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Influence of aging on gastric ulcer healing activities of the antioxidants α-tocopherol and probucol
European Journal of Pharmacology 601 (2008) 143–147
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Influence of aging on gastric ulcer healing activities of the antioxidants α-tocopherol and probucol Masashi Ishihara, Ryoji Kojima, Mikio Ito ⁎ Laboratory of Analytical Pharmacology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan
a r t i c l e
i n f o
Article history: Received 14 April 2008 Received in revised form 8 October 2008 Accepted 9 October 2008 Available online 21 October 2008 Keywords: Gastric ulcer Aging α-Tocopherol Probucol Antioxidant
a b s t r a c t In the present study, we compared the effects of α-tocopherol and probucol, antioxidants, on the healing of acetic acid-induced gastric ulcers in 8-, 48- and 96-week-old rats. The repeated oral administration of α-tocopherol (16 mg/kg twice daily) and probucol (1000 mg/kg twice daily) for 14 consecutive days markedly accelerated the gastric ulcer healing in 48- and 96-week-old rats as well as 8-week-old ones. The ulcer healing effects of both drugs were not significantly different among the rats at three different ages. The superoxide dismutase (SOD) activity in the ulcerated region of 8-, 48- and 96-week-old rats was markedly lower than that in the unulcerated region. In contrast, the thiobarbituric acid (TBA)-reactive substance content, an index of lipid peroxidation, in the ulcerated region of rats at three different ages markedly increased, as compared to that in the unulcerated region. The SOD activity tended to decrease with aging, while the TBA-reactive substance content gradually increased. The repeated administration of α-tocopherol and probucol accelerated the ulcer healing and inhibited the increase in the TBA-reactive substance content in the ulcerated region. These results suggest that α-tocopherol and probucol promote the ulcer healing by their potent antioxidant activities in 48- and 96-week-old rats as well as 8-week-old rats. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Aging causes gastrointestinal functional changes. Aggressive factors such as gastric acid secretion (Khalil et al., 1988; Majumdar et al., 1988; Ohno et al., 1988) and pepsin activity (Ohno et al., 1988) have been shown to decrease with aging in humans and animals. Defensive factors such as gastric mucosal blood flow (Masuda et al., 1991), gastric mucosal prostaglandin (PG) content (Cryer et al., 1992; Goto et al., 1992; Lee and Feldman, 1994), and bicarbonate secretion (Kim et al., 1990) have been reported to show an age-related decrease in humans and animals. Cimetidine and omeprazole are thought to cause hypergastrinemia through the elevation of intragastric pH due to antisecretory action (Peters et al., 1983; Larsson et al., 1986). In addition to stimulating gastric acid secretion, gastrin has been shown to possess a trophic action; for example, it stimulates the proliferation of gastric mucosal cells (Willems et al., 1972; Hansen et al., 1976). We have already reported that cimetidine, a histamine H2 receptor antagonist, and omeprazole, a proton pump inhibitor, accelerate the healing of gastric ulcers by the trophic action of gastrin via the increase in gastrin secretion rather than by an antisecretory action (Ito et al., 1994a,b). Furthermore, we have shown that cimetidine and omeprazole have potent gastric ulcer healing actions in 8- and 48-week-old rats, mainly ⁎ Corresponding author. Tel.: +81 52 839 2692; fax: +81 52 834 8090. E-mail address: [email protected] (M. Ito). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.020
through potent serum gastrin-elevating actions, but both drugs are ineffective in 96-week-old rats which have lost their gastrin-elevating actions (Ishihara and Ito, 2002). Oxygen-derived free radicals have been postulated to play an important role in the pathogenesis of acute gastric mucosal injuries (Pery et al., 1986; Cochran et al., 1982; Pihan et al., 1987; Del Soldato et al., 1985) or in the delay of chronic gastric ulcer healing (Shii et al., 1992). In the previous study, we have reported that the following drugs possess a potent antioxidant action and are effective in acute gastric mucosal injury and/or in promoting the healing of chronic gastric ulcers: polaprezinc, an anti-ulcer drug (Ito et al.,1992); probucol, a lipid-lowering drug (Ito et al., 1998); quercetin, a flavonoid; α-tocopherol, a vitamin; nifedipine, a Ca2+-channel blocker; and tetracycline, an antibiotic (Suzuki et al., 1998). Little is known about the influence of aging on the gastric ulcer healing-promoting actions of the antioxidants mentioned above. Therefore, in the present study, we investigated the influence of aging on the gastric ulcer healing activities of α-tocopherol and probucol, antioxidants, using 8-, 48- and 96-weekold rats. 2. Materials and methods 2.1. Animals Three different age (8, 48 and 96 weeks of age) male Wistar rats (Clea Japan Inc.,Tokyo, Japan) were used in the experiments. The animals were housed in an air-conditioned room at 23± 1 °C. All experimental
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procedures described were approved by the Experimental Animal Research Committee of Faculty of Pharmacy, Meijo University. 2.2. Drugs Test compounds used were α-tocopherol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and probucol (Sinlestal ®, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan). Both drugs were suspended in 1% gum arabic. 2.3. Influence of aging on gastric ulcer healing activities of test compounds The rats of three different ages were allowed daily access to commercial food pellets from 9:00 to 10:00 a.m. and 5:00 to 6:00 p.m. throughout the experimental period from 3 days prior to ulcer induction (Ito et al., 1994a). Tap water was always available ad libitum. Gastric ulcers were induced in the rats of three different ages by exposing the serosal surface of the corpus wall to 100 μl of 100% acetic acid for 60 s in accordance with the method of Okabe et al. (1977). Test compound (α-tocopherol or probucol) was given orally twice daily (8:30 a.m. and 4:30 p.m.) to each rat of the three different ages at 0.5 ml per 100 g of body weight for 14 consecutive days from the first day after serosal application of acetic acid. Control animals were given the vehicle (0.5% gum arabic) orally twice daily instead of test compound. On the 15th day, the animals were killed with an overdose of ether. The stomachs were removed, filled with 10% formalin, and allowed to stand for 5 min. Stomachs were then cut open along the greater curvature. The longitudinal and abscissal lengths of the upper, opened part of the ulcer were measured with a micrometer, which was mounted on a stereoscopic microscope, and the product of both lengths (mm2) was expressed in terms of the ulcer index. After the ulcer size was measured, the stomach tissue was again immersed in 10% formalin for 24 h. The formalin-fixed tissue was then cut so that a little of the normal tissue surrounding the ulcer remained. Thereafter, the central part of the ulcer was cut vertically against the serosa along the long diameters. These tissues, cut in half, were embedded in paraffin and cut into 2- and 3-μm-thick sections, which were then
stained with hematoxylin and eosin (HE). Micrographic histological measurements of the stained preparations were performed as shown in Fig. 1. These histological measurements were carried out under a light microscopy of H.E.-stained preparations by Toyobo Image Analyzer (Osaka, Japan). The ulcer size and histological measurements were carried out by two independent observers. The healing effect of each test compound was evaluated by comparing the ulcer index, the defective area in the ulcerated region, the index for the decrease in the exposed floor, and the index for the mucosal regeneration of each test compound with the indexes of the respective control. 2.4. Changes in ulcer index, superoxide dismutase (SOD) activity and thiobarbituric acid (TBA)-reactive substance content in ulcerated region during the process of gastric ulcer healing In order to clarify changes in ulcer index, SOD activity and TBAreactive substance content in the ulcerated region during the process of gastric ulcer healing, gastric ulcers were induced in 8-week-old rats as mentioned above. On the 3rd, 7th, 10th and 15th days after serosal application of acetic acid, the animals were killed with an overdose of ether. After the ulcer size was measured as the ulcer index, gastric mucosa was collected from the ulcerated and unulcerated regions by scraping. The SOD activity and TBA-reactive substance content in the ulcerated and unulcerated regions were determined by the methods of Ohyanagi (1984) and Ohkawa et al. (1986), respectively. The SOD activity is expressed as nitrate units per mg protein. On the other hand, TBAreactive substance content is expressed as nmol of malondialdehyde per mg protein. TBA (Kanto Chemicals, Tokyo, Japan) and 1,1,3,3-tetramethoxypropane (Tokyo Kasei, Tokyo, Japan) were used for the TBA assay. Furthermore, the SOD activity and TBA-reactive substance content in ulcerated and unulcerated regions were determined on the 7th day after application of acetic acid to 8-, 48- and 96-week-old rats. 2.5. Effects of test compounds on ulcer index and TBA-reactive substance content in ulcerated region Gastric ulcers were induced in rats of three different ages. Test compound (α-tocopherol or probucol) was given orally, twice (8:30 a.m.
Fig. 1. Method used for histological measurements. Schematic drawings of the vertical section in the ulcerated region on the 15th day after local application of acetic acid.
M. Ishihara et al. / European Journal of Pharmacology 601 (2008) 143–147
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Fig. 2. Influence of aging on ulcer healing activities of α-tocopherol and probucol in acetic acid-induced gastric ulcers in rats. α-Tocopherol (16 mg/kg) or probucol (1000 mg/kg) was given orally, twice daily for consecutive 14 days beginning the first day after local application of acetic acid. Ulcer healing activities of both drugs were evaluated on the 15th day. Each column shows the mean ± S.E.M. for 8 to 10 rats. Significantly different from respective control, ⁎P b 0.05, ⁎⁎P b 0.01.
and 4:30 p.m.) to rats for 14 consecutive days from the first day after acetic acid application. Control animals were given the vehicle orally twice daily. On the 15th day, the animals were killed with an overdose of ether. The effects of test compounds on ulcer index and TBA-reactive substance content in the ulcerated and unulcerated regions were evaluated as mentioned above.
ulcerated region by 65%, 46% and 30%, respectively. In addition, in 8-, 48- and 96-week-old rats, probucol increased the index for the decrease in the exposed ulcer base by 32%, 42% and 30%, respectively, and increased the index for mucosal regeneration by 86%, 63% and 62%, respectively. Thus, both compounds had potent gastric ulcer healing actions in 48- and 96-week-old rats as well as in 8-week-old rats.
2.6. Data analysis The results obtained are expressed as the mean ± S.E.M. The data were analyzed by one-way analysis of variance, and the statistical significance among groups was determined by Duncan's multiplerange test. 3. Results 3.1. Influence of aging on ulcer healing activities of α-tocopherol and probucol Repeated oral administration of α-tocopherol or probucol for 14 consecutive days markedly accelerated the healing of gastric ulcers in 8-, 48- and 96-week-old rats (Fig. 2). Namely, in 8-, 48- and 96-weekold rats, α-tocopherol (16 mg/kg twice daily) decreased the ulcer index by 46%, 50% and 56%, respectively, compared to respective control and decreased the defective area in the ulcerated region by 67%, 67% and 36%, respectively. In addition, in 8-, 48- and 96-weekold rats, α-tocopherol increased the index for the decrease in the exposed ulcer base by 35%, 47% and 33%, respectively, and increased the index for mucosal regeneration by 76%, 69% and 57%, respectively. On the other hand, in 8-, 48- and 96-week-old rats, probucol (1000 mg/kg twice daily) decreased the ulcer index by 49%, 47% and 43%, respectively, and decreased the defective area in the
3.2. Influence of aging on SOD activity and TBA-reactive substance content in ulcerated region The ulcer index (ulcer size), SOD activity and TBA-reactive substance content in the ulcerated region were evaluated on the 3rd, 7th, 10th and 15th days after local application of acetic acid in 8week-old rats (Fig. 3A). The ulcer index and TBA-reactive substance content showed the maximal values on the 7th day and then decreased over time. On the contrary, the SOD activity showed the minimal value on the 7th day and then increased as the day went on. Thus, there is a negative relationship between the time-related changes in the ulcer size or the TBA-reactive substance content and the SOD activity after the application of acetic acid. The SOD activity and TBA-reactive substance content in the ulcerated region were determined on the 7th day after the application of acetic acid in 8-, 48and 92-week-old rats (Fig. 3B). The TBA-reactive substance content in the ulcerated region in the 8-, 48- and 96-week-old rats increased by 4.2, 6.3 and 8.0 times higher than that in the respective unulcerated region in rats of each age. On the contrary, the SOD activity in the ulcerated region in 8-, 48- and 96-week-old rats decreased by 64%, 74% and 76% than that in the respective unulcerated region in rats of each age. Thus, the TBA-reactive substance content in the ulcerated region gradually increased with aging, while the SOD activity tended to decrease with aging.
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Fig. 3. Changes in ulcer index, superoxide dismutase (SOD) activity and thiobarbituric acid (TBA)-reactive substance content in ulcerated regions after local application of acetic acid in 8-week-old rats (A) and influence of aging on the SOD activity and TBA-reactive substance content in ulcerated regions (B) in acetic acid ulcers in rats. In the experiment shown in A, each parameter was evaluated on the 3rd, 7th, 10th and 15th days after local application of acetic acid. In the experiment shown in B, both parameters were evaluated on the 7th day. Each column shows the mean ± S.E.M. for 8 to 10 rats. Significantly different from unulcerated, ⁎P b 0.05, ⁎⁎P b 0.01. Significantly different from 8-week, #P b 0.05, ##P b 0.01.
3.3. Influences of aging on effects of α-tocopherol and probucol on ulcer index and TBA-reactive substance content in the ulcerated region Repeated oral administration of α-tocopherol (16 mg/kg twice daily) to 8-, 48- and 96-week-old rats for 14 consecutive days decreased the ulcer index by 37%, 55% and 44%, respectively (Fig. 4, left). On the other hand, in 8-, 48- and 96-week-old rats, probucol (1000 mg/kg twice daily, p.o.) decreased the ulcer index by 53%, 58% and 42%, respectively. The TBAreactive substance content in the ulcerated region of 8-, 48- and 96-week old control rats was 3.8, 4.7 and 5.5 times higher than that in the respective
unulcerated region of each age control rat (Fig. 4, right). α-Tocopherol inhibited the increase in the TBA-reactive substance content in the ulcerated region in 8-, 48- and 96-week old rats by 44%, 64% and 70%, respectively. On the other hand, in 8-, 48- and 96-week-old rats, probucol inhibited the increase in the TBA-reactive substance content in the ulcerated region by 75%, 65% and 46%, respectively. Thus, α-tocopherol and probucol accelerated the ulcer healing and inhibited the increase in TBA-reactive substance content in the ulcerated region, and the degree of these effects of both compounds was not significantly different among the rats of three different ages.
Fig. 4. Influence of aging on ulcer healing and lipid peroxide-preventing activities of α-tocopherol and probucol in acetic acid-induced gastric ulcers in rats. α-Tocopherol (16 mg/kg) or probucol (1000 mg/kg) was given orally, twice daily for consecutive 14 days beginning the first day after local application of acetic acid. Effects of both compounds on ulcer healing (ulcer index) and lipid peroxide (TBA-reactive substance content) in ulcerated regions were evaluated on the 15th day. Ulcer-healing activities of both drugs were evaluated on the 15th day. Each column shows the mean ± S.E.M. for 8 to 10 rats. Significantly different from respective control, ⁎P b 0.05, ⁎⁎P b 0.01.
M. Ishihara et al. / European Journal of Pharmacology 601 (2008) 143–147
4. Discussion We have already demonstrated in 8-week-old (young) rats that α-tocopherol (4, 8 and 16 mg/kg) (Suzuki et al., 1998) and probucol (250, 500 and 1000 mg/kg) (Ito et al., 1998), given orally, twice daily for 14 consecutive days from the day after acetic acid injection (the injection of 20% v/v in a volume of 0.05 ml into the submucosal layer at the junction of the fundus and antrum), dose-dependently, promoted the gastric ulcer healing and inhibited the increase in the TBA-reactive substance content in the ulcerated mucosa. As mentioned in the Introduction, we have already reported that cimetidine and omeprazole mainly accelerate the healing of gastric ulcers by the trophic action via the increase in gastrin secretion (Ito et al., 1994a,b). Furthermore, we have shown that cimetidine and omeprazole have potent gastric ulcer healing actions in 8- and 48-week-old rats through potent serum gastrin-elevating actions, but both drugs are ineffective in 96-week-old rats, which have lost their gastrin-elevating actions (Ishihara and Ito, 2002). In the present experiments, rats of three different ages were twice daily administered α-tocopherol and probucol at oral doses of 16 mg/kg and 1000 mg/kg, respectively. It is of interest that in extremely old (96week-old) rats as well as young (8-week-old) and aged (48-week-old) rats, both antioxidant compounds, unlike cimetidine and omeprazole, markedly promoted the gastric ulcer healing. In the present experiment, we compared the SOD activity and TBA reactive substance content in the ulcerated region of 8-, 48- and 96-week-old rats. The SOD activity in the ulcerated region of the rats of three different ages was markedly lower than that in the unulcerated region. On the other hand, the TBA-reactive substance content in the ulcerated region of 8-, 48- and 96-week-old rats markedly increased, compared to that in the unulcerated region of rats of the respective ages. The SOD activity in the ulcerated region tended to decrease with aging, while the TBA-reactive substance content in the ulcerated region gradually increased with aging. α-Tocopherol and probucol markedly promoted the gastric ulcer healing and inhibited the increase in the TBA-reactive substance content in the ulcerated region of rats of three different ages. These results strongly suggest that both antioxidant compounds promote the ulcer healing by their potent antioxidant activities in three different age rats. Free radical and lipid peroxide formation which can cause oxidative damage to cell membranes have been thought to be major risk factors for pathogenesis of various diseases such as diabetes mellitus (Gorogawa et al., 2002) and its complications (Jakus, 2000; West, 2000; Yoshida et al., 2005), hepatitis (Baskol et al., 2007), nephritis (Fu et al., 2007), myocardial ischemia (McCord, 1988), Alzheimer's disease (Vina et al., 2007) and UV-induced skin damage (Emri et al., 2006) as well as peptic ulcers. Therefore, antioxidants such as α-tocopherol and probucol may well be useful for the treatment of various diseases associated with oxidative stress. References Baskol, G., Baskol, M., Kocer, D., 2007. Oxidative stress and antioxidant defences in serum of patients with non-alcoholic steatohepatitis. Clin. Biol. 40, 776–780. Cochran, R.A., Stefanko, J., Moore, C., Saik, R., 1982. Cytotoxic-free radicals in stress ulcerations. Surg. Forum 28, 159–161. Cryer, B., Redfern, J.S., Lee, E., Feldman, M., 1992. Effect of aging on gastric and duodenal mucosal prostaglandin concentrations in humans: relationship with gastric acid secretion. Gastroenterology 102, 1118–1123. Del Soldato, P., Foschi, D., Beboni, G., Scarpignato, C., 1985. Oxygen free radicals interact with indomethacin to cause gastrointestinal injury. Agents Actions 17, 484–488. Emri, G., Horkay, I., Remenyik, E., 2006. The role of free radicals in the UV-induced skin damage. Orv. Hetil. 23, 731–735. Fu, H., Li, J., Li, Ox, Xia, L., Shao, L., 2007. Protective effect of ligustrazine on accelerated anti-glomerular basement membrane antibody nephritis in rats is based on its antioxidant properties. Eur. J. Pharmacol. 563, 197–202.
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Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Influence of aging on gastric ulcer healing activities of the antioxidants α-tocopherol and probucol Masashi Ishihara, Ryoji Kojima, Mikio Ito ⁎ Laboratory of Analytical Pharmacology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan
a r t i c l e
i n f o
Article history: Received 14 April 2008 Received in revised form 8 October 2008 Accepted 9 October 2008 Available online 21 October 2008 Keywords: Gastric ulcer Aging α-Tocopherol Probucol Antioxidant
a b s t r a c t In the present study, we compared the effects of α-tocopherol and probucol, antioxidants, on the healing of acetic acid-induced gastric ulcers in 8-, 48- and 96-week-old rats. The repeated oral administration of α-tocopherol (16 mg/kg twice daily) and probucol (1000 mg/kg twice daily) for 14 consecutive days markedly accelerated the gastric ulcer healing in 48- and 96-week-old rats as well as 8-week-old ones. The ulcer healing effects of both drugs were not significantly different among the rats at three different ages. The superoxide dismutase (SOD) activity in the ulcerated region of 8-, 48- and 96-week-old rats was markedly lower than that in the unulcerated region. In contrast, the thiobarbituric acid (TBA)-reactive substance content, an index of lipid peroxidation, in the ulcerated region of rats at three different ages markedly increased, as compared to that in the unulcerated region. The SOD activity tended to decrease with aging, while the TBA-reactive substance content gradually increased. The repeated administration of α-tocopherol and probucol accelerated the ulcer healing and inhibited the increase in the TBA-reactive substance content in the ulcerated region. These results suggest that α-tocopherol and probucol promote the ulcer healing by their potent antioxidant activities in 48- and 96-week-old rats as well as 8-week-old rats. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Aging causes gastrointestinal functional changes. Aggressive factors such as gastric acid secretion (Khalil et al., 1988; Majumdar et al., 1988; Ohno et al., 1988) and pepsin activity (Ohno et al., 1988) have been shown to decrease with aging in humans and animals. Defensive factors such as gastric mucosal blood flow (Masuda et al., 1991), gastric mucosal prostaglandin (PG) content (Cryer et al., 1992; Goto et al., 1992; Lee and Feldman, 1994), and bicarbonate secretion (Kim et al., 1990) have been reported to show an age-related decrease in humans and animals. Cimetidine and omeprazole are thought to cause hypergastrinemia through the elevation of intragastric pH due to antisecretory action (Peters et al., 1983; Larsson et al., 1986). In addition to stimulating gastric acid secretion, gastrin has been shown to possess a trophic action; for example, it stimulates the proliferation of gastric mucosal cells (Willems et al., 1972; Hansen et al., 1976). We have already reported that cimetidine, a histamine H2 receptor antagonist, and omeprazole, a proton pump inhibitor, accelerate the healing of gastric ulcers by the trophic action of gastrin via the increase in gastrin secretion rather than by an antisecretory action (Ito et al., 1994a,b). Furthermore, we have shown that cimetidine and omeprazole have potent gastric ulcer healing actions in 8- and 48-week-old rats, mainly ⁎ Corresponding author. Tel.: +81 52 839 2692; fax: +81 52 834 8090. E-mail address: [email protected] (M. Ito). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.020
through potent serum gastrin-elevating actions, but both drugs are ineffective in 96-week-old rats which have lost their gastrin-elevating actions (Ishihara and Ito, 2002). Oxygen-derived free radicals have been postulated to play an important role in the pathogenesis of acute gastric mucosal injuries (Pery et al., 1986; Cochran et al., 1982; Pihan et al., 1987; Del Soldato et al., 1985) or in the delay of chronic gastric ulcer healing (Shii et al., 1992). In the previous study, we have reported that the following drugs possess a potent antioxidant action and are effective in acute gastric mucosal injury and/or in promoting the healing of chronic gastric ulcers: polaprezinc, an anti-ulcer drug (Ito et al.,1992); probucol, a lipid-lowering drug (Ito et al., 1998); quercetin, a flavonoid; α-tocopherol, a vitamin; nifedipine, a Ca2+-channel blocker; and tetracycline, an antibiotic (Suzuki et al., 1998). Little is known about the influence of aging on the gastric ulcer healing-promoting actions of the antioxidants mentioned above. Therefore, in the present study, we investigated the influence of aging on the gastric ulcer healing activities of α-tocopherol and probucol, antioxidants, using 8-, 48- and 96-weekold rats. 2. Materials and methods 2.1. Animals Three different age (8, 48 and 96 weeks of age) male Wistar rats (Clea Japan Inc.,Tokyo, Japan) were used in the experiments. The animals were housed in an air-conditioned room at 23± 1 °C. All experimental
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procedures described were approved by the Experimental Animal Research Committee of Faculty of Pharmacy, Meijo University. 2.2. Drugs Test compounds used were α-tocopherol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and probucol (Sinlestal ®, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan). Both drugs were suspended in 1% gum arabic. 2.3. Influence of aging on gastric ulcer healing activities of test compounds The rats of three different ages were allowed daily access to commercial food pellets from 9:00 to 10:00 a.m. and 5:00 to 6:00 p.m. throughout the experimental period from 3 days prior to ulcer induction (Ito et al., 1994a). Tap water was always available ad libitum. Gastric ulcers were induced in the rats of three different ages by exposing the serosal surface of the corpus wall to 100 μl of 100% acetic acid for 60 s in accordance with the method of Okabe et al. (1977). Test compound (α-tocopherol or probucol) was given orally twice daily (8:30 a.m. and 4:30 p.m.) to each rat of the three different ages at 0.5 ml per 100 g of body weight for 14 consecutive days from the first day after serosal application of acetic acid. Control animals were given the vehicle (0.5% gum arabic) orally twice daily instead of test compound. On the 15th day, the animals were killed with an overdose of ether. The stomachs were removed, filled with 10% formalin, and allowed to stand for 5 min. Stomachs were then cut open along the greater curvature. The longitudinal and abscissal lengths of the upper, opened part of the ulcer were measured with a micrometer, which was mounted on a stereoscopic microscope, and the product of both lengths (mm2) was expressed in terms of the ulcer index. After the ulcer size was measured, the stomach tissue was again immersed in 10% formalin for 24 h. The formalin-fixed tissue was then cut so that a little of the normal tissue surrounding the ulcer remained. Thereafter, the central part of the ulcer was cut vertically against the serosa along the long diameters. These tissues, cut in half, were embedded in paraffin and cut into 2- and 3-μm-thick sections, which were then
stained with hematoxylin and eosin (HE). Micrographic histological measurements of the stained preparations were performed as shown in Fig. 1. These histological measurements were carried out under a light microscopy of H.E.-stained preparations by Toyobo Image Analyzer (Osaka, Japan). The ulcer size and histological measurements were carried out by two independent observers. The healing effect of each test compound was evaluated by comparing the ulcer index, the defective area in the ulcerated region, the index for the decrease in the exposed floor, and the index for the mucosal regeneration of each test compound with the indexes of the respective control. 2.4. Changes in ulcer index, superoxide dismutase (SOD) activity and thiobarbituric acid (TBA)-reactive substance content in ulcerated region during the process of gastric ulcer healing In order to clarify changes in ulcer index, SOD activity and TBAreactive substance content in the ulcerated region during the process of gastric ulcer healing, gastric ulcers were induced in 8-week-old rats as mentioned above. On the 3rd, 7th, 10th and 15th days after serosal application of acetic acid, the animals were killed with an overdose of ether. After the ulcer size was measured as the ulcer index, gastric mucosa was collected from the ulcerated and unulcerated regions by scraping. The SOD activity and TBA-reactive substance content in the ulcerated and unulcerated regions were determined by the methods of Ohyanagi (1984) and Ohkawa et al. (1986), respectively. The SOD activity is expressed as nitrate units per mg protein. On the other hand, TBAreactive substance content is expressed as nmol of malondialdehyde per mg protein. TBA (Kanto Chemicals, Tokyo, Japan) and 1,1,3,3-tetramethoxypropane (Tokyo Kasei, Tokyo, Japan) were used for the TBA assay. Furthermore, the SOD activity and TBA-reactive substance content in ulcerated and unulcerated regions were determined on the 7th day after application of acetic acid to 8-, 48- and 96-week-old rats. 2.5. Effects of test compounds on ulcer index and TBA-reactive substance content in ulcerated region Gastric ulcers were induced in rats of three different ages. Test compound (α-tocopherol or probucol) was given orally, twice (8:30 a.m.
Fig. 1. Method used for histological measurements. Schematic drawings of the vertical section in the ulcerated region on the 15th day after local application of acetic acid.
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Fig. 2. Influence of aging on ulcer healing activities of α-tocopherol and probucol in acetic acid-induced gastric ulcers in rats. α-Tocopherol (16 mg/kg) or probucol (1000 mg/kg) was given orally, twice daily for consecutive 14 days beginning the first day after local application of acetic acid. Ulcer healing activities of both drugs were evaluated on the 15th day. Each column shows the mean ± S.E.M. for 8 to 10 rats. Significantly different from respective control, ⁎P b 0.05, ⁎⁎P b 0.01.
and 4:30 p.m.) to rats for 14 consecutive days from the first day after acetic acid application. Control animals were given the vehicle orally twice daily. On the 15th day, the animals were killed with an overdose of ether. The effects of test compounds on ulcer index and TBA-reactive substance content in the ulcerated and unulcerated regions were evaluated as mentioned above.
ulcerated region by 65%, 46% and 30%, respectively. In addition, in 8-, 48- and 96-week-old rats, probucol increased the index for the decrease in the exposed ulcer base by 32%, 42% and 30%, respectively, and increased the index for mucosal regeneration by 86%, 63% and 62%, respectively. Thus, both compounds had potent gastric ulcer healing actions in 48- and 96-week-old rats as well as in 8-week-old rats.
2.6. Data analysis The results obtained are expressed as the mean ± S.E.M. The data were analyzed by one-way analysis of variance, and the statistical significance among groups was determined by Duncan's multiplerange test. 3. Results 3.1. Influence of aging on ulcer healing activities of α-tocopherol and probucol Repeated oral administration of α-tocopherol or probucol for 14 consecutive days markedly accelerated the healing of gastric ulcers in 8-, 48- and 96-week-old rats (Fig. 2). Namely, in 8-, 48- and 96-weekold rats, α-tocopherol (16 mg/kg twice daily) decreased the ulcer index by 46%, 50% and 56%, respectively, compared to respective control and decreased the defective area in the ulcerated region by 67%, 67% and 36%, respectively. In addition, in 8-, 48- and 96-weekold rats, α-tocopherol increased the index for the decrease in the exposed ulcer base by 35%, 47% and 33%, respectively, and increased the index for mucosal regeneration by 76%, 69% and 57%, respectively. On the other hand, in 8-, 48- and 96-week-old rats, probucol (1000 mg/kg twice daily) decreased the ulcer index by 49%, 47% and 43%, respectively, and decreased the defective area in the
3.2. Influence of aging on SOD activity and TBA-reactive substance content in ulcerated region The ulcer index (ulcer size), SOD activity and TBA-reactive substance content in the ulcerated region were evaluated on the 3rd, 7th, 10th and 15th days after local application of acetic acid in 8week-old rats (Fig. 3A). The ulcer index and TBA-reactive substance content showed the maximal values on the 7th day and then decreased over time. On the contrary, the SOD activity showed the minimal value on the 7th day and then increased as the day went on. Thus, there is a negative relationship between the time-related changes in the ulcer size or the TBA-reactive substance content and the SOD activity after the application of acetic acid. The SOD activity and TBA-reactive substance content in the ulcerated region were determined on the 7th day after the application of acetic acid in 8-, 48and 92-week-old rats (Fig. 3B). The TBA-reactive substance content in the ulcerated region in the 8-, 48- and 96-week-old rats increased by 4.2, 6.3 and 8.0 times higher than that in the respective unulcerated region in rats of each age. On the contrary, the SOD activity in the ulcerated region in 8-, 48- and 96-week-old rats decreased by 64%, 74% and 76% than that in the respective unulcerated region in rats of each age. Thus, the TBA-reactive substance content in the ulcerated region gradually increased with aging, while the SOD activity tended to decrease with aging.
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Fig. 3. Changes in ulcer index, superoxide dismutase (SOD) activity and thiobarbituric acid (TBA)-reactive substance content in ulcerated regions after local application of acetic acid in 8-week-old rats (A) and influence of aging on the SOD activity and TBA-reactive substance content in ulcerated regions (B) in acetic acid ulcers in rats. In the experiment shown in A, each parameter was evaluated on the 3rd, 7th, 10th and 15th days after local application of acetic acid. In the experiment shown in B, both parameters were evaluated on the 7th day. Each column shows the mean ± S.E.M. for 8 to 10 rats. Significantly different from unulcerated, ⁎P b 0.05, ⁎⁎P b 0.01. Significantly different from 8-week, #P b 0.05, ##P b 0.01.
3.3. Influences of aging on effects of α-tocopherol and probucol on ulcer index and TBA-reactive substance content in the ulcerated region Repeated oral administration of α-tocopherol (16 mg/kg twice daily) to 8-, 48- and 96-week-old rats for 14 consecutive days decreased the ulcer index by 37%, 55% and 44%, respectively (Fig. 4, left). On the other hand, in 8-, 48- and 96-week-old rats, probucol (1000 mg/kg twice daily, p.o.) decreased the ulcer index by 53%, 58% and 42%, respectively. The TBAreactive substance content in the ulcerated region of 8-, 48- and 96-week old control rats was 3.8, 4.7 and 5.5 times higher than that in the respective
unulcerated region of each age control rat (Fig. 4, right). α-Tocopherol inhibited the increase in the TBA-reactive substance content in the ulcerated region in 8-, 48- and 96-week old rats by 44%, 64% and 70%, respectively. On the other hand, in 8-, 48- and 96-week-old rats, probucol inhibited the increase in the TBA-reactive substance content in the ulcerated region by 75%, 65% and 46%, respectively. Thus, α-tocopherol and probucol accelerated the ulcer healing and inhibited the increase in TBA-reactive substance content in the ulcerated region, and the degree of these effects of both compounds was not significantly different among the rats of three different ages.
Fig. 4. Influence of aging on ulcer healing and lipid peroxide-preventing activities of α-tocopherol and probucol in acetic acid-induced gastric ulcers in rats. α-Tocopherol (16 mg/kg) or probucol (1000 mg/kg) was given orally, twice daily for consecutive 14 days beginning the first day after local application of acetic acid. Effects of both compounds on ulcer healing (ulcer index) and lipid peroxide (TBA-reactive substance content) in ulcerated regions were evaluated on the 15th day. Ulcer-healing activities of both drugs were evaluated on the 15th day. Each column shows the mean ± S.E.M. for 8 to 10 rats. Significantly different from respective control, ⁎P b 0.05, ⁎⁎P b 0.01.
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4. Discussion We have already demonstrated in 8-week-old (young) rats that α-tocopherol (4, 8 and 16 mg/kg) (Suzuki et al., 1998) and probucol (250, 500 and 1000 mg/kg) (Ito et al., 1998), given orally, twice daily for 14 consecutive days from the day after acetic acid injection (the injection of 20% v/v in a volume of 0.05 ml into the submucosal layer at the junction of the fundus and antrum), dose-dependently, promoted the gastric ulcer healing and inhibited the increase in the TBA-reactive substance content in the ulcerated mucosa. As mentioned in the Introduction, we have already reported that cimetidine and omeprazole mainly accelerate the healing of gastric ulcers by the trophic action via the increase in gastrin secretion (Ito et al., 1994a,b). Furthermore, we have shown that cimetidine and omeprazole have potent gastric ulcer healing actions in 8- and 48-week-old rats through potent serum gastrin-elevating actions, but both drugs are ineffective in 96-week-old rats, which have lost their gastrin-elevating actions (Ishihara and Ito, 2002). In the present experiments, rats of three different ages were twice daily administered α-tocopherol and probucol at oral doses of 16 mg/kg and 1000 mg/kg, respectively. It is of interest that in extremely old (96week-old) rats as well as young (8-week-old) and aged (48-week-old) rats, both antioxidant compounds, unlike cimetidine and omeprazole, markedly promoted the gastric ulcer healing. In the present experiment, we compared the SOD activity and TBA reactive substance content in the ulcerated region of 8-, 48- and 96-week-old rats. The SOD activity in the ulcerated region of the rats of three different ages was markedly lower than that in the unulcerated region. On the other hand, the TBA-reactive substance content in the ulcerated region of 8-, 48- and 96-week-old rats markedly increased, compared to that in the unulcerated region of rats of the respective ages. The SOD activity in the ulcerated region tended to decrease with aging, while the TBA-reactive substance content in the ulcerated region gradually increased with aging. α-Tocopherol and probucol markedly promoted the gastric ulcer healing and inhibited the increase in the TBA-reactive substance content in the ulcerated region of rats of three different ages. These results strongly suggest that both antioxidant compounds promote the ulcer healing by their potent antioxidant activities in three different age rats. Free radical and lipid peroxide formation which can cause oxidative damage to cell membranes have been thought to be major risk factors for pathogenesis of various diseases such as diabetes mellitus (Gorogawa et al., 2002) and its complications (Jakus, 2000; West, 2000; Yoshida et al., 2005), hepatitis (Baskol et al., 2007), nephritis (Fu et al., 2007), myocardial ischemia (McCord, 1988), Alzheimer's disease (Vina et al., 2007) and UV-induced skin damage (Emri et al., 2006) as well as peptic ulcers. Therefore, antioxidants such as α-tocopherol and probucol may well be useful for the treatment of various diseases associated with oxidative stress. References Baskol, G., Baskol, M., Kocer, D., 2007. Oxidative stress and antioxidant defences in serum of patients with non-alcoholic steatohepatitis. Clin. Biol. 40, 776–780. Cochran, R.A., Stefanko, J., Moore, C., Saik, R., 1982. Cytotoxic-free radicals in stress ulcerations. Surg. Forum 28, 159–161. Cryer, B., Redfern, J.S., Lee, E., Feldman, M., 1992. Effect of aging on gastric and duodenal mucosal prostaglandin concentrations in humans: relationship with gastric acid secretion. Gastroenterology 102, 1118–1123. Del Soldato, P., Foschi, D., Beboni, G., Scarpignato, C., 1985. Oxygen free radicals interact with indomethacin to cause gastrointestinal injury. Agents Actions 17, 484–488. Emri, G., Horkay, I., Remenyik, E., 2006. The role of free radicals in the UV-induced skin damage. Orv. Hetil. 23, 731–735. Fu, H., Li, J., Li, Ox, Xia, L., Shao, L., 2007. Protective effect of ligustrazine on accelerated anti-glomerular basement membrane antibody nephritis in rats is based on its antioxidant properties. Eur. J. Pharmacol. 563, 197–202.
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