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Abamectin attenuates gastric mucosal damage induced by ethanol through activation of vagus nerve in rats
Alcohol 30 (2003) 61–65
Abamectin attenuates gastric mucosal damage induced by ethanol through activation of vagus nerve in rats Ming-Yie Liua,b,*, James Po-Jung Chianga, Dur-Zong Hsua,b, Jou-Fang Dengc a
Graduate Institute of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan 704 b Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan 704 c Division of Clinical Toxicology, Department of Medicine, National Yang Ming University and Veterans General Hospital-Taipei, Taipei, Taiwan 112 Received 6 September 2002; received in revised form 16 April 2003; accepted 18 April 2003
Abstract Some type A gamma-aminobutyric acid (GABAA) receptor agonists are effective in protecting against the formation of stomach lesions induced by ethanol. Natural product abamectin, one of the existing GABAA receptor agonists, might protect against the development of gastric ulcers induced by ethanol. We investigated the protective effect of abamectin against the formation of gastric mucosal lesions induced by ethanol in rats. Abamectin (3 mg/kg, p.o.) was given to rats 1 h before administration of ethanol [4 ml of a 30% (volume/ volume) solution]. Mucosal lipid peroxidation (LPO), nitric oxide (NO) levels, and ulcer index were measured 3 h after gastric surgery (vagotomy vs. sham vagotomy) in treated versus control subjects. Abamectin attenuated ethanol-induced gastric ulceration, decreased LPO regeneration, and increased NO production in the gastric mucosa of rats in the sham vagotomy group. However, this protective effect of abamectin against ethanol-induced gastric lesions was not observed in rats in the group that underwent vagotomy. These results support the suggestion that administration of abamectin ameliorated the ethanol-induced gastric mucosal injury through elevation of NO production. Activation of the vagus nerve may be involved in the abamectin-associated gastric protection against the effects of ethanol in rats. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: Ethanol; Abamectin; Lipid peroxidation; Nitric oxide; Vagus nerve
1. Introduction Since the 19th century (Beaumont, 1833), it has been known that exposure of the gastric mucosa to concentrated alcohol can induce acute gastritis. Although some evidence seems to indicate that the neuropharmacological action of ethanol is mediated through gamma-aminobutyric acid [(GABA); a central inhibitory synaptic neurotransmitter], the exact mechanism of ethanol-induced gastric damage has not been clarified (Khadour et al., 1996). Oral pretreatment with benzodiazepines, which are GABAergic compounds, prevents ulceration of the gastric mucosa induced by acidified ethanol (Erdo¨ et al., 1989; File & Pearce, 1981). It has been reported further that such a protective action is not due to the sedative effect of benzodiazepines (Najim & Karim, 1991). Thus, activation of the GABA receptor may be effective in protecting the gastric mucosa against damage induced by ethanol. * Corresponding author. Graduate Institute of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan 704. Tel.: ⫹886-2353535, ext. 5805; fax: ⫹886-6-2752484. E-mail address: [email protected] (M.-Y. Liu). Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00094-6
Abamectin, an analog of ivermectin, is a mixture of avermectins containing at least 80% of avermectin B1a and less than 20% of avermectin B1b. Avermectins are produced by the soil bacterium Streptomyces avermitilis (Agarwal, 1998; Campbell et al., 1983). As a safe chemical in mammals, abamectin has been used as an anthelmintic agent in both animals and human beings. In addition, abamectin is believed to be a GABAergic agent (Kaplan et al., 1994). Both lipid peroxidation (LPO) and glutathione (GSH) are crucial parameters in evaluating gastric mucosal injury (Hung & Hsu, 1998). Mucosal LPO plays a significant role in the pathogenesis of ethanol-induced gastric mucosal lesions. Oxygen radicals have been implicated in the direct damage of cell membranes after administration of ethanol (Kvietys et al., 1990; Szelenyi & Brune, 1988; Terano et al., 1989). Glutathione, an important endogenous sulfhydryl compound in the cellular defense system (Poli et al., 1993), is markedly reduced in gastric mucosa after ethanol challenge (ElDakhakhny et al., 2000). Furthermore, mucosal nitric oxide (NO), in the gastric defense system, is released after activation by mild irritants to induce cytoprotection (Ko & Cho, 1999). Results of a previous study from our laboratory indicate that abamectin ingestion can increase serum NO production (Hsu et al., 2001). However, whether abamectin can
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protect against gastric mucosal damage induced by ethanol by stimulating NO production is unknown. To explore this question, we examined the effect of abamectin on gastric mucosa in rats treated with acidified ethanol. 2. Materials and methods 2.1. Reagents Abamectin (98% purity) was a gift from Novartis Crop Protection Taiwan Co. Ltd. (Taipei, Taiwan). Ethanol (99.8% purity) was obtained from Merck (Darmstadt, Germany). 2.2. Animals Male, specific-pathogen free, Wistar rats (each weighing between 200 and 300 g) were obtained from and housed in our institution’s Laboratory Animal Center. The animal care and experimental protocols were in accordance with the institutional guidelines, which are approved by the National Laboratory Animal Breeding and Research Center in Taiwan. Ethyl ether was used to anesthetize animals before the gastric surgery. Rats were deprived of food but allowed free access to tap water for 24 h before experiments. Results of preliminary experiments demonstrated that mineral oil could be used as a solvent for the abamectin (range, 1 to 10 mg/kg) in the study of gastric mucosal injury in rats, and that mineral oil, with or without abamectin (3 mg/kg), did not induce injury to the gastric mucosa in rats. Therefore, we chose to evaluate a 3-mg/kg dose of abamectin (in mineral oil) as a drug to protect against the development of gastric ulcers induced by ethanol. Study animals were assigned to one of two groups: those that underwent vagotomy (vagotomy group) and those that did not undergo vagotomy (sham vagotomy group). Animals in each of the two groups were assigned to five subgroups: two control (nontreated and mineral oil control) and three treated (abamectin, ethanol, and abamectin plus ethanol). Forty rats (four rats in each group) were used. Rats were gavaged with abamectin (3 mg/kg, suspended in 1 ml of mineral oil) under light ether anesthesia 1 h before gastric surgery. 2.3. Gastric surgery procedures The stomachs of animals were surgically exposed for ligation of the pylorus and lower portion of the esophagus. Gastric bilateral vagotomy or sham vagotomy was performed in these groups of rats (Hung et al., 2000). A small incision was made on the forestomach, and the stomach contents were expelled. A polypropylene tube was inserted through the incision, secured, and rinsed with saline (at 37ºC). An isotonic solution (7 ml), containing 30% ethanol plus 150 mM HCl (acidified ethanol), was instilled into the stomach and mixed, and 3 ml of the solution was taken as an initial sample. The abdominal wound was subsequently sutured. After 3 h, the final gastric sample was collected for determination of gastric hemoglobin concentration.
2.4. Assay of mucosal glutathione level Tissue samples were homogenized in ice-cold trichloroacetic acid (1 g of tissue plus 10 ml of 10% trichloroacetic acid) in a tissue homogenizer. Briefly, after centrifugation at 3,000 rpm for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 M Na2HPO4 2H2O solution. A 0.2-ml solution of dithiobisnitrobenzoate (1% sodium citrate, 0.4 mg/ml) was added, and the absorbance at 412 nm was measured immediately (Yegen et al., 1992). 2.5. Measurement of lipid peroxidation levels in the serum and tissues Gastric mucosa was homogenized in TRIS HCl (20 mM, pH 7.4). Tissue homogenate (1 ml) was centrifuged at 2,500g for 10 min at 4ºC. Supernatant (200 µl) was taken for LPO measurement by using a commercial assay kit (CalbiochemNovabiochem Co., Darmstadt, Germany) and a spectrophotometer reading of 586 nm. 2.6. Measurement of mucosal nitrite levels Briefly, the amounts of nitrite in mucosa were measured following the Griess reaction by incubating a 100-µl sample with 100 µl of Griess reagent at room temperature for 20 min. The absorbance at 550 nm was measured with a spectrophotometer (Green et al., 1981). Nitrite concentration was calculated by comparison with a standard solution of known sodium nitrite concentrations. 2.7. Morphological studies of gastric mucosa The stomach was opened along the greater curvature. The length and width of the ulcers on the gastric mucosa were measured with a planimeter (1 × 1 mm). The ulcer areas were measured by using the following formula: Ulcer index (mm2) ⫽ length (mm) × width (mm) × π/4
2.8. Evaluation of gastric mucosal hemorrhage To evaluate the level of gastric mucosal hemorrhage, luminal hemoglobin was measured spectrophotometrically (DU 640B; Backman, Fullerton, CA, USA) at 376 nm (Holzer et al., 1989). The luminal hemoglobin content was calculated as Fv × FHb ⫺ (7-Iv) × IHb Fv and Iv are the volumes of the final and initial samples, respectively, whereas FHb and IHb are the luminal hemoglobin concentrations in the final and initial samples, respectively. 2.9. Statistical analysis Data obtained from the experiments were expressed as mean ⫾ S.E.M. Significant differences between groups were
M.-Y. Liu et al. / Alcohol 30 (2003) 61–65
63
evaluating by using an analysis of variance (ANOVA). A P value ⬍.05 was considered statistically significant.
LPO, NO levels, and ulcer index in rats treated with acidified ethanol (Fig. 2).
3. Results
4. Discussion
3.1. Effect of ethanol on gastric mucosa in rats
Abamectin pretreatment attenuated gastric ulceration induced by ethanol in intact rats (sham vagotomy group), but not in rats that underwent vagotomy (vagotomy group). The vagus nerve may play a role in abamectin-associated gastric protection in rats that are treated with acidified ethanol. The involvement of GABAergic mechanisms in the gastric damage induced by ethanol has been investigated, and quite different findings are reported. CNS-administered GABAA receptors agonists have been shown to aggravate (Goto & Watanabe, 1982) or alleviate (Bhargava et al., 1985) gastric ulceration in various experimental models. It is thus suggested that the vagus nerve does not play a role in the gastric effects of GABAergic compounds (Erdo¨ et al., 1989). However, in the current study, we found that orally administered abamectin showed a protective effect against ethanolinduced gastric damage, which supports findings obtained in a previous study (File & Pearce, 1981). Furthermore, the protective effect of abamectin in gastric mucosal injury was abolished in rats that underwent vagotomy (vagotomy group). Therefore, it seems that the vagus nerve is involved in the abamectin-exerted gastric protection. Although the vagus nerve may play a vital role in the maintenance of gastric mucosal integrity against ethanol, the exact mechanism is still unclear. It has been reported that NO production is regulated by activity of the vagus nerve (Saperas et al., 1995) and is capable of protecting the stomach from injury by ethanol (Tepperman & Soper, 1994). We proposed that abamectin would attenuate ethanol-induced gastric mucosal injury through elevation of NO production, which resulted from activation of the vagus nerve. However,
When results for treatment with ethanol alone versus no treatment (control) were compared, ethanol caused significant injury to the gastric mucosa and altered the gastric mucosal indicators measured: LPO (23.95 ⫾ 4.75 vs. 11.82 ⫾ 0.48 µM malondialdehyde per gram of tissue), GSH (0.66 ⫾ 0.05 vs. 1.18 ⫾ 0.11 mmol per gram of tissue), nitrite levels (49.74 ⫾ 5.76 vs. 102.02 ⫾ 15.78 nmol per gram of tissue), ulcer area (77.55 ⫾ 39.66 vs. 2.2 ⫾ 0.93 mm2), and hemorrhage (1.96 ⫾ 0.47 vs. 0.19 ⫾ 0.03 mg of hemoglobin per stomach). 3.2. Effect of abamectin on gastric mucosal damage induced by ethanol in intact rats (sham vagotomy group) To examine the effect of abamectin on gastric mucosal lesions induced by ethanol, gastric mucosal LPO, NO levels, and ulcer index were determined for rats in the sham vagotomy group. Oral administration of abamectin reversed the gastric mucosal LPO, NO levels, and ulcer index in rats treated with acidified ethanol (all P ⬍ .05) (Fig. 1). 3.3. Effect of abamectin on gastric mucosal damage induced by ethanol in rats that underwent vagotomy (vagotomy group) To examine involvement of the vagus nerve in abamectinassociated gastric protection, the effect of abamectin on gastric lesions induced by ethanol was assessed for rats in the vagotomy group. Abamectin failed to affect gastric mucosal
Fig. 1. Effect of abamectin (ABM) on the gastric mucosal injury induced by ethanol (EtOH) in intact rats (sham vagotomy group). Abamectin (3 mg/kg, p.o.) was given 1 h before administration of ethanol [4 ml of a 30% (volume/volume) solution]. Mucosal lipid peroxidation (LPO) (A), nitrite levels (B), and ulcer index (C) were determined 3 h after gastric surgery. Data are reported as means ⫾ S.E.M. (n ⫽ 4). The differences among those treatments with different letters were statistically significant (P ⬍ .05). MDA ⫽ Malondialdehyde.
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Fig. 2. Effect of abamectin (ABM) on the gastric mucosal injury induced by ethanol (EtOH) in rats that underwent vagotomy (vagotomy group). Abamectin (3 mg/kg, p.o.) was given 1 h before administration of ethanol [4 ml of a 30% (volume/volume) solution]. Mucosal lipid peroxidation (LPO) (A), nitrite levels (B), and ulcer index (C) were determined 3 h after gastric surgery. Data are reported as means ⫾ S.E.M. (n ⫽ 4). The differences among those treatments with different letters were statistically significant (P ⬍ .05). MDA ⫽ Malondialdehyde.
findings obtained for rats in the vagotomy group in the current study revealed that abamectin failed to have an effect on the decreased NO production in ethanol-treated rats, and this may be because the activity of the vagus nerve was completely abolished. Nitric oxide has been reported to reduce the generation of oxyradicals, which is suggested as an offensive factor in gastric mucosal damage (Kanner et al., 1991). Although its mechanism for scavenging radicals has not been clearly delineated, the ability of NO to scavenge superoxide anion has been documented both in vitro (Rubanyi et al., 1991) and in vivo (Gaboury et al., 1993). It also protects against H2O2-induced gastric damage by chelating stored Fe3⫹ or reduced Fe2⫹ to form iron-nitrosyl compounds (Kim & Kim, 1998). It has been suggested that abamectin might protect the gastric mucosa against ethanol by activating the NO pathway. In summary, administration of abamectin improved ethanol-induced gastric mucosal injury through elevation of NO production. Activation of the vagus nerve may be involved in the abamectin-associated gastric protection against ethanol in rats.
Acknowledgments This research was supported by a grant (NSC89-2320B006-026) from the National Science Council of Taiwan and a grant (VGH 92-198) from the Veterans General Hospital-Taipei.
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Bhargava, K. P., Gupta, G. P., & Gupta, M. B. (1985). Central GABAergic mechanism in stress-induced gastric ulceration. Br J Pharmacol 84, 619–623. Campbell, W. C., Fisher, M. H., Stapley, E. O., Albers-Schonberg, G., & Jacob, T. A. (1983). Ivermectin: a potent antiparasitic agent. Science 221, 823–828. El-Dakhakhny, M., Barakat, M., El-Halim, M. A., & Aly, S. M. (2000). Effects of Nigella sativa oil on gastric secretion and ethanol induced ulcer in rats. J Ethnopharmacol 72, 299–304. Erdo¨, S. L., Ezer, E., Matuz, J., Wolff, J. R., & Amenta, F. (1989). GABAA receptors in the rat stomach may mediate mucoprotective effects. Eur J Pharmacol 165, 79–86. File, S. E., & Pearce, J. B. (1981). Benzodiazepines reduce gastric ulcers induced in rats by stress. Br J Pharmacol 74, 593–599. Gaboury, J., Woodman, R. C., Granger, D. N., Reinhardt, P., & Kubes, P. (1993). Nitric oxide prevents leukocyte adherence: role of superoxide. Am J Physiol 265(3 Pt 2), H862–H867. Goto, Y., & Watanabe, K. (1982). Possible involvement of brain GABA metabolism in the pathogenesis of experimental ulcers in the rat. In C. J. Pfeiffer (Ed.), Drugs and Peptic Ulcer, Vol. 2 (p. 203). New York: CRC Press. Green, L. C., Ruiz de Luzuriaga, K., Wagner, D. A., Rand, W., Istfan, N., Young, V. R., & Tannenbaum, S. R. (1981). Nitrate biosynthesis in man. Proc Natl Acad Sci U S A 78, 7764–7768. Holzer, P., Pabst, M. A., & Lippe, I. T. H. (1989). Intragastric capsaicin protects against aspirin-induced lesion formation and bleeding in the rat gastric mucosa. Gastroenterology 96, 1425–1433. Hsu, D. Z., Hsu, C. H., Huang, B. M., & Liu, M. Y. (2001). Abamectin effects on aspartate aminotransferase and nitric oxide in rats. Toxicology 165, 189–193. Hung, C. R., Cheng, J. T., & Shih, C. S. (2000). Gastric mucosal damage induced by arecoline seizure in rats. Life Sci 66, 2337–2349. Hung, C. R., & Hsu, D. Z. (1998). Roles of histamine receptors and oxyradicals in aggravation of acid-induced gastric haemorrhagic ulcers in endotoxaemic rats. Inflammopharmacology 6, 339–355. Kanner, J., Harel, S., & Granit, R. (1991). Nitric oxide as an antioxidant. Arch Biochem Biophys 289, 130–136. Kaplan, R. M., Courtney, C. H., Kunkle, W. E., Zeng, Q. Y., Jernigan, A. D., & Eagleson, J. S. (1994). Efficacy of injectable abamectin against gastrointestinal tract nematodes and lungworms of cattle. Am J Vet Res 55, 353–357. Khadour, F., Karim, K., & Najim, R. (1996). Drugs acting at central benzodiazepine receptors attenuate ethanol-induced gastric lesions in rats. Eur J Pharmacol 298, 271–277.
M.-Y. Liu et al. / Alcohol 30 (2003) 61–65 Kim, H., & Kim, K. H. (1998). Effect of nitric oxide on hydrogen peroxide–induced damage in isolated rabbit gastric glands. Pharmacology 57, 323–330. Ko, J. K., & Cho, C. H. (1999). Co-regulation of mucosal nitric oxide and prostaglandin in gastric adaptive cytoprotection. Inflamm Res 48, 471–478. Kvietys, P. R., Twohig, B., Danzell, J., & Specian, R. D. (1990). Ethanolinduced injury to the rat gastric mucosa. Role of neutrophils and xanthine oxidase–derived radicals. Gastroenterology 98, 909–920. Najim, R. A., & Karim, K. H. (1991). Protection against ethanol-induced gastric damage by drugs acting at the GABA–benzodiazepine receptor complex. Psychopharmacology (Berl) 103, 110–114. Poli, G., Albano, E., & Dianzani, M. U. (1993). Free Radicals: From Basic Science to Medicine. New York: Springer-Verlag. Rubanyi, G. M., Ho, E. H., Cantor, E. H., Lumma, W. C., & Botelho, L. H. (1991). Cytoprotective function of nitric oxide: inactivation of
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superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 181, 1392–1397. Saperas, E., Mourelle, M., Santos, J., Moncada, S., & Malagelada, J. R. (1995). Central vagal activation by an analogue of TRH stimulates gastric nitric oxide release in rats. Am J Physiol 268(6 Pt 1), G895–G899. Szelenyi, I., & Brune, K. (1988). Possible role of oxygen free radicals in ethanol-induced gastric mucosal damage in rats. Dig Dis Sci 33, 865–871. Tepperman, B. L., & Soper, B. D. (1994). Nitric oxide synthase induction and cytoprotection of rat gastric mucosa from injury by ethanol. Can J Physiol Pharmacol 72, 1308–1312. Terano, A., Hiraishi, H., Ota, S., Shiga, J., & Sugimoto, T. (1989). Role of superoxide and hydroxyl radicals in rat gastric mucosal injury induced by ethanol. Gastroenterol Jpn 24, 488–493. Yegen, B. C., Alican, I., Yalcin, A. S., & Oktay, S. (1992). Calcium channel blockers prevent stress-induced ulcers in rats. Agents Actions 35, 130–134.
Abamectin attenuates gastric mucosal damage induced by ethanol through activation of vagus nerve in rats Ming-Yie Liua,b,*, James Po-Jung Chianga, Dur-Zong Hsua,b, Jou-Fang Dengc a
Graduate Institute of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan 704 b Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan 704 c Division of Clinical Toxicology, Department of Medicine, National Yang Ming University and Veterans General Hospital-Taipei, Taipei, Taiwan 112 Received 6 September 2002; received in revised form 16 April 2003; accepted 18 April 2003
Abstract Some type A gamma-aminobutyric acid (GABAA) receptor agonists are effective in protecting against the formation of stomach lesions induced by ethanol. Natural product abamectin, one of the existing GABAA receptor agonists, might protect against the development of gastric ulcers induced by ethanol. We investigated the protective effect of abamectin against the formation of gastric mucosal lesions induced by ethanol in rats. Abamectin (3 mg/kg, p.o.) was given to rats 1 h before administration of ethanol [4 ml of a 30% (volume/ volume) solution]. Mucosal lipid peroxidation (LPO), nitric oxide (NO) levels, and ulcer index were measured 3 h after gastric surgery (vagotomy vs. sham vagotomy) in treated versus control subjects. Abamectin attenuated ethanol-induced gastric ulceration, decreased LPO regeneration, and increased NO production in the gastric mucosa of rats in the sham vagotomy group. However, this protective effect of abamectin against ethanol-induced gastric lesions was not observed in rats in the group that underwent vagotomy. These results support the suggestion that administration of abamectin ameliorated the ethanol-induced gastric mucosal injury through elevation of NO production. Activation of the vagus nerve may be involved in the abamectin-associated gastric protection against the effects of ethanol in rats. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: Ethanol; Abamectin; Lipid peroxidation; Nitric oxide; Vagus nerve
1. Introduction Since the 19th century (Beaumont, 1833), it has been known that exposure of the gastric mucosa to concentrated alcohol can induce acute gastritis. Although some evidence seems to indicate that the neuropharmacological action of ethanol is mediated through gamma-aminobutyric acid [(GABA); a central inhibitory synaptic neurotransmitter], the exact mechanism of ethanol-induced gastric damage has not been clarified (Khadour et al., 1996). Oral pretreatment with benzodiazepines, which are GABAergic compounds, prevents ulceration of the gastric mucosa induced by acidified ethanol (Erdo¨ et al., 1989; File & Pearce, 1981). It has been reported further that such a protective action is not due to the sedative effect of benzodiazepines (Najim & Karim, 1991). Thus, activation of the GABA receptor may be effective in protecting the gastric mucosa against damage induced by ethanol. * Corresponding author. Graduate Institute of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan 704. Tel.: ⫹886-2353535, ext. 5805; fax: ⫹886-6-2752484. E-mail address: [email protected] (M.-Y. Liu). Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00094-6
Abamectin, an analog of ivermectin, is a mixture of avermectins containing at least 80% of avermectin B1a and less than 20% of avermectin B1b. Avermectins are produced by the soil bacterium Streptomyces avermitilis (Agarwal, 1998; Campbell et al., 1983). As a safe chemical in mammals, abamectin has been used as an anthelmintic agent in both animals and human beings. In addition, abamectin is believed to be a GABAergic agent (Kaplan et al., 1994). Both lipid peroxidation (LPO) and glutathione (GSH) are crucial parameters in evaluating gastric mucosal injury (Hung & Hsu, 1998). Mucosal LPO plays a significant role in the pathogenesis of ethanol-induced gastric mucosal lesions. Oxygen radicals have been implicated in the direct damage of cell membranes after administration of ethanol (Kvietys et al., 1990; Szelenyi & Brune, 1988; Terano et al., 1989). Glutathione, an important endogenous sulfhydryl compound in the cellular defense system (Poli et al., 1993), is markedly reduced in gastric mucosa after ethanol challenge (ElDakhakhny et al., 2000). Furthermore, mucosal nitric oxide (NO), in the gastric defense system, is released after activation by mild irritants to induce cytoprotection (Ko & Cho, 1999). Results of a previous study from our laboratory indicate that abamectin ingestion can increase serum NO production (Hsu et al., 2001). However, whether abamectin can
62
M.-Y. Liu et al. / Alcohol 30 (2003) 61–65
protect against gastric mucosal damage induced by ethanol by stimulating NO production is unknown. To explore this question, we examined the effect of abamectin on gastric mucosa in rats treated with acidified ethanol. 2. Materials and methods 2.1. Reagents Abamectin (98% purity) was a gift from Novartis Crop Protection Taiwan Co. Ltd. (Taipei, Taiwan). Ethanol (99.8% purity) was obtained from Merck (Darmstadt, Germany). 2.2. Animals Male, specific-pathogen free, Wistar rats (each weighing between 200 and 300 g) were obtained from and housed in our institution’s Laboratory Animal Center. The animal care and experimental protocols were in accordance with the institutional guidelines, which are approved by the National Laboratory Animal Breeding and Research Center in Taiwan. Ethyl ether was used to anesthetize animals before the gastric surgery. Rats were deprived of food but allowed free access to tap water for 24 h before experiments. Results of preliminary experiments demonstrated that mineral oil could be used as a solvent for the abamectin (range, 1 to 10 mg/kg) in the study of gastric mucosal injury in rats, and that mineral oil, with or without abamectin (3 mg/kg), did not induce injury to the gastric mucosa in rats. Therefore, we chose to evaluate a 3-mg/kg dose of abamectin (in mineral oil) as a drug to protect against the development of gastric ulcers induced by ethanol. Study animals were assigned to one of two groups: those that underwent vagotomy (vagotomy group) and those that did not undergo vagotomy (sham vagotomy group). Animals in each of the two groups were assigned to five subgroups: two control (nontreated and mineral oil control) and three treated (abamectin, ethanol, and abamectin plus ethanol). Forty rats (four rats in each group) were used. Rats were gavaged with abamectin (3 mg/kg, suspended in 1 ml of mineral oil) under light ether anesthesia 1 h before gastric surgery. 2.3. Gastric surgery procedures The stomachs of animals were surgically exposed for ligation of the pylorus and lower portion of the esophagus. Gastric bilateral vagotomy or sham vagotomy was performed in these groups of rats (Hung et al., 2000). A small incision was made on the forestomach, and the stomach contents were expelled. A polypropylene tube was inserted through the incision, secured, and rinsed with saline (at 37ºC). An isotonic solution (7 ml), containing 30% ethanol plus 150 mM HCl (acidified ethanol), was instilled into the stomach and mixed, and 3 ml of the solution was taken as an initial sample. The abdominal wound was subsequently sutured. After 3 h, the final gastric sample was collected for determination of gastric hemoglobin concentration.
2.4. Assay of mucosal glutathione level Tissue samples were homogenized in ice-cold trichloroacetic acid (1 g of tissue plus 10 ml of 10% trichloroacetic acid) in a tissue homogenizer. Briefly, after centrifugation at 3,000 rpm for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 M Na2HPO4 2H2O solution. A 0.2-ml solution of dithiobisnitrobenzoate (1% sodium citrate, 0.4 mg/ml) was added, and the absorbance at 412 nm was measured immediately (Yegen et al., 1992). 2.5. Measurement of lipid peroxidation levels in the serum and tissues Gastric mucosa was homogenized in TRIS HCl (20 mM, pH 7.4). Tissue homogenate (1 ml) was centrifuged at 2,500g for 10 min at 4ºC. Supernatant (200 µl) was taken for LPO measurement by using a commercial assay kit (CalbiochemNovabiochem Co., Darmstadt, Germany) and a spectrophotometer reading of 586 nm. 2.6. Measurement of mucosal nitrite levels Briefly, the amounts of nitrite in mucosa were measured following the Griess reaction by incubating a 100-µl sample with 100 µl of Griess reagent at room temperature for 20 min. The absorbance at 550 nm was measured with a spectrophotometer (Green et al., 1981). Nitrite concentration was calculated by comparison with a standard solution of known sodium nitrite concentrations. 2.7. Morphological studies of gastric mucosa The stomach was opened along the greater curvature. The length and width of the ulcers on the gastric mucosa were measured with a planimeter (1 × 1 mm). The ulcer areas were measured by using the following formula: Ulcer index (mm2) ⫽ length (mm) × width (mm) × π/4
2.8. Evaluation of gastric mucosal hemorrhage To evaluate the level of gastric mucosal hemorrhage, luminal hemoglobin was measured spectrophotometrically (DU 640B; Backman, Fullerton, CA, USA) at 376 nm (Holzer et al., 1989). The luminal hemoglobin content was calculated as Fv × FHb ⫺ (7-Iv) × IHb Fv and Iv are the volumes of the final and initial samples, respectively, whereas FHb and IHb are the luminal hemoglobin concentrations in the final and initial samples, respectively. 2.9. Statistical analysis Data obtained from the experiments were expressed as mean ⫾ S.E.M. Significant differences between groups were
M.-Y. Liu et al. / Alcohol 30 (2003) 61–65
63
evaluating by using an analysis of variance (ANOVA). A P value ⬍.05 was considered statistically significant.
LPO, NO levels, and ulcer index in rats treated with acidified ethanol (Fig. 2).
3. Results
4. Discussion
3.1. Effect of ethanol on gastric mucosa in rats
Abamectin pretreatment attenuated gastric ulceration induced by ethanol in intact rats (sham vagotomy group), but not in rats that underwent vagotomy (vagotomy group). The vagus nerve may play a role in abamectin-associated gastric protection in rats that are treated with acidified ethanol. The involvement of GABAergic mechanisms in the gastric damage induced by ethanol has been investigated, and quite different findings are reported. CNS-administered GABAA receptors agonists have been shown to aggravate (Goto & Watanabe, 1982) or alleviate (Bhargava et al., 1985) gastric ulceration in various experimental models. It is thus suggested that the vagus nerve does not play a role in the gastric effects of GABAergic compounds (Erdo¨ et al., 1989). However, in the current study, we found that orally administered abamectin showed a protective effect against ethanolinduced gastric damage, which supports findings obtained in a previous study (File & Pearce, 1981). Furthermore, the protective effect of abamectin in gastric mucosal injury was abolished in rats that underwent vagotomy (vagotomy group). Therefore, it seems that the vagus nerve is involved in the abamectin-exerted gastric protection. Although the vagus nerve may play a vital role in the maintenance of gastric mucosal integrity against ethanol, the exact mechanism is still unclear. It has been reported that NO production is regulated by activity of the vagus nerve (Saperas et al., 1995) and is capable of protecting the stomach from injury by ethanol (Tepperman & Soper, 1994). We proposed that abamectin would attenuate ethanol-induced gastric mucosal injury through elevation of NO production, which resulted from activation of the vagus nerve. However,
When results for treatment with ethanol alone versus no treatment (control) were compared, ethanol caused significant injury to the gastric mucosa and altered the gastric mucosal indicators measured: LPO (23.95 ⫾ 4.75 vs. 11.82 ⫾ 0.48 µM malondialdehyde per gram of tissue), GSH (0.66 ⫾ 0.05 vs. 1.18 ⫾ 0.11 mmol per gram of tissue), nitrite levels (49.74 ⫾ 5.76 vs. 102.02 ⫾ 15.78 nmol per gram of tissue), ulcer area (77.55 ⫾ 39.66 vs. 2.2 ⫾ 0.93 mm2), and hemorrhage (1.96 ⫾ 0.47 vs. 0.19 ⫾ 0.03 mg of hemoglobin per stomach). 3.2. Effect of abamectin on gastric mucosal damage induced by ethanol in intact rats (sham vagotomy group) To examine the effect of abamectin on gastric mucosal lesions induced by ethanol, gastric mucosal LPO, NO levels, and ulcer index were determined for rats in the sham vagotomy group. Oral administration of abamectin reversed the gastric mucosal LPO, NO levels, and ulcer index in rats treated with acidified ethanol (all P ⬍ .05) (Fig. 1). 3.3. Effect of abamectin on gastric mucosal damage induced by ethanol in rats that underwent vagotomy (vagotomy group) To examine involvement of the vagus nerve in abamectinassociated gastric protection, the effect of abamectin on gastric lesions induced by ethanol was assessed for rats in the vagotomy group. Abamectin failed to affect gastric mucosal
Fig. 1. Effect of abamectin (ABM) on the gastric mucosal injury induced by ethanol (EtOH) in intact rats (sham vagotomy group). Abamectin (3 mg/kg, p.o.) was given 1 h before administration of ethanol [4 ml of a 30% (volume/volume) solution]. Mucosal lipid peroxidation (LPO) (A), nitrite levels (B), and ulcer index (C) were determined 3 h after gastric surgery. Data are reported as means ⫾ S.E.M. (n ⫽ 4). The differences among those treatments with different letters were statistically significant (P ⬍ .05). MDA ⫽ Malondialdehyde.
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Fig. 2. Effect of abamectin (ABM) on the gastric mucosal injury induced by ethanol (EtOH) in rats that underwent vagotomy (vagotomy group). Abamectin (3 mg/kg, p.o.) was given 1 h before administration of ethanol [4 ml of a 30% (volume/volume) solution]. Mucosal lipid peroxidation (LPO) (A), nitrite levels (B), and ulcer index (C) were determined 3 h after gastric surgery. Data are reported as means ⫾ S.E.M. (n ⫽ 4). The differences among those treatments with different letters were statistically significant (P ⬍ .05). MDA ⫽ Malondialdehyde.
findings obtained for rats in the vagotomy group in the current study revealed that abamectin failed to have an effect on the decreased NO production in ethanol-treated rats, and this may be because the activity of the vagus nerve was completely abolished. Nitric oxide has been reported to reduce the generation of oxyradicals, which is suggested as an offensive factor in gastric mucosal damage (Kanner et al., 1991). Although its mechanism for scavenging radicals has not been clearly delineated, the ability of NO to scavenge superoxide anion has been documented both in vitro (Rubanyi et al., 1991) and in vivo (Gaboury et al., 1993). It also protects against H2O2-induced gastric damage by chelating stored Fe3⫹ or reduced Fe2⫹ to form iron-nitrosyl compounds (Kim & Kim, 1998). It has been suggested that abamectin might protect the gastric mucosa against ethanol by activating the NO pathway. In summary, administration of abamectin improved ethanol-induced gastric mucosal injury through elevation of NO production. Activation of the vagus nerve may be involved in the abamectin-associated gastric protection against ethanol in rats.
Acknowledgments This research was supported by a grant (NSC89-2320B006-026) from the National Science Council of Taiwan and a grant (VGH 92-198) from the Veterans General Hospital-Taipei.
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