Original Paper
Digestion 1992;53:45-53
Second Department of Surgery, and Critical Care and Emergency Medicine, Yokohama City University School of Medicine, Yokohama, Japan
Effect of Teprenone on Acute Gastric Mucosal Lesions Induced by Cold-Restraint Stress
KeyWords
Abstract
Teprenone Acute gastric mucosal lesions Defensive factors Lesion-enhancing factors Obstructive jaundice
The effects of intragastric administration of teprenone on acute gastric mucosal lesions induced by cold-restraint stress was investigated using a model of obstructive jaundice. Rats received teprenone 200 mg/kg/day for a week before stress; nontreated rats served as controls. Teprenone suppressed stress-induced depressions in defensive factors (blood flow, transmucosal potential difference, hexosamine content and lectin staining of carbohydrate residues) and suppressed in creases in lesion-enhancing factors (gastric mucosal lysosomal enzyme activity and thiobarbituric acid reactants showing lipid peroxidation). Intragastric pH did not change signifi cantly with teprenone but the ulcer index decreased. These results showed that teprenone protects gastric mucosa against stress, even in the presence of obstructive jaundice.
Recently, various investigators have at tempted to evaluate the pathogenic mecha nism of acute gastric mucosal lesions (AGML). Defensive factors, such as gastric mucosal blood flow, mucus, bicarbonate and others, are thought to play an important role in the etiology of AGML, and various drugs which maintain or accelerate defensive fac tors have been developed recently to prevent peptic ulcers, including AGML. One such
Received: February 10, 1992 Received in revised form:
drug is teprenone, a new polyisoprenoid, which was derived from vitamin A. Experi mental ulcers induced by aspirin, indomethacin, ethanol or cold-restraint stress have been inhibited by teprenone independently of any inhibition of acid secretion [1-3], Clinically, many authors have demonstrated the ability of teprenone to prevent AGML in patients with renal failure, bronchial asthma, liver cir rhosis or rheumatoid arthritis, probably by
Chikara Kunisaki, MD Department of Surgery. Fujisawa City Hospital 2-6-1. Fujisawa. Kanagawa 251 (Japan)
© 1992 S. Karger AG. Basel 0012-2823/92/ 0532-0045$2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:15 PM
Cliikara Kunisakia Mitsugi Sugiyam ah
Materials and Methods Animal Preparation Our research was conducted with male Wistar rats weighing 250-300 g divided into two groups, a tepre none group and a nontreated group. Teprenone as an emulsion in 5% gum arabic and 0.6% Tween 80 was instilled intragastrically in a dose of 200 mg/kg body weight twice per day for 1 week from the 7th day after ligation of the common bile duct to stimulate obstruc tive jaundice. The dose chosen was based on earlier studies [1,3. 5], Cold-restraint stress was induced 2 weeks after ligation of the bile duct using cold water immersion. Rats were deprived of food for 24 h before the experiments but had free access to water. Three hours after the final instillation of teprenone, the experimental protocol was started, and at 1. 2, 3 and 6 h after immersion rats were anesthetized with so dium pentobarbital (50 mg/kg). and the abdominal wall was opened to investigate the following parame ters. Defensive Factors Gastric mucosal blood flow was measured at the greater curvature of the corpus using a hydrogen gas clearance method. The data were expressed as percent
46
Kunisaki/Sugivama
ages of the values before stress in the nontreated group. The gastric mucosal hexosamine level was mea sured as a representative substance in mucus, using Neuhaus’ method [6], Specimens of about 20-25 mmwerc excised from the greater curvature of the corpus. Transmucosal electric potential difference (PD) was measured using bridges made with polyethylene tubes filled with saturated KC1 in 3% agar: one end of the bridge was placed at the gastric fundic mucosa and the other end was positioned at the peritoneum. The electrodes were inserted into the stomach through the opened forestomach and the PD was measured imme diately at the greater curvature. Lectin stains were used to detect changes in the dis tribution, quantity and quality of carbohydrate resi dues in the gastric mucosa, utilizing specific lectins to bind specific carbohydrate residues. Specimens cut from the greater curvature were fixed in 4% para formaldehyde phosphate buffer (pH 7.4) and embed ded in paraffin. The sections were processed by routine methods including the avidin-biotin complex tech nique. Arachis liypogaea agglutinin. Ricinus communis agglutinin (RCA-1), Ulex europaeus agglutinin, Triticum vulgaris agglutinin and concanavalin A were used as representative lectins in this research. Lesion-Enhancing Factors Intragastric pH was measured at the greater curva ture of the corpus through the forestomach, using glass electrodes. Gastric mucosal lysosomal enzymes were also mea sured. It has been reported that p-N-acetyl-D-glucosaminidase and |l-glucuronidase. as gastric mucosal glycosidases. are representative lysosomal enzymes, predominant in mucosa. We considered these enzymes to be lesion-promoting factors because, at an optimal acid pH. they are capable of degrading every' known constituent of cells, including mucus granules. Lyso somal enzymes were measured biochemically using Larusso's method [7], Lysosomal membrane integrity, or latent enzyme activity, was calculated by assaying enzyme activity in the absence or presence of the deter gent Triton X. and expressed as a percentage according to the following equation: Latent enzyme activity = total activity - free activity total activity
-------------------------------------X 100 (%).
The reaction of lipid peroxides with thiobarbituric acid (TBA) has been widely adopted as a sensitive
Effect ofTeprenone on Experimentally Induced AGML
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:15 PM
promoting the elaboration of glycoprotein, phospholipid and bicarbonate and by main taining blood flow; these observations suggest that teprenone is capable of gastric mucosal protection. In addition, teprenone has been reported to stimulate mucus synthesis and secretion in vitro [4], However. the absorptive and metabolic characteristics of this agent are not understood precisely; in particular, it is not known whether teprenone is absorbed from gut lumen without bile acid, as in the presence of obstructive jaundice. We used an experimental protocol with cold-restraint stress, simulating clinical surgical stress to elucidate the metabolism of this agent and its effect on experimentally induced AGML. We employed a model of obstructive jaundice, which is frequently associated with AGML clinically.
m ean ± $ f
assay method for lipid peroxidation, although its speci ficity is rather low. To evaluate the degree of change in lipid peroxidation after cold-restraint stress, TBA reac tants in gastric mucosa were measured using Yagi’s method [8]. Ulcer Index Stomachs were removed and cut open along the greater curvature, and lesions such as erosions or ulcers were measured macroscopically (length and width in mm). In this study, the ulcer index for each group w'as expressed as the sum of all lesion areas (length X width, mm-). Statistical A nalysis The results reported in this research are expressed as the mean ± SE. Data were analyzed using the unpaired Student’s t test.
Results
Defensive Factors Gastric mucosal blood flow decreased sig nificantly (p < 0.01) from prestress levels to 41.5 ± 4.8%. 33.3 ± 3.9%, 25.3 ± 5.0%and 15.4 ± 0.7% at l, 2. 3 and 6 h. respectively.
after stress in the nontreated group (fig. I). Oral administration of teprcnone signifi cantly reduced the decrease in gastric mucosal blood flow after stress at each time period. Gastric mucosal hexosamine levels de creased remarkably in the nontreated group from 12.3 ± 0.51 pg/mg (prestress) to 8.9 ± 0.64 and 8.0 ± 0.64 pg/mg at 3 and 6 h after stress, respectively. In the teprenone group, this decrease was inhibited, although not sig nificantly (fig. 2). Transmucosal PD decreased after stress in a manner similar to that of other defensive factors in both groups. In the nontreated group. PD decreased significantly compared with values in the teprenone group, to -13.7 ± 0.9 and - 10.7 ± l .2 mV at 3 and 6 h after stress, respectively (p < 0.01: fig. 3). Lectin stains with RCA-l. the brown reac tive products were observed in the parietal, mucous neck and foveolar cells in the non treated gastric mucosa. In rats with obstruc tive jaundice, reactive products diminished or disappeared in these cells. In the teprenone
47
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Fig. 1. Effect of teprcnone on gastric mucosal blood How. De crease in blood flow was reduced significantly by teprenone. * p < 0.05 and ** < 0.01 vs. nontreated group.
Fig. 3. Effect of teprenonc on transmucosal PD. As with other defensive factors. PD decrease was reduced significantly by teprenone. * p < 0.01 vs. nontreated group.
48
Kunisaki/Sugiyama
Effect of Teprenonc on Experimentally Induced AGML
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Fig. 2. Effect of teprenonc on gastric mucosal hexosamine levels. Decrease in hexosamine level was reduced by teprenone. but not sig nificantly. *p < 0.05 vs. nontreated group.
UEA-1
Control
w'
Teprenone
pre
6h
group, the staining observed in cells remained at prestress levels, and the decrease in staining was reduced even after stress (fig. 4). With other lectins, similar changes were observed not only in mucus-producing cells but also in parietal cells. Lesion-Enhancing Factors
Intragastric pH increased after stress in both groups, but not significantly. Teprenone did not affect acid secretion, and no signifi cant difference was detected between the two groups (fig. 5). Latent enzyme activity of P-N-acetyl-Dglucosaminidase decreased after stress, as the
instability of the lysosomal membrane in creased. reaching 24.8 ± 3.4% after 3 h and gradually tending to recover. Similarly, latent enzyme activity of p-glucuronidase decreased to 17.2 ± 1.4% at 2 h and 15.6 ± 2.6% at 3 h after stress (p < 0.05) and to 18.2 ± 2.5% at 6 h after stress (p < 0.01; fig. 6). TBA reactants increased significantly from prestress levels to 192.8 ± 9.3 nmol/g wet weight at 30 min after stress, and gradually recovered to prestress values at 3 h after stress in the nontreated group. In the teprenone group, the values in the course of this study tended to decrease but not significantly (fig. 7).
49
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Fig. 4. Differences between the teprenone and nontreated groups in the distribution and quantity of carbohydrate residue comprising the glycoprotein were assessed histologically with representative lectins. In the teprenone group, the brown reactive products were more evident in mucous neck cells or parietal cells, as compared to those in the nontreated group. With other lectins, similar results were obtained. X 100.
Fig. 5. Effect of teprenonc on intragastric pH. Teprenone did not affect acid secretion, as observed in previous studies.
The ulcer index at 6 h after stress was sig nificantly reduced in the teprenone group compared with the nontreated group (22.6 ± 2.1 vs. 14.8 ± 1.6 mm2; p < 0.02; fig. 8).
Discussion
In 1981, teprenone was developed as a new acyclic polyisoprenoid derivative of vitamin A by Murakami et al. [1, 9], Many authors
50
Kunisaki/Sugjyama
have found that this agent has the potential to suppress experimental AG ML induced byethanol [2] or aspirin [10]. and Shay's coldrestraint, indomethacin and others forms of ulcer [1], without affecting gastric acid secre tion. It has been demonstrated that teprenone stimulates mucus elaboration and secretion [4], maintains gastric mucosal blood flow, en hances synthesis of mucosal phospholipid [5] and acts cooperatively with endogenous pros taglandin. Some evidence suggests the oppo-
Effect of Teprenonc on Experimentally Induced AGML
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Fig. 6. Changes in gastric mu cosal glycosidases due to teprenone administration were evaluated. The increment in p-glucuronidase activity was suppressed signifi cantly in the teprenone group. A similar tendency was observed with N-acctyl-p-D-glucosaminidase. * p < 0.05 and ** p < 0.01 vs. nontreated group.
site results with prostaglandin [3. 4], and this point remains controversial. The mechanism of teprenone has not been clarified yet, but oral administration may permit lymphatic ab sorption of teprenone. Nakazawa et al. [11] reported that after food intake, the bioavail ability of teprenone was enhanced as com pared to that in the fasting state, probably due to a stimulation of gastric blood flow and secretion of bile and pancreatic juice, includ ing bile acids, fatty acids and monoglycerides. These constituents are necessary for mixed micelle formation and the biosynthesis of chylomicra, which are essential for lymphatic transport. In obstructive jaundice, lymphatic absorption of teprenone is reduced physiolog ically, and the benefits of the drug may de crease or disappear clinically. In our research, the absorption of teprenone in experimental obstructive jaundice and its effect on experi mentally induced AGML were assessed. We used cold-restraint stress as a model of clinical
Fig. 8. Ulcer index was calculated at 6 h after coldrestraint stress in both groups. In the reprenone group, the index was suppressed due to an acceleration of maintenance of defensive mechanisms, p vs. nontreated group.
51
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Fig. 7. Changes in TBA reac tants were measured biochemical ly. Increases in TBA reactants were reduced by the addition of teprenonc but not significantly.
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Kunisaki/Sugivama
by ischemia or as a promotor of gastric muco sal blood flow. Other studies have also indi cated that teprenone has a direct function as a free radical scavenger, but this question re quires further investigation. The increment of lysosomal enzyme activity was also sup pressed by the addition of teprenone, possibly because the drug maintained the stability of the lysosomal membrane by decreasing free radical activity induced by ischemia-repcrfu sion or by inhibiting ischemia. In short, isch emia induced by various kinds of stress acti vates free radicals, which injure lysosomal membranes in the course of lipid peroxida tion. Furthermore, activated lysosomal en zymes degrade all kinds of cell constituents by entering cells during ischemia. In a normal physiological state, lysomal enzymes in gas tric mucosa are distributed chiefly in surface epithelial and mucous neck cells, but they do not participate in cell kinetics [12]. In macro phages or neutrophils, lysosomal enzymes act by wrapping or fusion systems but, in an isch emic state, lysosomal enzymes may enter the cells as the instability of lysosomal mem branes increases, and may act even in gastric mucosal cells. We believe that these phenom ena contribute to the development of AGML. Teprenone may exert antiulcer effects by breaking this chain of events, for example, by accelerating the synthesis of mucus or phos pholipid. maintaining gastric mucosal blood flow, and suppressing the activity of free radi cals. In our research, the mechanism of ab sorptive access could not be defined precisely, but teprenone may act without micele forma tion to bile acid in the presence of obstructive jaundice. With regard to this point, further research is needed to evaluate the function of teprenone, including its effect on free radi cals.
Effect ofTcprcnonc on Experimentally Induced AGML
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AGML after surgery. In this experimental model, gastric defensive factors such as gas tric mucosal blood flow, hexosamine levels and PD decreased after stress. Acid secretion was depressed, probably due to a hypofunction of parietal cells, induced by ischemia. Gastric mucosal glycosidases, which we re garded as lesion-enhancing factors in terms of entering cells and the blood stream and de stroying or damaging ischemic cells and or gans, were also activated due to increasing instability of lysosomal membranes after stress, with a peak at 2-3 h. Previously, we had evaluated these parameters biochemi cally and electron microscopically with acid phosphatase and found that gastric mucosal glycosidases were activated and had a role in damaging mucosal cells after stress induced by burn or cold restraint. Oxidative stress induced by ischemia-repcrfusion has been re ported to involve free radicals originating from the xanthine-xanthine oxidase system in endothelial cells or NADPH oxidase in neu trophils, with a role in damaging cells. We concluded that a reduction in defensive fac tors triggered AGML formation and that an increase in lesion-enhancing factors, exclud ing acid secretion, accelerated AGML devel opment: the existence of but not an increase in gastric acid appeared to be necessary for the evolution of AGML. We evaluated the effects of teprenone in view of the balance between gastric defensive factors and lesionpromoting factors, and found that, even in the presence of obstructive jaundice, teprenone stimulates secretion and biosynthesis of gly coprotein and maintains gastric mucosal blood flow to some extent. With regard to lesion-promoting factors, although acid secre tion did not change with the administration of teprenone, the activation of free radicals was suppressed, probably due to the drug's direct function as a scavenger or its indirect function as an inhibitor of lipid peroxidation induced
References 4 Terano A. Hiraishi H. Ohta S. Suginioto T: Geranylgeranylacetone, a novel anliulccr drug, stimulates mu cus synthesis and secrcliion in rat gastric cultured cells. Digestion 1986:32:206-210. 5 Nishizawa Y. Sakurai H, Oketani K. Horie T. Yamato C. Moriga M: Ef fects of taurocholic acid/HCI alone or after pretreatment with geranylgeranylacclone on phospholipid me tabolism in rat gastric mucosa. Biochem Pharmacol 1987:23:4111 — 4117. 6 Neuhaus OW, Letzring M: Determi nation of hexosamines in conjunc tion with electrophoresis on starch. Anal Chem 1957;29:1230-1233. 7 Nagorney DM. Larrusso NF. Dozois RR: Development application of methodology for assessing the role of lysosomes in experimental ulcerogenesis in the guinea pig. Gas troenterology 1983:85:548-556. 8 Yagi K: A simple fluorometr.c assay for iodoperoxide in blood plasma. Biochem Med 1976:15:217-219.
9 Murakami M, Oketani K, Fujisaki H, Wakabayashi T, Ohgo T: Effects of the antiulccr drug geranylgeranylacetonc on aspirin-induced gas tric ulcers in rats. Jap J Pharmacol 1982;32:299-306. 10 Terano A. Shiga J. Hiraishi H, Ohta S, Sugimoto T: Protective action of tetraprenvlacetone against ethanolinduced damage in rat gastric muco sa. Digestion 1986:35:182—188. 11 Nakazawa S. Tsuboi Y, Tsukamoto Y. Yoshino J. Okada M: Scrum and stomach tissue levels of geranvlgeranylacctone in patients, int J Clin Pharmac Ther Toxicol 1983:6:267270. 12 Katsuoka K. Yakeoka Y. Hirano S: Electron microscopic observation of surface mucous cells in the mouse gastric mucosa during physiological degeneration and extrusion. Arch Histol Jap 1985:3:327-339.
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1 Murakami M. Okelani K. Fujisaki H, Wakabayashi T, Ohgo T: Anli ulcer cfTecI of geranylgeranylacetone. a new acyclic polyisoprenoid on experimentally induced gastric and duodenal ulcers in rats. Arzneini ittelforschu ng 1981:31:799804. 2 Oketani K. Murakami M. Fujisaki H. Wakabayashi T: Effect of gcranylgcranylacetone on aspirin-in duced changes in gastric glycopro teins. Jap J Pharmacol 1983:33: 593-601. 3 Bilski J. Murty VLN, Nadziejko C. Sarosiek J. Aono M. Moriga M. Slomiany A, Slomiany BL: Protec tion against alcohol-induced gastric mucosal injury by geranylgeranvlacetone; Effect ofindomethacin. Di gestion 1988:41:22-33.
Digestion 1992;53:45-53
Second Department of Surgery, and Critical Care and Emergency Medicine, Yokohama City University School of Medicine, Yokohama, Japan
Effect of Teprenone on Acute Gastric Mucosal Lesions Induced by Cold-Restraint Stress
KeyWords
Abstract
Teprenone Acute gastric mucosal lesions Defensive factors Lesion-enhancing factors Obstructive jaundice
The effects of intragastric administration of teprenone on acute gastric mucosal lesions induced by cold-restraint stress was investigated using a model of obstructive jaundice. Rats received teprenone 200 mg/kg/day for a week before stress; nontreated rats served as controls. Teprenone suppressed stress-induced depressions in defensive factors (blood flow, transmucosal potential difference, hexosamine content and lectin staining of carbohydrate residues) and suppressed in creases in lesion-enhancing factors (gastric mucosal lysosomal enzyme activity and thiobarbituric acid reactants showing lipid peroxidation). Intragastric pH did not change signifi cantly with teprenone but the ulcer index decreased. These results showed that teprenone protects gastric mucosa against stress, even in the presence of obstructive jaundice.
Recently, various investigators have at tempted to evaluate the pathogenic mecha nism of acute gastric mucosal lesions (AGML). Defensive factors, such as gastric mucosal blood flow, mucus, bicarbonate and others, are thought to play an important role in the etiology of AGML, and various drugs which maintain or accelerate defensive fac tors have been developed recently to prevent peptic ulcers, including AGML. One such
Received: February 10, 1992 Received in revised form:
drug is teprenone, a new polyisoprenoid, which was derived from vitamin A. Experi mental ulcers induced by aspirin, indomethacin, ethanol or cold-restraint stress have been inhibited by teprenone independently of any inhibition of acid secretion [1-3], Clinically, many authors have demonstrated the ability of teprenone to prevent AGML in patients with renal failure, bronchial asthma, liver cir rhosis or rheumatoid arthritis, probably by
Chikara Kunisaki, MD Department of Surgery. Fujisawa City Hospital 2-6-1. Fujisawa. Kanagawa 251 (Japan)
© 1992 S. Karger AG. Basel 0012-2823/92/ 0532-0045$2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:15 PM
Cliikara Kunisakia Mitsugi Sugiyam ah
Materials and Methods Animal Preparation Our research was conducted with male Wistar rats weighing 250-300 g divided into two groups, a tepre none group and a nontreated group. Teprenone as an emulsion in 5% gum arabic and 0.6% Tween 80 was instilled intragastrically in a dose of 200 mg/kg body weight twice per day for 1 week from the 7th day after ligation of the common bile duct to stimulate obstruc tive jaundice. The dose chosen was based on earlier studies [1,3. 5], Cold-restraint stress was induced 2 weeks after ligation of the bile duct using cold water immersion. Rats were deprived of food for 24 h before the experiments but had free access to water. Three hours after the final instillation of teprenone, the experimental protocol was started, and at 1. 2, 3 and 6 h after immersion rats were anesthetized with so dium pentobarbital (50 mg/kg). and the abdominal wall was opened to investigate the following parame ters. Defensive Factors Gastric mucosal blood flow was measured at the greater curvature of the corpus using a hydrogen gas clearance method. The data were expressed as percent
46
Kunisaki/Sugivama
ages of the values before stress in the nontreated group. The gastric mucosal hexosamine level was mea sured as a representative substance in mucus, using Neuhaus’ method [6], Specimens of about 20-25 mmwerc excised from the greater curvature of the corpus. Transmucosal electric potential difference (PD) was measured using bridges made with polyethylene tubes filled with saturated KC1 in 3% agar: one end of the bridge was placed at the gastric fundic mucosa and the other end was positioned at the peritoneum. The electrodes were inserted into the stomach through the opened forestomach and the PD was measured imme diately at the greater curvature. Lectin stains were used to detect changes in the dis tribution, quantity and quality of carbohydrate resi dues in the gastric mucosa, utilizing specific lectins to bind specific carbohydrate residues. Specimens cut from the greater curvature were fixed in 4% para formaldehyde phosphate buffer (pH 7.4) and embed ded in paraffin. The sections were processed by routine methods including the avidin-biotin complex tech nique. Arachis liypogaea agglutinin. Ricinus communis agglutinin (RCA-1), Ulex europaeus agglutinin, Triticum vulgaris agglutinin and concanavalin A were used as representative lectins in this research. Lesion-Enhancing Factors Intragastric pH was measured at the greater curva ture of the corpus through the forestomach, using glass electrodes. Gastric mucosal lysosomal enzymes were also mea sured. It has been reported that p-N-acetyl-D-glucosaminidase and |l-glucuronidase. as gastric mucosal glycosidases. are representative lysosomal enzymes, predominant in mucosa. We considered these enzymes to be lesion-promoting factors because, at an optimal acid pH. they are capable of degrading every' known constituent of cells, including mucus granules. Lyso somal enzymes were measured biochemically using Larusso's method [7], Lysosomal membrane integrity, or latent enzyme activity, was calculated by assaying enzyme activity in the absence or presence of the deter gent Triton X. and expressed as a percentage according to the following equation: Latent enzyme activity = total activity - free activity total activity
-------------------------------------X 100 (%).
The reaction of lipid peroxides with thiobarbituric acid (TBA) has been widely adopted as a sensitive
Effect ofTeprenone on Experimentally Induced AGML
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:15 PM
promoting the elaboration of glycoprotein, phospholipid and bicarbonate and by main taining blood flow; these observations suggest that teprenone is capable of gastric mucosal protection. In addition, teprenone has been reported to stimulate mucus synthesis and secretion in vitro [4], However. the absorptive and metabolic characteristics of this agent are not understood precisely; in particular, it is not known whether teprenone is absorbed from gut lumen without bile acid, as in the presence of obstructive jaundice. We used an experimental protocol with cold-restraint stress, simulating clinical surgical stress to elucidate the metabolism of this agent and its effect on experimentally induced AGML. We employed a model of obstructive jaundice, which is frequently associated with AGML clinically.
m ean ± $ f
assay method for lipid peroxidation, although its speci ficity is rather low. To evaluate the degree of change in lipid peroxidation after cold-restraint stress, TBA reac tants in gastric mucosa were measured using Yagi’s method [8]. Ulcer Index Stomachs were removed and cut open along the greater curvature, and lesions such as erosions or ulcers were measured macroscopically (length and width in mm). In this study, the ulcer index for each group w'as expressed as the sum of all lesion areas (length X width, mm-). Statistical A nalysis The results reported in this research are expressed as the mean ± SE. Data were analyzed using the unpaired Student’s t test.
Results
Defensive Factors Gastric mucosal blood flow decreased sig nificantly (p < 0.01) from prestress levels to 41.5 ± 4.8%. 33.3 ± 3.9%, 25.3 ± 5.0%and 15.4 ± 0.7% at l, 2. 3 and 6 h. respectively.
after stress in the nontreated group (fig. I). Oral administration of teprcnone signifi cantly reduced the decrease in gastric mucosal blood flow after stress at each time period. Gastric mucosal hexosamine levels de creased remarkably in the nontreated group from 12.3 ± 0.51 pg/mg (prestress) to 8.9 ± 0.64 and 8.0 ± 0.64 pg/mg at 3 and 6 h after stress, respectively. In the teprenone group, this decrease was inhibited, although not sig nificantly (fig. 2). Transmucosal PD decreased after stress in a manner similar to that of other defensive factors in both groups. In the nontreated group. PD decreased significantly compared with values in the teprenone group, to -13.7 ± 0.9 and - 10.7 ± l .2 mV at 3 and 6 h after stress, respectively (p < 0.01: fig. 3). Lectin stains with RCA-l. the brown reac tive products were observed in the parietal, mucous neck and foveolar cells in the non treated gastric mucosa. In rats with obstruc tive jaundice, reactive products diminished or disappeared in these cells. In the teprenone
47
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Fig. 1. Effect of teprcnone on gastric mucosal blood How. De crease in blood flow was reduced significantly by teprenone. * p < 0.05 and ** < 0.01 vs. nontreated group.
Fig. 3. Effect of teprenonc on transmucosal PD. As with other defensive factors. PD decrease was reduced significantly by teprenone. * p < 0.01 vs. nontreated group.
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Kunisaki/Sugiyama
Effect of Teprenonc on Experimentally Induced AGML
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Fig. 2. Effect of teprenonc on gastric mucosal hexosamine levels. Decrease in hexosamine level was reduced by teprenone. but not sig nificantly. *p < 0.05 vs. nontreated group.
UEA-1
Control
w'
Teprenone
pre
6h
group, the staining observed in cells remained at prestress levels, and the decrease in staining was reduced even after stress (fig. 4). With other lectins, similar changes were observed not only in mucus-producing cells but also in parietal cells. Lesion-Enhancing Factors
Intragastric pH increased after stress in both groups, but not significantly. Teprenone did not affect acid secretion, and no signifi cant difference was detected between the two groups (fig. 5). Latent enzyme activity of P-N-acetyl-Dglucosaminidase decreased after stress, as the
instability of the lysosomal membrane in creased. reaching 24.8 ± 3.4% after 3 h and gradually tending to recover. Similarly, latent enzyme activity of p-glucuronidase decreased to 17.2 ± 1.4% at 2 h and 15.6 ± 2.6% at 3 h after stress (p < 0.05) and to 18.2 ± 2.5% at 6 h after stress (p < 0.01; fig. 6). TBA reactants increased significantly from prestress levels to 192.8 ± 9.3 nmol/g wet weight at 30 min after stress, and gradually recovered to prestress values at 3 h after stress in the nontreated group. In the teprenone group, the values in the course of this study tended to decrease but not significantly (fig. 7).
49
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Fig. 4. Differences between the teprenone and nontreated groups in the distribution and quantity of carbohydrate residue comprising the glycoprotein were assessed histologically with representative lectins. In the teprenone group, the brown reactive products were more evident in mucous neck cells or parietal cells, as compared to those in the nontreated group. With other lectins, similar results were obtained. X 100.
Fig. 5. Effect of teprenonc on intragastric pH. Teprenone did not affect acid secretion, as observed in previous studies.
The ulcer index at 6 h after stress was sig nificantly reduced in the teprenone group compared with the nontreated group (22.6 ± 2.1 vs. 14.8 ± 1.6 mm2; p < 0.02; fig. 8).
Discussion
In 1981, teprenone was developed as a new acyclic polyisoprenoid derivative of vitamin A by Murakami et al. [1, 9], Many authors
50
Kunisaki/Sugjyama
have found that this agent has the potential to suppress experimental AG ML induced byethanol [2] or aspirin [10]. and Shay's coldrestraint, indomethacin and others forms of ulcer [1], without affecting gastric acid secre tion. It has been demonstrated that teprenone stimulates mucus elaboration and secretion [4], maintains gastric mucosal blood flow, en hances synthesis of mucosal phospholipid [5] and acts cooperatively with endogenous pros taglandin. Some evidence suggests the oppo-
Effect of Teprenonc on Experimentally Induced AGML
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Fig. 6. Changes in gastric mu cosal glycosidases due to teprenone administration were evaluated. The increment in p-glucuronidase activity was suppressed signifi cantly in the teprenone group. A similar tendency was observed with N-acctyl-p-D-glucosaminidase. * p < 0.05 and ** p < 0.01 vs. nontreated group.
site results with prostaglandin [3. 4], and this point remains controversial. The mechanism of teprenone has not been clarified yet, but oral administration may permit lymphatic ab sorption of teprenone. Nakazawa et al. [11] reported that after food intake, the bioavail ability of teprenone was enhanced as com pared to that in the fasting state, probably due to a stimulation of gastric blood flow and secretion of bile and pancreatic juice, includ ing bile acids, fatty acids and monoglycerides. These constituents are necessary for mixed micelle formation and the biosynthesis of chylomicra, which are essential for lymphatic transport. In obstructive jaundice, lymphatic absorption of teprenone is reduced physiolog ically, and the benefits of the drug may de crease or disappear clinically. In our research, the absorption of teprenone in experimental obstructive jaundice and its effect on experi mentally induced AGML were assessed. We used cold-restraint stress as a model of clinical
Fig. 8. Ulcer index was calculated at 6 h after coldrestraint stress in both groups. In the reprenone group, the index was suppressed due to an acceleration of maintenance of defensive mechanisms, p vs. nontreated group.
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Fig. 7. Changes in TBA reac tants were measured biochemical ly. Increases in TBA reactants were reduced by the addition of teprenonc but not significantly.
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by ischemia or as a promotor of gastric muco sal blood flow. Other studies have also indi cated that teprenone has a direct function as a free radical scavenger, but this question re quires further investigation. The increment of lysosomal enzyme activity was also sup pressed by the addition of teprenone, possibly because the drug maintained the stability of the lysosomal membrane by decreasing free radical activity induced by ischemia-repcrfu sion or by inhibiting ischemia. In short, isch emia induced by various kinds of stress acti vates free radicals, which injure lysosomal membranes in the course of lipid peroxida tion. Furthermore, activated lysosomal en zymes degrade all kinds of cell constituents by entering cells during ischemia. In a normal physiological state, lysomal enzymes in gas tric mucosa are distributed chiefly in surface epithelial and mucous neck cells, but they do not participate in cell kinetics [12]. In macro phages or neutrophils, lysosomal enzymes act by wrapping or fusion systems but, in an isch emic state, lysosomal enzymes may enter the cells as the instability of lysosomal mem branes increases, and may act even in gastric mucosal cells. We believe that these phenom ena contribute to the development of AGML. Teprenone may exert antiulcer effects by breaking this chain of events, for example, by accelerating the synthesis of mucus or phos pholipid. maintaining gastric mucosal blood flow, and suppressing the activity of free radi cals. In our research, the mechanism of ab sorptive access could not be defined precisely, but teprenone may act without micele forma tion to bile acid in the presence of obstructive jaundice. With regard to this point, further research is needed to evaluate the function of teprenone, including its effect on free radi cals.
Effect ofTcprcnonc on Experimentally Induced AGML
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AGML after surgery. In this experimental model, gastric defensive factors such as gas tric mucosal blood flow, hexosamine levels and PD decreased after stress. Acid secretion was depressed, probably due to a hypofunction of parietal cells, induced by ischemia. Gastric mucosal glycosidases, which we re garded as lesion-enhancing factors in terms of entering cells and the blood stream and de stroying or damaging ischemic cells and or gans, were also activated due to increasing instability of lysosomal membranes after stress, with a peak at 2-3 h. Previously, we had evaluated these parameters biochemi cally and electron microscopically with acid phosphatase and found that gastric mucosal glycosidases were activated and had a role in damaging mucosal cells after stress induced by burn or cold restraint. Oxidative stress induced by ischemia-repcrfusion has been re ported to involve free radicals originating from the xanthine-xanthine oxidase system in endothelial cells or NADPH oxidase in neu trophils, with a role in damaging cells. We concluded that a reduction in defensive fac tors triggered AGML formation and that an increase in lesion-enhancing factors, exclud ing acid secretion, accelerated AGML devel opment: the existence of but not an increase in gastric acid appeared to be necessary for the evolution of AGML. We evaluated the effects of teprenone in view of the balance between gastric defensive factors and lesionpromoting factors, and found that, even in the presence of obstructive jaundice, teprenone stimulates secretion and biosynthesis of gly coprotein and maintains gastric mucosal blood flow to some extent. With regard to lesion-promoting factors, although acid secre tion did not change with the administration of teprenone, the activation of free radicals was suppressed, probably due to the drug's direct function as a scavenger or its indirect function as an inhibitor of lipid peroxidation induced
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1 Murakami M. Okelani K. Fujisaki H, Wakabayashi T, Ohgo T: Anli ulcer cfTecI of geranylgeranylacetone. a new acyclic polyisoprenoid on experimentally induced gastric and duodenal ulcers in rats. Arzneini ittelforschu ng 1981:31:799804. 2 Oketani K. Murakami M. Fujisaki H. Wakabayashi T: Effect of gcranylgcranylacetone on aspirin-in duced changes in gastric glycopro teins. Jap J Pharmacol 1983:33: 593-601. 3 Bilski J. Murty VLN, Nadziejko C. Sarosiek J. Aono M. Moriga M. Slomiany A, Slomiany BL: Protec tion against alcohol-induced gastric mucosal injury by geranylgeranvlacetone; Effect ofindomethacin. Di gestion 1988:41:22-33.
