Role of prostaglandins generated by cyclooxygenase-1 and cyclooxygenase-2 in healing of ischemia–reperfusion-induced gastric lesions

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Abstract

In this study, ischemia–reperfusion produced in rats by clamping the celiac artery for 0.5 h followed by 1 h of reperfusion was used to develop a new model of superficial gastric erosions progressing to deeper ulcers. Ischemia alone resulted in an immediate fall in gastric blood flow but no gross mucosal lesions were observed. When ischemia was followed by reperfusion, gastric erosive lesions occurred, reached a maximum at 12 h and then declined after 24 h. These acute erosions progressed into deeper lesions 24 h after ischemia–reperfusion and reached a peak after 3 days. Gastric blood flow and the mucosal generation of prostaglandin E2 were significantly suppressed immediately following ischemia–reperfusion, but with the healing of deeper gastric ulcers, both gastric blood flow and prostaglandin E2 generation were gradually restored. Cyclooxygenase-1 mRNA was detected by reverse transcription-polymerase chain reaction in intact gastric mucosa and throughout the recovery of the mucosa from acute ischemia–reperfusion lesions, whereas cyclooxygenase-2 mRNA, was recorded only after ischemia–reperfusion. NS-398 and rofecoxib, selective inhibitors of cyclooxyganase-2, failed to affect prostaglandin E2 generation in the non-ulcerated gastric mucosa but inhibited it significantly in the ulcer area. The two cyclooxygenase-2 inhibitors as well as resveratrol, a specific cyclooxygenase-1 inhibitor and indomethacin and meloxicam, non-specific inhibitors of cyclooxygenase, augmented acute gastric erosions induced by ischemia–reperfusion and delayed significantly the progression of these lesions into deeper ulcers at each time interval after ischemia–reperfusion. We conclude that prostaglandins generated by both cyclooxygenase-1 and cyclooxygenase-2 contribute to the healing of gastric lesions induced by ischemia–reperfusion.

Introduction

Ischemia followed by reperfusion is known to induce gastric lesions predominantly due to excessive formation of reactive oxygen species, neutrophil activation and microvascular dysfunction Itoh and Guth, 1985, Yoshikawa et al., 1992, but the involvement of endogenous prostaglandins, which are an important component of the gastric mucosal defense (Robert, 1979), in the mechanism of the healing of these lesions has been little examined. Previous studies documented that ischemia weakens the gastric mucosal barrier and increases the acid back diffusion, thus predisposing the mucosa to damage Itoh and Guth, 1985, Kawai et al., 1994. After reperfusion, the generation of reactive oxygen species and the activation of neutrophils were found to increase the lipid peroxidation, which in combination with the aggressive action of gastric acid resulted in cellular death and mucosal damage Andrews et al., 1994, Wada et al., 1996.

It is generally accepted that there are two isoforms of cyclooxygenase: constitutive cyclooxygenase-1, which produces PG for physiological reactions including the maintenance of mucosal integrity, gastric microcirculation, secretory activity and motor functions, and inducible cyclooxygenase-2, which is triggered by various cytokines, growth factors and endotoxins Xie et al., 1991, Eberhart and Dubois, 1995, Feng et al., 1995. While prostaglandins produced by cyclooxygenase-1 appear to contribute to the physiological control of mucosal integrity, cyclooxygenase-2 products have been implicated in inflammatory reactions Xie et al., 1991, Feng et al., 1995. Recently, an increased cyclooxygenase-2 expression was shown in gastric mucosa exposed to ischemia–reperfusion Kishimoto et al., 1998a, Kishimoto et al., 1998b but no attempt has been made to compare the effects of specific cyclooxygenase-1 and cyclooxygenase-2 inhibitors with the effects of classic non-specific inhibitors of cyclooxygenase such as indomethacin on the healing of ischemia–reperfusion-induced damage and to determine which cyclooxygenase isoform contributes to spontaneous recovery of gastric mucosa from lesions induced by ischemia–reperfusion.

Using the model of gastric lesions induced by ischemia–reperfusion as proposed recently by Wada et al. (1996), we examined the involvement of prostaglandins in the appearance of acute gastric erosions and their progression to deeper ulcers and their role in gastric blood flow induced by ischemia–reperfusion in rats treated with non-selective and selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors. Among the cyclooxygenase inhibitors tested, we used resveratol, which is a phenolic product derived from grapes and which has been shown to inhibit selectively cyclooxygenase-1 and hydroxyperoxidase of cyclooxygenase-1 with an ED50 of 3.7 μm (Jang et al.,1997). We also determined whether addition of minute doses of exogenous PG to these inhibitors could influence the healing of gastric lesions and gastric blood flow in rats subjected to ischemia–reperfusion. In addition, we attempted to assess the expression of cyclooxygenase-1 and cyclooxygenase-2 mRNA, as determined with the reverse transcription-polymerase chain reaction, and to measure the changes in plasma gastrin and interleukin-1β levels in intact gastric mucosa and in mucosa exposed to ischemia–reperfusion and showing gastric lesions. Since proinflammatory cytokines such as interleukin-1β influence gastric secretion and ulcer healing (Robert et al., 1991), our rationale was to study the expression of interleukin-1β mRNA and plasma levels of this cytokine along with the time course of recovery from ischemia–reperfusion damage. Lastly, we attempted to determine the involvement of gastrin in the healing of ischemia–reperfusion damage since this hormone exhibits protective activity against lesions induced by corrosive substances such as absolute ethanol (Konturek et al., 1995). Moreover, prolonged hypergastrinemia contributes to the healing of pre-existing ulcers in the rat stomach (Li and Helander, 1996).

Section snippets

Production of lesions induced by ischemia–reperfusion

Ischemia–reperfusion erosions were produced in 120 rats by the method originally described by Wada et al. (1996). Briefly, under pentobarbital anesthesia (50 mg/kg i.p.), the abdomen was opened and the celiac artery was identified and clamped with a small device for 30 min, followed by removal of the clamp to obtain reperfusion. Erosions were measured immediately after 30 min of ischemia, after 60 min of reperfusion (time 0) and then after 3, 12 or 24 h after the termination of

Gastric lesions produced by ischemia–reperfusion and mucosal recovery from these lesions

Immediately after 30 min clamping of the celiac artery (ischemia), no gastric lesions were observed, but after 60 min of reperfusion, superficial bleeding gastric erosions were found in all tested stomachs. The area of these lesions was significantly increased at 3 h, peaked at 12 h after ischemia–reperfusion but then declined at 24 h after ischemia–reperfusion (Fig. 1). These superficial erosions progressed into smaller but deeper ulcerations whose area reached a maximum at day 3 after

Discussion

In this study a novel model was used to induce gastric lesions, namely, ischemia–reperfusion. Initially, acute erosions were produced that progressed into deeper chronic ulcers which, like naturally occurring ulcers in humans, appeared spontaneously and healed spontaneously within 15 days after ischemia–reperfusion. We confirmed the original observation of Wada et al. (1996) that the exposure of the gastric mucosa to ischemia induced by clamping of the celiac artery followed by reperfusion

Acknowledgements

This study was supported in part by the Interdisciplinary Centre for Clinical Research at the University of Erlangen, Nuremberg, Germany. The authors thank Mrs. Kinga Ogonowska for her expert technical assistance during the preparation of the manuscript.

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