absorrbance at 405 nm was measured with a spectrophotometer (Microplate reader model 3550, Bio-Rad, Hercules, CA, USA). The concentration of MPO was expressed as nanogram per mg protein using Bradford’s method [16]. 2.5. Effect of rolipram on mucosal content of PGE2 To determine the effect of rolipram on PGE2 synthesis, the animals were sacrificed 6 h after rolipram administration and the stomach was removed. A part of fundic mucosa (about 100 mg) was exised for determination of PGE2 synthesis according to the method described previously [17]. The samples were weighed, finely minced with scissors for 15 s, then suspended in 1.0 ml of 10 nM sodium phosphate buffer (pH 7.4). The samples were then incubated in a shaking bath (37 8C) for 29 min followed by centrifugation (9000 /g for 30 s). The supernatant was frozen and subsequent determination of PGE2 was performed by specific radioimmunoassays using PGE2 [125I] RIA kit (Dupont/NEN, Boston, MA, USA). 2.6. Data analysis
Indomethacin-induced gastric injury was produced by the intragastric administration of indomethacin (30 mg/ kg). Indomethacin was dissolved in 3% sodium bicarbonate solution and 1 ml of the solution was administrated. Rolipram (0.125, 0.25, 0.5 mg/kg, n /5 in each dose) or vehicle was intraperitoneally injected 30 min prior to indomethacin administration. Control rats were given only physiological saline. The animals were sacrificed by stunning and cervical dislocation 6 h after indomethacin administration and the stomach was removed. The gastric mucosal lesions were measured by two independent observers blind to the treatment. The ulcer index was calculated as the sum of the lengths of all lesions [15].
All data were expressed as mean9/S.E.M. Statistical analysis was performed using Mann /Whitney U -test. P value B/0.05 was considered to be statistically significant.
3. Results 3.1. Effect of rolipram on indomethacin-induced gastric mucosal injury Indomethacin caused multiple hemorrhagic erosions and bleeding developed in the glandular stomach 6 h
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Fig. 1. Macroscopic findings of the gastric mucosa 6 h after administration of indomethacin (30 mg/kg) with or without pretreatment with rolipram (0.5 mg/kg, i.p.). Control: treated with vehicle. Multiple hemorrhagic erosions in the glandular stomach of rats 6 h after administration of indomethacin. Pretreatment with rolipram at a dose of 0.5 mg/kg markedly reduced these erosions.
after indomethacin administration. In contrast, pretreatment of rolipram inhibited the gastric lesions (Fig. 1). The ulcer index in vehicle-treated rats was 46.59/2.5 mm. The ulcer index in rats pretreated with rolipram was significantly suppressed to 14.59/1.94 mm (0.125 mg/kg, P B/0.001), 7.49/0.93 mm (0.25 mg/kg, P B/ 0.001), or 3.69/1.57 mm (0.5 mg/kg, P B/0.001) in a dose dependent manner (Fig. 2). The protective effect of rolipram was confirmed histologically. Indomethacin administration resulted in large areas of epithelial crypt
loss, predominantly neutrophilic infiltrate throughout the mucosa and sub-mucosa, erosions, and mucosal bleeding. In contrast, pre-treatment with rolipram resulted in smaller erosions with few neutrophils (Fig. 3).
3.2. Effect of rolipram on myeloperoxidase activity in gastric mucosa Fig. 4 shows the gastric mucosal MPO concentration in rats 6 h after indomethacin administration. The MPO activity in vehicle-treated rats was 15.89/2.77 ng/mg protein. The MPO concentration was suppressed by rolipram pretreatment to 9.179/1.11 ng/mg protein (0.125 mg/kg, P B/0.05), 7.349/2.35 ng/mg protein (0.25 mg/kg, P B/0.05), or 0.629/0.61 ng/mg protein (0.5 mg/kg, P B/0.05) compared with vehicle-treated rats.
3.3. Effect of rolipram on mucosal content of PGE2
Fig. 2. Ulcer index of rats 6 h after administration of indomethacin with or without pretreatment with rolipram (0.125, 0.25, 0.5 mg/kg, i.p.), (n /5 in each group) * P B/0.001, significant difference compared with vehicle treated group.
The concentration of PGE2 was 37.29/14.5 pg/mg in vehicle-treatment rats. Rolipram administration did not interfere with reduction of gastric PGE2 concentration (Fig. 5).
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Fig. 3. Microscopic findings of gastric mucosa 6 h after administration of indomethacin with or without pretreatment with rolipram (0.5 mg/kg, i.p.). Original magnification 100 and 200 / (H and E staining). Hemorrhagic mucosal erosions and inflammatory cell infiltration developed in the glandular stomach of rats 6 h after the administration of indomethacin. Pretreatment of rolipram at a dose of 0.5 mg/kg markedly inhibited these changes.
Fig. 4. Effect of rolipram on MPO concentration in the gastric mucosa (n /5 in each group). * P B/0.05, significant difference compared with vehicle treated group.
Fig. 5. Effect of rolipram on PGE2 concentration in the gastric mucosa. NS, statistically no significant difference.
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4. Discussion The present study demonstrated for the first time that rolipram prevents indomethacin-induced gastric mucosal injury. Rolipram significantly inhibited the increase in MPO concentration and neutrophil accumulation index in the gastric mucosa 6 h after indomethacin administration. Various mechanisms and factors, such as stimulation of gastric acid, inflammatory cell infiltration, cytokines, mucosal blood flow, and free radicals have been known to contribute to the development of NSAIDs-induced gastric mucosal damage [18]. Recently, it has been reported that neutrophil depletion by intraperitoneal injection of anti-neutrophil serum significantly attenuates the gastric mucosal injury induced by indomethacin [19]. Also, it has been proved that immunoneutralization of the CD11b/CD18 adherence complex on neutrophils inhibited indomethacininduced gastric injury [20]. Therefore, neutrophilsmediated inflammation is one of the critical factors in the development of indomethacin-induced gastric mucosal injury. Rolipram has initially been developed and studied in clinical trials as an antidepressant [21]. Recently, the potential usefulness of specific type IV phosphodiesterase inhibitor, rolipram, as a novel antiinflammatory agent has been reported [22,23]. Agent that elevate the concentration of intracellular cAMP can inhibit inflammatory cell activities such as cytokine production, chemotaxis, cytotoxicity and cell aggregation [24 /26]. Rolipram inhibits neutrophil activation and TNF-a mediated neutrophil adhesion to the vascular endothelium. And it reduces the synthesis and release of reactive oxygen species. In addition, rolipram has been reported to inhibit experimentally-induced colonic lesion in animal model [27]. These findings led us to examine the effect of rolipram on indomethacininduced gastric mucosal lesion. In this study, we clearly demonstrated that the rolipram ameliorated the development of gastric mucosal injury induced by indomethacin administration and highest dose tested caused an approximately 90% reduction in gastric mucosal lesion. Histopathological findings showed the infiltration of inflammatory cells in submucosal layer was dramatically suppressed by rolipram. Further, MPO activity increases in the gastric mucosa 6 h after indomethacin administration in rats treated with vehicle solution, and the increase in the MPO concentration is significantly inhibited by treatment with rolipram. These results indicate that the inhibition of neutrophil infiltration by rolipram may be one of the protective factors decreasing indomethacin-induced gastric mucosal injury. It has been reported that PGE2 prevented gastric mucosal damage by indomethacin in human and animals [28]. However, the protective effect of rolipram did not depend on gastric mucosal prostaglandin synthesis, since pretreatment with rolipram, which reduced gastric
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damage had no effect on the gastric mucosal prostaglandin concentration. On the other hand, phosphodiesterase inhibitor is known to inhibit TNF-a production in vitro and in vivo [29]. The predominant phosphodiesterase isoenzyme family in monocytes, a main source of TNF-a production, is the type IV phosphodiesterase. The specific type IV phosphodiesterase inhibitor, rolipram, is a 500-fold more potent inhibitor of TNF synthesis compared with nonspecific phoshodiesterase inhibitor, pentoxifylline, in human mononuclear cells [30]. TNF-a is a proinflammatory cytokine and has recently been shown to be a crucial mediator of NSAIDs induced gastric mucosal injury [31]. Also, TNF-a is a cytokine that strongly stimulates neutrophil adherence by inducing the synthesis and expression of adhesion molecules on endothelial cells and neutrophils [32,33]. Furthermore, studies on experimental models have shown that intravenous administration of TNF-a produces extensive neutrophil infiltration within the micorvasculature of the digestive tract. In addition, portal infusion of TNF-a caused gastric and small intestinal damage in rats [34]. It has been also shown that NSAIDs administration increases TNF-a concentrations in both humans and experimental animals [35,36]. These evidences indicate that the anti-ulcer effect of rolipram might be caused by inhibition of TNF-a production.
5. Conclusion Rolipram prevented indomethacin-induced gastric damage in rats. The effect of rolipram is not related to the synthesis of prostaglandins but depended on inhibition of neutrophil infiltration to gastric mucosa. These results indicate that agents which modulates cAMP could be clinically useful for the therapy of NSAIDsinduced gastropathy.
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