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Mechanisms involved in the gastroprotective activity of esculin on acute gastric lesions in mice
Chemico-Biological Interactions 188 (2010) 246–254
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Mechanisms involved in the gastroprotective activity of esculin on acute gastric lesions in mice Emiliano Ricardo Vasconcelos Rios a,∗ , Nayrton Flávio Moura Rocha a , Edith Teles Venâncio a , Brinell Arcanjo Moura a , Mariana Lima Feitosa a , Gilberto Santos Cerqueira a , Pedro Marcos Gomes Soares a , David John Woods c , Francisca Cléa Florenc¸o de Sousa a , Luzia Kalyne Almeida Moreira Leal a,b , Marta Maria de Franc¸a Fonteles a,b a
Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Rua Coronel Nunes de Melo, 1127, Brazil Pharmacy Department, Faculty of Dentistry, Nursing and Pharmacy, Federal University of Ceará, Rua Capitão Francisco Pedro, 1210, Brazil c School of Pharmacy, University of Otago, PO Box 913, Dunedin, New Zealand b
a r t i c l e
i n f o
Article history: Received 23 May 2010 Received in revised form 20 July 2010 Accepted 21 July 2010 Available online 1 August 2010 Keywords: Esculin Gastric lesion Gastroprotection Ethanol Indomethacin
a b s t r a c t This work describes the gastroprotective actions of esculin (6,7-dihydroxycoumarin-6-o-glucoside) against indomethacin- or ethanol-induced lesions and verifies the role of nitric oxide, ATP-dependent K+ channels, prostaglandins, transient receptor potential vanilloid 1 and antioxidant effects in the gastroprotective mechanism of esculin in the ethanol-induced gastric lesion model. The intragastric administration of esculin at doses of 12.5, 25 and 50 mg/kg was able to protect the gastric mucosa against ethanol (0.2 mL/animal p.o.), and esculin at doses of 25 and 50 mg/kg protected against indomethacininduced lesions (20 mg/kg p.o.). Administration of l-NAME (10 mg/kg i.p.), glibenclamide (10 mg/kg i.p.) or indomethacin (10 mg/kg p.o.), but not capsazepine (5 mg/kg p.o.), was able to reduce the gastroprotection promoted by esculin (25 mg/kg) on the ethanol-induced lesions. Measurements of nitrite, a NO metabolite, were increased in the group that was pretreated with esculin. In terms of antioxidant activity as a gastroprotective mechanism of esculin, the results show that pre-treatment with esculin decreased the amount of GSH, increased SOD activity, did not interfere with the CAT activity and decreased both the MPO activity and the MDA amount. In conclusion, pre-treatment with esculin confers significant gastroprotective and antioxidant activity and leads to a reduction in gastric injury; the mechanisms underlying these effects include stimulation of endogenous prostaglandins, nitric oxide synthesis, opening of KATP channels and reduction of free radicals or modulation of antioxidant enzyme systems. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The gastric mucous membrane is continuously exposed to potentially harmful agents, such as HCl, pepsin, bile acids, food seasonings, bacterial products and drugs. These agents are involved in the pathogenesis of gastric injury by promoting an increase in the secretion of gastric acid and pepsin, decreasing gastric blood flow, suppressing the output of endogenous prostaglandins, inhibiting the cellular proliferation and growth of the mucous membrane and altering gastric motility [1]. In physiological conditions, there is a balance between the aggressive factors (HCl, pepsin, bile and pancreatic enzymes)
∗ Corresponding author at: Department of Physiology and Pharmacology, Federal University of Ceará, Rua Cel. Nunes de Melo 1127, CEP: 60430-270, Fortaleza, Ceará, Brazil. Tel.: +55 85 3366 8337; fax: +55 85 3366 8333. E-mail address: [email protected] (E.R.V. Rios). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.07.020
and the gastroprotective factors (mucus-bicarbonate, blood flow, prostaglandins and glutathione). Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDS), smoking, alcohol, trauma, sepsis, shock, Helicobacter pylori, and stress have been shown to contribute to gastric ulcer formation [2]. Indomethacin-induced gastric lesions are characterized by significant oxidative injury, reduced mucosal blood flow and reduced secretion of mucus/bicarbonate, mainly due to inhibition of PG secretion [3]. Ethanol-induced gastric lesions occur mainly due to intense infiltration in the sub-mucosa that promotes formation of ROS, decreased mucus, depletion of sulfhydryl groups and decreased blood flow, resulting in damage to the gastric mucosa [4]. This model has fundamental importance for scientific research due to the fact that we can utilize it to evaluate possible mechanisms by which substances can act to promote gastroprotection. The generation of ROS and pro-oxidative events are involved in the etiology of gastric lesions in these two models. Several antioxidants exhibit gastroprotective activity, such as quercetin [5] and curcumin [6].
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Esculin (6,7-dihydroxycoumarin-6-o-glucoside) is a coumarinic derivative found in Aesculus hippocastanum L. (Horse-chestnut). Their seeds have long been used to treat inflammatory and vascular problems. In Brazilian folk medicine, the tea prepared from the crushed seeds is used to protect against kidney stones and stomach pain. Esculin is known to be a 5- and 12-lipoxygenase inhibitor and to inhibit the production of leukotrienes and 5hydroxyeicosatetraenoic acid through the lipoxygenase pathway [7]. In 2007, Zhao et al. used the dopamine-induced cytotoxicity model in human neuroblastoma SH-SY5Y cells to demonstrate that esculin inhibited dopamine-induced caspase-3 cleavage and decreased cell death, overproduction of ROS, morphological changes of nuclei and damage to antioxidant enzymes [8]. Esculin scavenges hydroxyl radicals and inhibits lipid peroxidation in the rat liver [9] and displays anti-inflammatory activity in both zymosan- and carrageenan-induced paw edema in mice [10]. The gastroprotective effect of esculin was also observed in rats by Martin et al. [11] in cold-restraint stress and pylorus ligation-induced ulcer models; however, neither its effect on indomethacin-induced lesions nor the gastroprotective mechanisms involved were investigated in this study. Thus, this study was developed to better characterize the potential gastroprotective actions of esculin in mice and to investigate the possible mechanisms involved.
opened along the greater curvature for examination. The total and injured stomach areas (glandular face) were measured by the Image J computer program and expressed in terms of the percent (%) of lesioned gastric area.
2. Materials and methods
To study the possible mechanisms of action, the experiments were conducted using esculin (25 mg/kg; intermediate dose between the effective doses tested) and the following drugs: l-NAME, an inhibitor of NO-synthase activity (10 mg/kg, i.p.); glibenclamide, a blocker of KATP channels (10 mg/kg, i.p.); indomethacin (10 mg/kg, p.o.); and capsazepine (CZP), a TRPV-1 antagonist (5 mg/kg, i.p.). l-NAME or glibenclamide were administered 15 min before administration of esculin (25 mg/kg, p.o.), l-arginine (600 mg/kg, p.o.) or diazoxide (3 mg/kg, p.o.). Indomethacin (10 mg/kg, p.o.) was administrated 2 h before administration of esculin (25 mg/kg, p.o.) or misoprostol (50 g/kg, p.o.). Capsazepine (5 mg/kg, i.p.) was given 30 min before administration of esculin (25 mg/kg, p.o.) or capsaicin (CPS) (0.3 mg/kg, p.o.). After 1 h of oral administration was induced gastric injury with ethanol. All animals received absolute ethanol (0.2 mL) for the lesion induction. Thirty minutes after the administration of ethanol, the mice were killed and the stomach was removed for examination as previously described. The dose selections for these drugs were based on our pilot experiments and on literature findings [13–16].
2.1. Animals Male Swiss mice (25–35 g) were used in this study. Animals were kept in a temperature-controlled room at 25 ± 2 ◦ C with a 12-h light/dark cycle, with food and water ad libitum. The study was approved by the Ethics Committee for Animal Research at the Federal University of Ceará in Brazil, and it was conducted in accordance with the National Institute of Health in Bethesda, USA. 2.2. Drugs Capsazepine, capsaicin, cyproheptadine, diazoxide, esculin, glibenclamide, indomethacin, l-arginine, NG -nitro-l-arginine methyl ester (l-NAME), sodium nitrite and Tween 80 were purchased from Sigma–Aldrich® (St. Louis, MO, USA), N-acetyll-cysteine (NAC) from União Química® (São Paulo, SP, Brazil), Ranitidine from GlaxoSmithKline® (Rio de Janeiro, RJ, Brazil) and Misoprostol from Pfizer® (São Paulo, SP, Brazil). 2.3. Study of the gastroprotective activity of esculin in the ethanol-induced gastric lesion model The acute gastric lesions were induced by intragastric administration of absolute ethanol in accordance with a previously described method [3]. Male Swiss mice were randomly divided into five groups and fasted for 15 h before the experiment, but mice had free access to water. The ethanol groups were orally administered (0.2 mL/animal) 60 min after the treatment with esculin (12.5, 25 and 50 mg/kg, p.o – ESC12.5, ESC25 and ESC50, respectively). These doses were chosen based on results from previous studies by the same group with coumarin and umbelliferone (unpublished data) and from the study by Martin et al. [11]. The esculin was dissolved in 3% Tween 80 in distilled water. Control mice were similarly treated with Tween. Cyproheptadine, a non-selective antagonist of 5-HT and histamine receptors (10 mg/kg; p.o. – CYP10), was used as a reference drug. All experimental groups consisted of eight mice. These treatments were performed by gavage with a metal orogastric tube. Thirty minutes after the administration of ethanol, the mice were killed by cervical dislocation, and the stomach was removed and
2.4. Histopathological assessment Histological evaluation was performed on the glandular face of the stomach. Tissue samples were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Sections about 4 mm thick were cut and stained with hematoxylin and eosin. The mucosal injury evaluation was performed under light microscopy by an experienced histologist blinded to the treatment regimen. The histopathological changes were assessed according to the following criteria that were previously described by Laine and Weinstein [12]: (1) edema (score 0–4), (2) hemorrhagic damage (score 0–4), (3) inflammatory infiltration (score 0–3), and (4) epithelial cell loss (score 0–3). 2.5. Evaluation of the role of nitric oxide (NO), ATP-dependent K+ channels (KATP ), prostaglandins and transient receptor potential vanilloid 1 (TRPV1) in the gastroprotective effect of esculin on the ethanol-induced gastric lesion model
2.6. Measurement of total nitrite levels The amount of stable nitrite, the end product of NO metabolism, in the gastric mucosa was determined by a colorimetric assay as described by Green et al. [17]. Briefly, 100 L of gastric mucosa homogenate was mixed with an equal volume of Griess reagent that consists of equal parts of 1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride (NEED), 5% H3 PO4 and distilled water and incubated at room temperature for 10 min. The absorbance was read at 540 nm on a microplate reader (UVM-340, Asys Hitech, Netherlands). The amount of nitrite was calculated from a NaNO2 standard curve. 2.7. Study of the gastroprotective action of esculin in the indomethacin-induced gastric lesion model Male Swiss mice were randomly divided into four groups and treated orally with vehicle (controls), esculin (25 and 50 mg/kg p.o.) or ranitidine, a H2 histamine receptor antagonist (20 mg/kg; p.o.). After 60 min, gastric lesions were induced in all groups by
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E.R.V. Rios et al. / Chemico-Biological Interactions 188 (2010) 246–254 Table 1 Scale by attribution of scores for degree of ulceration [19]. Injuiy
Scores
Discoloration of mucosa Edema Hemorrhages Number of petechia Until 10 More than 10 Intensity of ulceration Ulcers or erosion up to 1 mm Ulcers or erosion larger than 1 mm Perforated ulcers
1 1 1 2 3 N×2 N×3 N×4
N number of stomach lesions.
indomethacin (20 mg/kg, p.o.) suspended in 0.5% carboxymethylcellulose in distilled water. After 7 h, as per Bhargava’s method [18], the animals were sacrificed by cervical dislocation, and the stomachs were removed and immersed in 5% formalin for 15 min and then opened by the great curvature and washed with saline solution for examination of the lesions. The degree of lesion formation was graded according to an arbitrary scale by attribution of scores as presented in Table 1 [19].
assayed. In a dark chamber, 1 mL of the reactant (50 mM phosphate buffer, 100 nM EDTA and 13 mM l-methionine, pH 7.8) was mixed with 30 L of the sample, 150 L of 75 M NBT and 300 L of 2 M riboflavin. The tubes containing the resulting solution were exposed to fluorescent light bulbs (15 W) for 15 min and then read using a spectrophotometer at 560 nm.
2.10. Measurement of catalase (CAT) activity Catalase activity is proportional to the rate of output of O2 and H2 O. Hydrogen peroxide (H2 O2 ) is used as the substrate and is hydrolyzed according to the Maehly and Chance method [23]. The activity of the enzyme is measured at 230 nm using spectrophotometry (Beckman DU) by reading the variation of the absorbance between the first and sixth minutes and then expressing the results in M/min/g of protein. The reaction environment was prepared with H2 O2 (18 mL), 1 M Tris HCl buffer 5 mM EDTA pH 8 (1 mL) and Milli-Q H2 O (0.8 mL). Immediately, 980 L of the reaction environment was added to 20 L of the homogenate (10%) in a quartz cuvette.
2.11. Measurement of the myeloperoxidase (MPO) activity 2.8. Evaluation of the antioxidant effect of esculin in gastroprotection and measurement of the amount of reduced glutathione To assess the changes in the gastric mucosal amount of GSH (a non-protein sulfhydryl compound), its content was measured according to the method described by Sedlak and Lindsay [20] with slight modifications. Briefly, esculin (25 mg/kg, p.o.), N-acetylcysteine (300 mg/kg, p.o.) or vehicle (control) was intragastrically administered to mice 1 h before administration of saline (healthy) or absolute ethanol (0.2 mL/animal) (ulcerated). Thirty minutes after treatment, the animals were sacrificed by cervical dislocation and their stomachs were removed. For the GSH assay, the glandular segment from each stomach was homogenized in ice-cold 0.02 M EDTA solution (at 10%). Aliquots (400 L) of tissue homogenate were mixed with 320 L of distilled water and 80 L of 50% (w/v) trichloroacetic acid in glass tubes and centrifuged at 3000 rpm for 15 min. Subsequently, supernatants (400 L) were mixed with 800 L Tris buffer (0.4 M, pH 8.9) and 5,5-dithio-bis(2nitrobenzoic acid) (DTNB; 0.01 M) was added. After shaking the reaction mixture for 3 min, its absorbance was measured at 412 nm within 5 min of the addition of DTNB against a blank with no homogenate. The absorbance values were extrapolated from a glutathione standard curve and the GSH content was expressed in g GSH/g of protein. The concentration of proteins was measured using the method described by Bradford [21]. 2.9. Measurement of the superoxide dismutase (SOD) activity SOD activity was measured according to Sun et al. [22]. The activity of the enzyme was evaluated by measuring its capacity to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT). In this assay, the photochemical reduction of riboflavin generates O2− that reduces the NBT to produce formazan salt, which absorbs at a wavelength of 560 nm. In the presence of SOD, the reduction of the NBT is inhibited because the enzyme converts the superoxide radical to peroxide. The results are expressed as the quantity of SOD necessary to inhibit the rate of reduction of the NBT by 50% in units of the enzyme per gram of protein. Homogenates (10% of tissue in buffer phosphate) were centrifuged (10 min, 3600 rpm, 4 ◦ C), and the supernatant was removed and centrifuged a second time (20 min, 12,000 rpm, 4 ◦ C). The resulting supernatant was
The animals were divided into groups of eight and were sacrificed 30 min after administration of the ethanol. Stomach samples were removed and immediately frozen in liquid nitrogen. Samples were then homogenized in a solution of 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer pH 6.0 (1 mL per 50 mg of the tissue) and centrifuged at 4000 rpm for 15 min at 4 ◦ C. The amount of the enzyme in the supernatant (30 L) was analyzed by spectrophotometry after addition of 200 L of phosphate buffer (50 mM, pH 6), containing 0.167 mg/ml of dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The absorbance at 470 nm was measured at time 0 and 5 min [24].
2.12. Measurement of the membrane lipids peroxidation The rate of lipoperoxidation in the gastric mucous membrane was estimated by determination of malondialdehyde (MDA) using the Thiobarbituric Acid Reactive Substances (TBARS) test. The stomachs were washed with saline to minimize the interference of hemoglobin with free radicals and to remove blood adhered to the mucous membrane. The stomachs were homogenized to 10% of tissue with potassium phosphate buffer. Then, 250 L was removed and stored at 37 ◦ C for 1 h, after which 400 L of 35% perchloric acid was added, and the mixture was centrifuged at 14,000 rpm for 20 min at 4 ◦ C. The supernatant was removed, mixed with 400 L of 0.6% thiobarbituric acid and incubated at 95–100 ◦ C for 1 h. After cooling, the absorbance at 532 nm was measured. A standard curve was generated using 1,1,3,3-tetrametoxypropane. The results were expressed as nmol of MDA/mg of protein. The concentration of proteins was measured using the method described by Bradford [21].Measurement of total protein in the stomach sample after ethanol-induced lesions The method is based on the interaction of the Coomassie Blue G250 dye with proteins. At the pH of the reaction, the interaction between proteins of high molecular weight and the dye causes a shift in the dye to the anionic form, which absorbs strongly at 595 nm. Solutions of albumin standard, distilled water, buffer and samples were added to the wells. For sample preparation, 2 L of sample and 38 L of buffer were added to each well. Then, 200 L Bradford’s solution (diluted 5×) was added to each well. After 5 min, a reading was taken at the wavelength of 595 nm [21].
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Fig. 1. Effects of esculin on the gastroprotection in mice (n = 8/group) subjected to ethanol and indomethacin-induced gastric lesion. The results are presented as mean ± SEM. * p < 0.05 and *** p < 0.001, significant difference compared with Vehicle group. ANOVA followed by Newman–Keuls as the post hoc test and Kruskal–Wallis test followed by Dunnˇıs test as the post hoc test were used for the ethanol and indomethacin-induced model, respectively.
2.14. Statistical analysis Values were expressed as mean ± SEM. For statistical analysis, one-way analysis of variance (ANOVA) was used, followed by the Student Newman–Keuls post hoc test. For histopathological assessment and indomethacin-induced lesions, the Kruskal–Wallis test was used, followed by Dunn’s test. Probability (p) values less than 0.05 were considered significant.
3. Results 3.1. Study of the gastroprotective effect of esculin in the ethanol-induced ulcer model The administration of absolute ethanol produced lesions in the gastric mucosa that were reduced in the animals pretreated with 12.5 mg/kg (4.381 ± 1.129%; p < 0.001), 25 mg/kg (5.163 ± 1.043%; p < 0.001), or 50 mg/kg esculin (3.938 ± 0.7326%; p < 0.001), or with 10 mg/kg cyproheptadine (4.108 ± 0.611%; p < 0.001), when compared to the control group (18.31 ± 1.647%) (Fig. 1).
3.2. Study of gastroprotective effect of esculin in indomethacin-induced ulcer As demonstrated in Fig. 1, the administration of indomethacin produced lesions in the gastric mucosa (9.500 ± 0.422 scores), which were reduced in the animals pretreated with ESC 25 (3.571 ± 0.481 scores; p < 0.001), ESC 50 (4.857 ± 0.508 scores; p < 0.05), or 20 mg/kg ranitidine (5.125 ± 0.441 scores; p < 0.05), when compared to the control animals that were pretreated only with the vehicle.
3.3. Histopathological assessment after ethanol-induced lesions Histopathological analyses of the gastric mucosa are shown in Fig. 2 and Table 1. Animals pretreated with esculin (25 mg/kg, p.o.) (Fig. 2B) showed less macro- and microscopic mucosal damage when compared with the control group treated only with ethanol (Fig. 2A), in which severe lesions were characterized by hemorrhagic injury, edema and loss of epithelial cells. Pre-treatment with NAC also inhibited lesions promoted by ethanol (Fig. 2C), and the animals in this treatment group showed similar characteristics to the group without lesions (Fig. 2D). Esculin significantly decreased the ethanol-induced lesions to a similar extent as NAC (Table 2).
3.4. Evaluation of the role of the nitric oxide (NO) metabolic pathway in the gastroprotective effects of esculin in the ethanol-induced ulcer model The ethanol pre-treatment produced significant gastric lesions (17.590 ± 1.570%) that were reduced by ESC 25 (5.163 ± 1.043%; p < 0.001). Similarly, l-arginine (3.489 ± 0.922%; p < 0.001) was able to protect the mucosa from injury induced by ethanol. Previous administration of l-NAME (10 mg/kg) reduced the gastroprotection of ESC 25 (34.68 ± 3.899%; p < 0.001) compared to ESC 25 alone. A similar effect was caused by l-NAME plus l-ARG (19.00 ± 2.184%; p < 0.001) when compared to the l-ARG group (Fig. 3). 3.5. Evaluation of the role of KATP on the gastroprotective effects of esculin in the ethanol-induced gastric lesion model in mice The effects of oral administration of esculin on ethanol-induced lesions in glibenclamide-pretreated mice are demonstrated in Fig. 3. Pre-treatment with this KATP blocker significantly decreased the gastroprotective effect of esculin (26.57 ± 3.175%; p < 0.001) compared to the ESC 25 group (5.163 ± 1.043%) and the control group (17.59 ± 1.570%). The same result was shown with glibenclamide plus diazoxide (18.42 ± 3.329%, p < 0.001) compared to diazoxide alone (4.291 ± 1.088%). 3.6. Evaluation of the involvement of prostaglandins in the gastroprotective effects of esculin in the ethanol-induced lesion model The data in Fig. 3 show that the ethanol-induced lesions were significantly reduced by pre-treatment with ESC 25 (5.615 ± 1.039%; p < 0.001) or misoprostol (10.61 ± 1.06%; p < 0.01) compared to control (19.73 ± 1.698%). This effect was reduced by previous indomethacin administration (15.01 ± 0.7557%; p < 0.001) compared to the ESC 25 group. A similar result was shown for indomethacin plus misoprostol (22.51 ± 2.328%; p < 0.001) compared to misoprostol alone (Fig. 3). 3.7. Evaluation of the involvement of TRPV-1 (Transient Receptor Potential Vanilloid-1) in the gastroprotective effects of esculin in the ethanol-induced lesion model Fig. 3 also shows the effect of capsazepine on gastroprotection promoted by esculin. The ethanol-induced lesions were reduced by pre-treatment with 25 mg/kg esculin (4.095 ± 0.937%; p < 0.001) or capsaicin (5.230 ± 0.814%; p < 0.01) when compared to control (15.09 ± 1.245%). No change in the effect of esculin was observed in the group that was pre-treated with capsazepine (4.963 ± 1.042%).
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Fig. 2. Macro- and microscopic effects on the gastric mucosa of mice subjected to gastric lesions induced by ethanol pretreated or not with esculin. Photomicrographs of stomachs opened along the greater curvature (A–D) and microscopic analysis with magnification of 100× and 400× for each group (A–D 100× and 400×). (A) Animals treated with vehicle (saline 0.9–3% Tween 80, po), showing normal morphology, (B) treatment with ethanol (0.2 mL/animal), presenting inflammatory infiltration, edema, moderate hemorrhage and severe loss of epithelial cells, (C) treatment only with esculin (25 mg/kg, p.o.), showing histological aspects similar to group D, (D) pre-treatment with NAC (300 mg/kg, p.o.) before administration of ethanol, showing preservation of the gastric mucosa.
The capsazepine was able to antagonize the gastroprotection promoted by capsaicin (12.00 ± 3.135%; p < 0.01) compared to capsaicin alone. 3.8. Nitrite content in the injured stomach after ethanol-induced lesions To increase understanding of the role of nitric oxide in the gastroprotective action of esculin, the concentration of nitrite in the tissue was measured as an indirect way of assessing the amount of tissue NO. In corroboration with previous results, the group pretreated with esculin (25 mg/kg, p.o.) showed a signifi-
cantly higher concentration of nitrite (24.47 ± 4.15 mM; p < 0.001) than the group pretreated with vehicle (3% Tween 80 in distilled water, p.o; 7.50 ± 0.32 mM). No statistical difference was observed between the vehicle-treated group (11.79 ± 0.98 mM) and the ulcerated group. As standards, two groups were pre-treated with Nacetylcysteine (NAC; 300 mg/kg) or l-arginine (l-ARG; 600 mg/kg). The group pre-treated with NAC did not show a statistically significant difference (8.16 ± 0.40 mM) compared to the lesioned group, whereas the group treated with l-ARG (19.61 ± 2.55 mM; p < 0.001) showed a higher nitrite concentration compared to the injured control and was similar to the group pre-treated with esculin (Fig. 4).
Table 2 Scores of histopathological changes in the gastric mucosa of mice subjected to gastric lesions induced by ethanol pretreated or not with esculin. Animals were treated orally with vehicle (saline 0.9–3% Tween 80), esculin (ESC 25 mg/kg), or N-acetylcysteine (NAC 300 mg/kg), 60 min before administration of ethanol (0.2 mL, p.o.). Histopathological changes of glandular portion of stomach were assessed as described by Laine and Weinstein [12]: 1 – edema (score 0–4), 2 – hemorrhagic damage (score 0–4), 3 – Inflammatory infiltration (0–3), and 4 – loss epithelial cells (score 0–3). The results are presented as median with minimum and maximum values shown in parentheses (n = 4). * p < 0.05, ** p < 0.01 vs. vehicle + ethanol. We used the nonparametric Kruskal–Wallis followed by Dunn’s test as post hoc. Treatment
Vehicle Vehicle + ethanol ESC (25 mg/kg) + ethanol NAC (300 mg/kg) + ethanol
Microscopic scores Edema (score 0–4)
Hemorrhagic damage (score 0–4)
Inflammatory infiltration (score 0–3)
Loss epithelial cell (score 0–3)
0 3 (2–4) 0* 0,5 (0–2)
0 3,5 (2–4) 0** 0**
0 1 0** 0**
0 3 (2–3) 0 (0–1)* 0 (0–1)*
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Fig. 3. Effects of pre-treatment with l-NAME, glibenclamide, indomethacin or capsazepine on the gastroprotective effect of esculin in mice (n = 8/group). The results are presented as mean ± SEM. ** p < 0.01, *** p < 0.001, significant difference compared with saline group, ## p < 0.01, ### p < 0.001, significant difference compared with ESC 25 group and ++ p < 0.01, +++ p < 0.001, significant difference compared with l-ARG, DIAZ or MISO group. ANOVA followed by Newman–Keuls as the post hoc test was used.
3.9. Evaluation of the involvement of the antioxidant effect in the gastroprotection promoted by esculin 3.9.1. Evaluation of the involvement of glutathione (GSH) metabolism in the gastroprotective effects of esculin Fig. 5 shows the influence of pre-treatment with esculin or NAC on the gastric content of GSH in ethanol-induced ulcers in healthy animals. Animals treated with NAC (300 mg/kg) before ethanol administration showed a significant increase in the GSH levels (649.4 ± 16.170 g/g of protein; p < 0.001) when compared with the saline plus ethanol group (285.4 ± 22.130 g/g of protein). However, in the group treated with esculin (263.6 ± 14.510 g/g of protein), the GSH content showed no change when compared with the saline plus ethanol group. Healthy animals (399.5 ± 55.29 g/g of protein; p < 0.01) showed a higher GSH content than the control.
Fig. 4. Effect of esculin 25 mg/kg on nitrite content of gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to stressor agent. The results are presented as mean ± SEM. *** p < 0.001, significant difference compared with vehicle + absolute ethanol group. ANOVA followed by Newman–Keuls as the post hoc test was used.
3.9.2. Measurement of the superoxide dismutase (SOD) activity Ethanol reduced the SOD activity in ulcerated stomachs pretreated with vehicle (332.90 ± 42.86 U/g of protein; p < 0.05) when compared with controls without lesions (534.30 ± 30.69 U/g of protein). Pre-treatment with esculin (25 mg/kg) increased the activity of the enzyme (642.90 ± 57.39 U/g of protein; p < 0.01 and p < 0.001) compared with the lesioned group and the group without lesions. Similarly, N-acetylcysteine increased SOD activity (748.60 ± 76.86 U/g of protein; p < 0.001) compared with lesioned and non-lesioned controls (Fig. 5). 3.9.3. Measurement of the catalase (CAT) activity Ethanol-induced gastric lesions increased the CAT activity in the animals pre-treated only with the vehicle (3% Tween 80 in distilled water) (129.90 ± 8.53 g/min/g of protein; p < 0.01) when compared with non-ulcerated healthy controls that received vehicle alone (93.08 ± 7.58 g/min/g of protein). Pre-treatment
Fig. 5. Effect of esculin 25 mg/kg on enzymes activity (SOD and CAT) and nonprotein sulfhydryl (GSH) content of gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to stressor agent. The results are presented as mean ± SEM. ** p < 0.01 and * p < 0.05, significant difference compared with vehicle (non-lesioned) group and ### p < 0.001 significant difference with vehicle + absolute ethanol group. ANOVA followed by Newman–Keuls as the post hoc test was used.
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Fig. 6. Effect of esculin 25 mg/kg on lipoperoxidation level (MDA account) and MPO activity in the gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to stressor agent. The results are presented as mean ± SEM. *** p < 0.001, significant difference compared with vehicle (non-lesioned) group and ### p < 0.001 significant difference with vehicle + absolute ethanol group. ANOVA followed by Newman–Keuls as the post hoc test was used.
with esculin (25 mg/kg) did not alter the activity of the enzyme (79.19 ± 5.06 g/min/g of protein) compared with the healthy group, but CAT activity diminished significantly (p < 0.001) when compared with the ulcerated group. Similarly, the group treated with N-acetylcysteine showed diminished CAT activity (81.97 ± 7.00 g/min/g of protein; p < 0.001) when compared with the ulcerated controls (Fig. 5). 3.9.4. Measurement of the myeloperoxidase (MPO) activity MPO activity in ulcerated stomachs pre-treated only with the vehicle (3% Tween 80 in distilled water) (20.29 ± 2.318 U of MPO/min/mg of tissue, p < 0.001) was higher when compared with the healthy group without lesions (7.39 ± 0.98 U of MPO/min/mg of tissue). In the stomachs of the animals pre-treated with esculin (25 mg/kg), the activity of the enzyme was decreased (12.08 ± 0.89 U of MPO/min/mg of tissue, p < 0.001) compared with the ulcerated group. Similarly, treatment with N-acetylcysteine (300 mg/kg) decreased activity (10.16 ± 1.22 U of MPO/min/mg of tissue; p < 0.001) compared with ulcerated controls (Fig. 6). 3.9.5. Measurement of membrane lipid peroxidation Lipoperoxidation in the lesioned stomachs pretreated only with vehicle (3% Tween 80 in distilled water) (86.15 ± 10.76 mol of MDA/mg of tissue; p < 0.001) was higher when compared with the non-lesioned group (26.77 ± 4.13 mol of MDA/mg of tissue). In the stomachs of the animals pretreated with esculin (25 mg/kg) (42.03 ± 5.28 mol of MDA/mg of tissue; p < 0.001) or N-acetylcysteine (300 mg/kg) (44.54 ± 7.39 mol of MDA/mg of tissue; p < 0.001), a decrease in lipoperoxidation was observed compared with the lesioned group, and this was a similar result to that seen in the healthy group (Fig. 6). 4. Discussion The present work describes the gastroprotective action of esculin against both indomethacin- and ethanol-induced lesions in mice and demonstrates the possible mechanisms involved. Several mechanisms are involved in the production of gastric lesions and the precise gastroprotective mechanism of esculin has not been identified. To investigate the gastroprotective effects of esculin, different pharmacological tools were used. Although the mechanism(s) of ethanol-induced gastric ulcers are not fully understood, it is well documented in the literature that the pathogenesis in animals is multifactorial, involving super-
ficial aggressive cellular necrosis and the release of tissue-derived mediators, which act on the gastric microvasculature to trigger a series of events that lead to mucosal and possibly submucosal tissue damage [19]. Ethanol-induced gastric ulceration in rats and mice is considered to be a reliable tool to study the pathogenesis of acute gastric mucosal ulceration [3]. In this study, esculin (12.5, 25 and 50 mg/kg) was shown to effectively protect the gastric mucosa against lesions induced by ethanol, but the effect was not dose-dependent and was similar to that exhibited by cyproheptadine, which is frequently used as a reference in these models [16,25]. From microscopic analysis, ethanol-induced lesions were characterized by hemorrhage, edema, inflammatory infiltrate, and loss of epithelial cells; this result is consistent with other studies [25,26]. Our findings showed that esculin was able to maintain the integrity of the gastric mucosa against the damaging effects of ethanol by a reduction in all histopathological parameters that were analyzed. In the indomethacin-induced lesion model, pre-treatment with esculin at two doses (25 and 50 mg/kg) also promoted protection against gastric damage, similar to ranitidine. These results do not necessarily mean that the protective action of the drug is directly linked to increased synthesis or decreased metabolism of PGs. Protection from this type of injury depends on several factors, and thus antisecretory drugs such as ranitidine, an H2receptor antagonist, are often used as reference drugs in this model due to a reduction of gastric lesions caused by decreasing acid secretion [27].Several pathological mechanisms are implicated in ethanol-induced gastric injury. The important role of nitric oxide (NO) as a crucial mediator of gastrointestinal mucosal defense is well described [28]. NO is involved in the prevention and healing of injuries in the gastrointestinal tract via several mechanisms, including the following: by regulating the capillary flow in the gastrointestinal tract wall, by acting as a cytoprotective and an anti-inflammatory factor, and by facilitating the protective effects of prostaglandins (PGs) in the stomach. These defense mechanisms can be stimulated and/or facilitated by PGs, which seem to inhibit acid secretion, stimulate mucus, bicarbonate and phospholipid secretion, increase mucosal blood flow, and accelerate epithelial restitution and mucosal healing [16]. The ATP-dependent K+ channels are involved with NO and PGs in gastroprotection, but the mechanisms remain unclear [29]. Another important factor in the generation of the lesion is the formation of free radicals and alteration in the activity of antioxidant enzymes that are promoted by ethanol. NO modulates several elements of gastric mucosal defense, including blood flow [30], neutrophil adhesion [31] and mucus secretion [32,33]. In the present study, the evaluation of mechanisms of action of esculin was performed using the dose of 25 mg/kg. These results showed that the gastroprotection of esculin and l-arginine against ethanol-induced gastric damage was reversed after pre-treatment with a non-selective inhibitor of NOsynthase, l-NAME; furthermore, stomachs pretreated with esculin showed higher nitrite levels compared to the stomachs of animals pretreated only with vehicle or of the non-injured group. Even the group pretreated with l-NAME showed a large lesion area. These results suggest the possible involvement of NO synthesis in the gastroprotective mechanism of esculin. Nitric oxide is a crucial mediator of gastrointestinal mucosal defense; paradoxically, it also contributes to mucosal injury in several situations. In 2008, Medeiros et al. [26] showed the beneficial effects of nitric oxide in the gastric mucosal defense. However, agents that can scavenge nitric oxide or peroxynitrite are promising drugs for the prevention of nitric oxide-associated tissue injury. Indeed, the coupling of a nitric oxide source with non-steroidal anti-inflammatory drugs is considered to be a valid means of reduc-
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ing the gastrointestinal toxicity of these drugs without decreasing their efficacy [26]. Cytoprotection in the stomach, consisting of the secretion and circulation of mucus and bicarbonate secretion to the gastric lumen, is highly dependent on prostaglandins [34]. To verify the role of prostaglandins in the gastroprotective role of esculin, mice were pretreated with indomethacin, a non-selective cyclooxygenase inhibitor. The results reveal that gastroprotection by esculin against ethanol-induced mucosal injury is reduced by indomethacin, suggesting a role for endogenous prostaglandins in gastroprotection. Recently, the involvement of KATP channels in several models of gastric protection was described [14,29]. The gastroprotection of esculin and diazoxide (3 mg/kg) against ethanol-induced gastric damage was reduced after the KATP channels were blocked with glibenclamide, showing the possible involvement of KATP channels in the gastroprotective mechanism of esculin. Capsaicin acts on the sensory neurons stimulating the receptors of membrane TRPV-1, and in small doses, it has a gastroprotective effect by stimulating gastric microcirculation. In the stomach, the afferent sensory nerves sensitive to capsaicin are involved in the local defense mechanism against the formation of gastric ulcers, and the oral administration of capsaicin exercises a protective effect on the gastric mucosa against injury caused by ethanol [35]. Based on these data, we investigated the role of the capsaicinsensitive afferent nerves in the gastroprotective effect of esculin in the model of gastric injury prompted by ethanol. For this, mice were pretreated with capsazepine, an antagonist of TRPV-1. The results reveal that the gastroprotection by esculin against ethanolinduced mucosa injury was similar when the TRPV-1 was blocked, suggesting a limited role for the capsaicin-sensitive afferent nerves in gastroprotection. On the other hand, the oxygen-derived free radicals have received increasing attention as a possible pathogenic factor in gastric mucosal injury associated with anti-inflammatory drugs and ethanol-induced ulcers. It is known that ethanol is able to deplete the levels of reduced glutathione in gastric tissue and alter the activity of antioxidant enzymes [16]. The results obtained in our experiments were consistent with those described in the literature for injured stomachs with ethanol. A decrease in the amount of GSH and an increase in both SOD activity and lipid peroxidation was observed for the group exposed to the deleterious action of ethanol [36]. With regard to the level of LPO, which was measured indirectly by the MDA amount, pre-treatment with esculin was able to reduce this rate, probably due to the elimination of toxic compounds, mainly by SOD. The decrease in the amount of GSH may be linked to its oxidation by toxic metabolites of ethanol, to a decrease in its synthesis caused by ethanol, or to consumption of GSH in the sequestration of free radicals or even with Glutathione Peroxidase activity (not investigated). Whereas esculin normalized the CAT activity, this may have occurred through a decrease in substrates for the enzyme [37]. The MPO is described as a marker of infiltration/aggregation of neutrophils and is often increased in ulcerogenic lesions [38]. It is present in neutrophils and catalyzes the breakdown of H2 O2 , generating ROS that is responsible for the antimicrobial activity of neutrophils. The data obtained for activity of this enzyme indicate a possible antioxidant mechanism promoted by esculin, as reduction was observed in MPO activity. The data concur with the antioxidant effects found previously; in addition, the data provide evidence of a possible anti-inflammatory mechanism that was not investigated and concur with the results of histopathological analyses. In conclusion, the results of this study indicate that the gastroprotective effect of esculin against gastric damage induced by ethanol and indomethacin is possibly mediated, in part, by endogenous prostaglandin synthesis, nitric oxide release, KATP channel
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opening and antioxidant effects, suggesting that esculin might have the potential for future development as a novel therapy to combat gastropathy. Esculin was shown to have good antioxidant activity, and its use may be investigated in the future to treat various diseases such as Parkinson’s and Alzheimer’s diseases. Conflict of interest statement None. Acknowledgements The authors are thankful to the CNPq and CAPES for financial support. References [1] W. Toma, J.S. Gracioso, F.D.P. Andrade, C.A. Hiruma-Lima, W. Vilegas, A.R.M.S. Brito, Antiulcerogenic activity of four extracts from the barks wood of Quassia amara L. (Simaroubaceae), Biol. Pharm. Bull. 25 (2002) 1151–1155. [2] G. Mózsik, T. Jávor, A biochemical and pharmacological approach to the genesis of ulcer disease. I. A model study of ethanol-induced injury to gastric mucosa in rats, Dig. Dis. Sci. 33 (1988) 92–105. [3] A. Robert, J.E. Nezamis, C. Lancaster, A.J. Hanchar, Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl, and thermal injury, Gastroenterology 77 (1979) 433–443. [4] Ch.V. Rao, S.K. Ojha, K. Radhakrishnan, R. Govindarajen, S. Rastogi, S. Mehrotra, P. Pushpangadan, Antiulcer activity of Utleria salicifolia rhizome extract, J. Ethnopharmacol. 91 (2004) 243–249. [5] Y. Suzuki, M. Ishihara, T. Segami, M. Ito, Anti-ulcer effects of antioxidants, quercetin, alpha-tocopherol, nifedipine and tetracycline in rats, Jpn. J. Pharmacol. 78 (1998) 435–441. [6] M. Tuorkey, K. Karolin, Anti-ulcer activity of curcumin on experimental gastric ulcer in rats and its effect on oxidative stress/antioxidant, IL-6 and enzyme activities, Biol. Environ. Sci. 22 (2009) 488–495. [7] T. Kaneko, S. Tahara, F. Takabayashi, Suppression of lipid hydroperoxideinduced oxidative damage to cellular DNA by esculetin, Biol. Pharm. Bull. 26 (2003) 840–844. [8] D.L. Zhao, L.B. Zou, S. Lin, J.G. Shi, H.B. Zhu, Anti-apoptotic effect of esculin on dopamine-induced cytotoxicity in the human neuroblastoma SH-SY5Y cell line, Neuropharmacology 53 (2007) 724–732. [9] T. Kaneko, S. Tahara, F. Takabayashi, Inhibitory effect of natural coumarin compounds, esculin and esculin, on oxidative DNA damage and formation of aberrant crypt foci and tumors induced by 1,2-dimethylhydrazine in rat colons, Biol. Pharm. Bull. 30 (2007) 2052–2057. [10] Z. Stefanova, H. Neychev, N. Ivanovska, I. Kostova, Effect of a total extract from Fraxinus ornus stem bark and esculin on zymosan- and carrageenan-induced paw oedema in mice, J. Ethnopharmacol. 46 (1995) 101–106. [11] M.J. Martin, E. Marhuenda, C. Alarcon de la Lastra, Esculin, ranitidine and carbenoxolone: different modes of action on gastric mucosa, Gen. Pharmacol. 22 (1991) 1001–1004. [12] L. Laine, W.M. Weinstein, Histology of alcoholic hemorrhagicgastritis: a prospective evaluation, Gastroenterology 94 (1988) 1254–1262. [13] G.O. Leite, A.R. Penha, C.N. Fernandes, H.H.F. Souza, J.G.M. Costa, A.R. Campos, Gastroprotective mechanism of Vanillosmopsis arborea bark essential oil, Fitoterapia 80 (2009) 77–80. [14] B.M. Peskar, K. Ehrlich, B.A. Peskar, Role of ATP-sensitive potassium channels in prostaglandin-mediated gastroprotection in the rat, J. Pharmacol. Exp. Ther. 301 (2002) 969–974. [15] M.M. Guedes, A.C.S. Carvalho, A.F. Lima, S.R.S. Lira, S.S. Queiroz, E.R. Silvera, F.A. Santos, V.S. Rao, Gastroprotective mechanisms of centipedic acid, a natural diterpenefrom egletes viscosa LESS, Biol. Pharm. Bull. 31 (2008) 1351– 1355. [16] N.F.M. Rocha, E.T. Venâncio, B.A. Moura, M.I.G. Silva, M.R.A. Neto, E.R.V. Rios, D.P. de Sousa, S.M.M. Vasconcelos, M.M.F. Fonteles, F.C.F. de Sousa, Gastroprotection of (−)-␣-bisabolol on acute gastric mucosal lesions in mice: the possible involved pharmacological mechanisms, Fund. Clin. Pharmacol. 24 (2010) 63–71. [17] L.C. Green, S.R. Tannenbaun, P. Goldman, Nitrate syntesis in Parkinsonˇıs disease using the model of the 6-hydroxydopamine and MPTP, Ann. N.Y. Acad. Sci. 899 (2000) 262–273. [18] K.P. Bhargava, M.B. Gupta, K.K. Tangri, Mechanism of ulcerogenic activity of indomethacin and oxyphenbutazone, Eur. J. Pharmacol. 22 (1973) 191–195. [19] A.S. Szabo, J.S. Trier, A. Brown, J. Schnoor, H.D. Homan, J.C. Bradford, A quantitative method for assessing the extent of experimental gastric erosions and ulcers, J. Pharmacol. Meth. 13 (1985) 59–66. [20] J. Sedlak, R.H. Lindsay, Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent, Anal. Biochem. 25 (1968) 192–205.
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Mechanisms involved in the gastroprotective activity of esculin on acute gastric lesions in mice Emiliano Ricardo Vasconcelos Rios a,∗ , Nayrton Flávio Moura Rocha a , Edith Teles Venâncio a , Brinell Arcanjo Moura a , Mariana Lima Feitosa a , Gilberto Santos Cerqueira a , Pedro Marcos Gomes Soares a , David John Woods c , Francisca Cléa Florenc¸o de Sousa a , Luzia Kalyne Almeida Moreira Leal a,b , Marta Maria de Franc¸a Fonteles a,b a
Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Rua Coronel Nunes de Melo, 1127, Brazil Pharmacy Department, Faculty of Dentistry, Nursing and Pharmacy, Federal University of Ceará, Rua Capitão Francisco Pedro, 1210, Brazil c School of Pharmacy, University of Otago, PO Box 913, Dunedin, New Zealand b
a r t i c l e
i n f o
Article history: Received 23 May 2010 Received in revised form 20 July 2010 Accepted 21 July 2010 Available online 1 August 2010 Keywords: Esculin Gastric lesion Gastroprotection Ethanol Indomethacin
a b s t r a c t This work describes the gastroprotective actions of esculin (6,7-dihydroxycoumarin-6-o-glucoside) against indomethacin- or ethanol-induced lesions and verifies the role of nitric oxide, ATP-dependent K+ channels, prostaglandins, transient receptor potential vanilloid 1 and antioxidant effects in the gastroprotective mechanism of esculin in the ethanol-induced gastric lesion model. The intragastric administration of esculin at doses of 12.5, 25 and 50 mg/kg was able to protect the gastric mucosa against ethanol (0.2 mL/animal p.o.), and esculin at doses of 25 and 50 mg/kg protected against indomethacininduced lesions (20 mg/kg p.o.). Administration of l-NAME (10 mg/kg i.p.), glibenclamide (10 mg/kg i.p.) or indomethacin (10 mg/kg p.o.), but not capsazepine (5 mg/kg p.o.), was able to reduce the gastroprotection promoted by esculin (25 mg/kg) on the ethanol-induced lesions. Measurements of nitrite, a NO metabolite, were increased in the group that was pretreated with esculin. In terms of antioxidant activity as a gastroprotective mechanism of esculin, the results show that pre-treatment with esculin decreased the amount of GSH, increased SOD activity, did not interfere with the CAT activity and decreased both the MPO activity and the MDA amount. In conclusion, pre-treatment with esculin confers significant gastroprotective and antioxidant activity and leads to a reduction in gastric injury; the mechanisms underlying these effects include stimulation of endogenous prostaglandins, nitric oxide synthesis, opening of KATP channels and reduction of free radicals or modulation of antioxidant enzyme systems. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The gastric mucous membrane is continuously exposed to potentially harmful agents, such as HCl, pepsin, bile acids, food seasonings, bacterial products and drugs. These agents are involved in the pathogenesis of gastric injury by promoting an increase in the secretion of gastric acid and pepsin, decreasing gastric blood flow, suppressing the output of endogenous prostaglandins, inhibiting the cellular proliferation and growth of the mucous membrane and altering gastric motility [1]. In physiological conditions, there is a balance between the aggressive factors (HCl, pepsin, bile and pancreatic enzymes)
∗ Corresponding author at: Department of Physiology and Pharmacology, Federal University of Ceará, Rua Cel. Nunes de Melo 1127, CEP: 60430-270, Fortaleza, Ceará, Brazil. Tel.: +55 85 3366 8337; fax: +55 85 3366 8333. E-mail address: [email protected] (E.R.V. Rios). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.07.020
and the gastroprotective factors (mucus-bicarbonate, blood flow, prostaglandins and glutathione). Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDS), smoking, alcohol, trauma, sepsis, shock, Helicobacter pylori, and stress have been shown to contribute to gastric ulcer formation [2]. Indomethacin-induced gastric lesions are characterized by significant oxidative injury, reduced mucosal blood flow and reduced secretion of mucus/bicarbonate, mainly due to inhibition of PG secretion [3]. Ethanol-induced gastric lesions occur mainly due to intense infiltration in the sub-mucosa that promotes formation of ROS, decreased mucus, depletion of sulfhydryl groups and decreased blood flow, resulting in damage to the gastric mucosa [4]. This model has fundamental importance for scientific research due to the fact that we can utilize it to evaluate possible mechanisms by which substances can act to promote gastroprotection. The generation of ROS and pro-oxidative events are involved in the etiology of gastric lesions in these two models. Several antioxidants exhibit gastroprotective activity, such as quercetin [5] and curcumin [6].
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Esculin (6,7-dihydroxycoumarin-6-o-glucoside) is a coumarinic derivative found in Aesculus hippocastanum L. (Horse-chestnut). Their seeds have long been used to treat inflammatory and vascular problems. In Brazilian folk medicine, the tea prepared from the crushed seeds is used to protect against kidney stones and stomach pain. Esculin is known to be a 5- and 12-lipoxygenase inhibitor and to inhibit the production of leukotrienes and 5hydroxyeicosatetraenoic acid through the lipoxygenase pathway [7]. In 2007, Zhao et al. used the dopamine-induced cytotoxicity model in human neuroblastoma SH-SY5Y cells to demonstrate that esculin inhibited dopamine-induced caspase-3 cleavage and decreased cell death, overproduction of ROS, morphological changes of nuclei and damage to antioxidant enzymes [8]. Esculin scavenges hydroxyl radicals and inhibits lipid peroxidation in the rat liver [9] and displays anti-inflammatory activity in both zymosan- and carrageenan-induced paw edema in mice [10]. The gastroprotective effect of esculin was also observed in rats by Martin et al. [11] in cold-restraint stress and pylorus ligation-induced ulcer models; however, neither its effect on indomethacin-induced lesions nor the gastroprotective mechanisms involved were investigated in this study. Thus, this study was developed to better characterize the potential gastroprotective actions of esculin in mice and to investigate the possible mechanisms involved.
opened along the greater curvature for examination. The total and injured stomach areas (glandular face) were measured by the Image J computer program and expressed in terms of the percent (%) of lesioned gastric area.
2. Materials and methods
To study the possible mechanisms of action, the experiments were conducted using esculin (25 mg/kg; intermediate dose between the effective doses tested) and the following drugs: l-NAME, an inhibitor of NO-synthase activity (10 mg/kg, i.p.); glibenclamide, a blocker of KATP channels (10 mg/kg, i.p.); indomethacin (10 mg/kg, p.o.); and capsazepine (CZP), a TRPV-1 antagonist (5 mg/kg, i.p.). l-NAME or glibenclamide were administered 15 min before administration of esculin (25 mg/kg, p.o.), l-arginine (600 mg/kg, p.o.) or diazoxide (3 mg/kg, p.o.). Indomethacin (10 mg/kg, p.o.) was administrated 2 h before administration of esculin (25 mg/kg, p.o.) or misoprostol (50 g/kg, p.o.). Capsazepine (5 mg/kg, i.p.) was given 30 min before administration of esculin (25 mg/kg, p.o.) or capsaicin (CPS) (0.3 mg/kg, p.o.). After 1 h of oral administration was induced gastric injury with ethanol. All animals received absolute ethanol (0.2 mL) for the lesion induction. Thirty minutes after the administration of ethanol, the mice were killed and the stomach was removed for examination as previously described. The dose selections for these drugs were based on our pilot experiments and on literature findings [13–16].
2.1. Animals Male Swiss mice (25–35 g) were used in this study. Animals were kept in a temperature-controlled room at 25 ± 2 ◦ C with a 12-h light/dark cycle, with food and water ad libitum. The study was approved by the Ethics Committee for Animal Research at the Federal University of Ceará in Brazil, and it was conducted in accordance with the National Institute of Health in Bethesda, USA. 2.2. Drugs Capsazepine, capsaicin, cyproheptadine, diazoxide, esculin, glibenclamide, indomethacin, l-arginine, NG -nitro-l-arginine methyl ester (l-NAME), sodium nitrite and Tween 80 were purchased from Sigma–Aldrich® (St. Louis, MO, USA), N-acetyll-cysteine (NAC) from União Química® (São Paulo, SP, Brazil), Ranitidine from GlaxoSmithKline® (Rio de Janeiro, RJ, Brazil) and Misoprostol from Pfizer® (São Paulo, SP, Brazil). 2.3. Study of the gastroprotective activity of esculin in the ethanol-induced gastric lesion model The acute gastric lesions were induced by intragastric administration of absolute ethanol in accordance with a previously described method [3]. Male Swiss mice were randomly divided into five groups and fasted for 15 h before the experiment, but mice had free access to water. The ethanol groups were orally administered (0.2 mL/animal) 60 min after the treatment with esculin (12.5, 25 and 50 mg/kg, p.o – ESC12.5, ESC25 and ESC50, respectively). These doses were chosen based on results from previous studies by the same group with coumarin and umbelliferone (unpublished data) and from the study by Martin et al. [11]. The esculin was dissolved in 3% Tween 80 in distilled water. Control mice were similarly treated with Tween. Cyproheptadine, a non-selective antagonist of 5-HT and histamine receptors (10 mg/kg; p.o. – CYP10), was used as a reference drug. All experimental groups consisted of eight mice. These treatments were performed by gavage with a metal orogastric tube. Thirty minutes after the administration of ethanol, the mice were killed by cervical dislocation, and the stomach was removed and
2.4. Histopathological assessment Histological evaluation was performed on the glandular face of the stomach. Tissue samples were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Sections about 4 mm thick were cut and stained with hematoxylin and eosin. The mucosal injury evaluation was performed under light microscopy by an experienced histologist blinded to the treatment regimen. The histopathological changes were assessed according to the following criteria that were previously described by Laine and Weinstein [12]: (1) edema (score 0–4), (2) hemorrhagic damage (score 0–4), (3) inflammatory infiltration (score 0–3), and (4) epithelial cell loss (score 0–3). 2.5. Evaluation of the role of nitric oxide (NO), ATP-dependent K+ channels (KATP ), prostaglandins and transient receptor potential vanilloid 1 (TRPV1) in the gastroprotective effect of esculin on the ethanol-induced gastric lesion model
2.6. Measurement of total nitrite levels The amount of stable nitrite, the end product of NO metabolism, in the gastric mucosa was determined by a colorimetric assay as described by Green et al. [17]. Briefly, 100 L of gastric mucosa homogenate was mixed with an equal volume of Griess reagent that consists of equal parts of 1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride (NEED), 5% H3 PO4 and distilled water and incubated at room temperature for 10 min. The absorbance was read at 540 nm on a microplate reader (UVM-340, Asys Hitech, Netherlands). The amount of nitrite was calculated from a NaNO2 standard curve. 2.7. Study of the gastroprotective action of esculin in the indomethacin-induced gastric lesion model Male Swiss mice were randomly divided into four groups and treated orally with vehicle (controls), esculin (25 and 50 mg/kg p.o.) or ranitidine, a H2 histamine receptor antagonist (20 mg/kg; p.o.). After 60 min, gastric lesions were induced in all groups by
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Scores
Discoloration of mucosa Edema Hemorrhages Number of petechia Until 10 More than 10 Intensity of ulceration Ulcers or erosion up to 1 mm Ulcers or erosion larger than 1 mm Perforated ulcers
1 1 1 2 3 N×2 N×3 N×4
N number of stomach lesions.
indomethacin (20 mg/kg, p.o.) suspended in 0.5% carboxymethylcellulose in distilled water. After 7 h, as per Bhargava’s method [18], the animals were sacrificed by cervical dislocation, and the stomachs were removed and immersed in 5% formalin for 15 min and then opened by the great curvature and washed with saline solution for examination of the lesions. The degree of lesion formation was graded according to an arbitrary scale by attribution of scores as presented in Table 1 [19].
assayed. In a dark chamber, 1 mL of the reactant (50 mM phosphate buffer, 100 nM EDTA and 13 mM l-methionine, pH 7.8) was mixed with 30 L of the sample, 150 L of 75 M NBT and 300 L of 2 M riboflavin. The tubes containing the resulting solution were exposed to fluorescent light bulbs (15 W) for 15 min and then read using a spectrophotometer at 560 nm.
2.10. Measurement of catalase (CAT) activity Catalase activity is proportional to the rate of output of O2 and H2 O. Hydrogen peroxide (H2 O2 ) is used as the substrate and is hydrolyzed according to the Maehly and Chance method [23]. The activity of the enzyme is measured at 230 nm using spectrophotometry (Beckman DU) by reading the variation of the absorbance between the first and sixth minutes and then expressing the results in M/min/g of protein. The reaction environment was prepared with H2 O2 (18 mL), 1 M Tris HCl buffer 5 mM EDTA pH 8 (1 mL) and Milli-Q H2 O (0.8 mL). Immediately, 980 L of the reaction environment was added to 20 L of the homogenate (10%) in a quartz cuvette.
2.11. Measurement of the myeloperoxidase (MPO) activity 2.8. Evaluation of the antioxidant effect of esculin in gastroprotection and measurement of the amount of reduced glutathione To assess the changes in the gastric mucosal amount of GSH (a non-protein sulfhydryl compound), its content was measured according to the method described by Sedlak and Lindsay [20] with slight modifications. Briefly, esculin (25 mg/kg, p.o.), N-acetylcysteine (300 mg/kg, p.o.) or vehicle (control) was intragastrically administered to mice 1 h before administration of saline (healthy) or absolute ethanol (0.2 mL/animal) (ulcerated). Thirty minutes after treatment, the animals were sacrificed by cervical dislocation and their stomachs were removed. For the GSH assay, the glandular segment from each stomach was homogenized in ice-cold 0.02 M EDTA solution (at 10%). Aliquots (400 L) of tissue homogenate were mixed with 320 L of distilled water and 80 L of 50% (w/v) trichloroacetic acid in glass tubes and centrifuged at 3000 rpm for 15 min. Subsequently, supernatants (400 L) were mixed with 800 L Tris buffer (0.4 M, pH 8.9) and 5,5-dithio-bis(2nitrobenzoic acid) (DTNB; 0.01 M) was added. After shaking the reaction mixture for 3 min, its absorbance was measured at 412 nm within 5 min of the addition of DTNB against a blank with no homogenate. The absorbance values were extrapolated from a glutathione standard curve and the GSH content was expressed in g GSH/g of protein. The concentration of proteins was measured using the method described by Bradford [21]. 2.9. Measurement of the superoxide dismutase (SOD) activity SOD activity was measured according to Sun et al. [22]. The activity of the enzyme was evaluated by measuring its capacity to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT). In this assay, the photochemical reduction of riboflavin generates O2− that reduces the NBT to produce formazan salt, which absorbs at a wavelength of 560 nm. In the presence of SOD, the reduction of the NBT is inhibited because the enzyme converts the superoxide radical to peroxide. The results are expressed as the quantity of SOD necessary to inhibit the rate of reduction of the NBT by 50% in units of the enzyme per gram of protein. Homogenates (10% of tissue in buffer phosphate) were centrifuged (10 min, 3600 rpm, 4 ◦ C), and the supernatant was removed and centrifuged a second time (20 min, 12,000 rpm, 4 ◦ C). The resulting supernatant was
The animals were divided into groups of eight and were sacrificed 30 min after administration of the ethanol. Stomach samples were removed and immediately frozen in liquid nitrogen. Samples were then homogenized in a solution of 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer pH 6.0 (1 mL per 50 mg of the tissue) and centrifuged at 4000 rpm for 15 min at 4 ◦ C. The amount of the enzyme in the supernatant (30 L) was analyzed by spectrophotometry after addition of 200 L of phosphate buffer (50 mM, pH 6), containing 0.167 mg/ml of dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The absorbance at 470 nm was measured at time 0 and 5 min [24].
2.12. Measurement of the membrane lipids peroxidation The rate of lipoperoxidation in the gastric mucous membrane was estimated by determination of malondialdehyde (MDA) using the Thiobarbituric Acid Reactive Substances (TBARS) test. The stomachs were washed with saline to minimize the interference of hemoglobin with free radicals and to remove blood adhered to the mucous membrane. The stomachs were homogenized to 10% of tissue with potassium phosphate buffer. Then, 250 L was removed and stored at 37 ◦ C for 1 h, after which 400 L of 35% perchloric acid was added, and the mixture was centrifuged at 14,000 rpm for 20 min at 4 ◦ C. The supernatant was removed, mixed with 400 L of 0.6% thiobarbituric acid and incubated at 95–100 ◦ C for 1 h. After cooling, the absorbance at 532 nm was measured. A standard curve was generated using 1,1,3,3-tetrametoxypropane. The results were expressed as nmol of MDA/mg of protein. The concentration of proteins was measured using the method described by Bradford [21].Measurement of total protein in the stomach sample after ethanol-induced lesions The method is based on the interaction of the Coomassie Blue G250 dye with proteins. At the pH of the reaction, the interaction between proteins of high molecular weight and the dye causes a shift in the dye to the anionic form, which absorbs strongly at 595 nm. Solutions of albumin standard, distilled water, buffer and samples were added to the wells. For sample preparation, 2 L of sample and 38 L of buffer were added to each well. Then, 200 L Bradford’s solution (diluted 5×) was added to each well. After 5 min, a reading was taken at the wavelength of 595 nm [21].
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Fig. 1. Effects of esculin on the gastroprotection in mice (n = 8/group) subjected to ethanol and indomethacin-induced gastric lesion. The results are presented as mean ± SEM. * p < 0.05 and *** p < 0.001, significant difference compared with Vehicle group. ANOVA followed by Newman–Keuls as the post hoc test and Kruskal–Wallis test followed by Dunnˇıs test as the post hoc test were used for the ethanol and indomethacin-induced model, respectively.
2.14. Statistical analysis Values were expressed as mean ± SEM. For statistical analysis, one-way analysis of variance (ANOVA) was used, followed by the Student Newman–Keuls post hoc test. For histopathological assessment and indomethacin-induced lesions, the Kruskal–Wallis test was used, followed by Dunn’s test. Probability (p) values less than 0.05 were considered significant.
3. Results 3.1. Study of the gastroprotective effect of esculin in the ethanol-induced ulcer model The administration of absolute ethanol produced lesions in the gastric mucosa that were reduced in the animals pretreated with 12.5 mg/kg (4.381 ± 1.129%; p < 0.001), 25 mg/kg (5.163 ± 1.043%; p < 0.001), or 50 mg/kg esculin (3.938 ± 0.7326%; p < 0.001), or with 10 mg/kg cyproheptadine (4.108 ± 0.611%; p < 0.001), when compared to the control group (18.31 ± 1.647%) (Fig. 1).
3.2. Study of gastroprotective effect of esculin in indomethacin-induced ulcer As demonstrated in Fig. 1, the administration of indomethacin produced lesions in the gastric mucosa (9.500 ± 0.422 scores), which were reduced in the animals pretreated with ESC 25 (3.571 ± 0.481 scores; p < 0.001), ESC 50 (4.857 ± 0.508 scores; p < 0.05), or 20 mg/kg ranitidine (5.125 ± 0.441 scores; p < 0.05), when compared to the control animals that were pretreated only with the vehicle.
3.3. Histopathological assessment after ethanol-induced lesions Histopathological analyses of the gastric mucosa are shown in Fig. 2 and Table 1. Animals pretreated with esculin (25 mg/kg, p.o.) (Fig. 2B) showed less macro- and microscopic mucosal damage when compared with the control group treated only with ethanol (Fig. 2A), in which severe lesions were characterized by hemorrhagic injury, edema and loss of epithelial cells. Pre-treatment with NAC also inhibited lesions promoted by ethanol (Fig. 2C), and the animals in this treatment group showed similar characteristics to the group without lesions (Fig. 2D). Esculin significantly decreased the ethanol-induced lesions to a similar extent as NAC (Table 2).
3.4. Evaluation of the role of the nitric oxide (NO) metabolic pathway in the gastroprotective effects of esculin in the ethanol-induced ulcer model The ethanol pre-treatment produced significant gastric lesions (17.590 ± 1.570%) that were reduced by ESC 25 (5.163 ± 1.043%; p < 0.001). Similarly, l-arginine (3.489 ± 0.922%; p < 0.001) was able to protect the mucosa from injury induced by ethanol. Previous administration of l-NAME (10 mg/kg) reduced the gastroprotection of ESC 25 (34.68 ± 3.899%; p < 0.001) compared to ESC 25 alone. A similar effect was caused by l-NAME plus l-ARG (19.00 ± 2.184%; p < 0.001) when compared to the l-ARG group (Fig. 3). 3.5. Evaluation of the role of KATP on the gastroprotective effects of esculin in the ethanol-induced gastric lesion model in mice The effects of oral administration of esculin on ethanol-induced lesions in glibenclamide-pretreated mice are demonstrated in Fig. 3. Pre-treatment with this KATP blocker significantly decreased the gastroprotective effect of esculin (26.57 ± 3.175%; p < 0.001) compared to the ESC 25 group (5.163 ± 1.043%) and the control group (17.59 ± 1.570%). The same result was shown with glibenclamide plus diazoxide (18.42 ± 3.329%, p < 0.001) compared to diazoxide alone (4.291 ± 1.088%). 3.6. Evaluation of the involvement of prostaglandins in the gastroprotective effects of esculin in the ethanol-induced lesion model The data in Fig. 3 show that the ethanol-induced lesions were significantly reduced by pre-treatment with ESC 25 (5.615 ± 1.039%; p < 0.001) or misoprostol (10.61 ± 1.06%; p < 0.01) compared to control (19.73 ± 1.698%). This effect was reduced by previous indomethacin administration (15.01 ± 0.7557%; p < 0.001) compared to the ESC 25 group. A similar result was shown for indomethacin plus misoprostol (22.51 ± 2.328%; p < 0.001) compared to misoprostol alone (Fig. 3). 3.7. Evaluation of the involvement of TRPV-1 (Transient Receptor Potential Vanilloid-1) in the gastroprotective effects of esculin in the ethanol-induced lesion model Fig. 3 also shows the effect of capsazepine on gastroprotection promoted by esculin. The ethanol-induced lesions were reduced by pre-treatment with 25 mg/kg esculin (4.095 ± 0.937%; p < 0.001) or capsaicin (5.230 ± 0.814%; p < 0.01) when compared to control (15.09 ± 1.245%). No change in the effect of esculin was observed in the group that was pre-treated with capsazepine (4.963 ± 1.042%).
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Fig. 2. Macro- and microscopic effects on the gastric mucosa of mice subjected to gastric lesions induced by ethanol pretreated or not with esculin. Photomicrographs of stomachs opened along the greater curvature (A–D) and microscopic analysis with magnification of 100× and 400× for each group (A–D 100× and 400×). (A) Animals treated with vehicle (saline 0.9–3% Tween 80, po), showing normal morphology, (B) treatment with ethanol (0.2 mL/animal), presenting inflammatory infiltration, edema, moderate hemorrhage and severe loss of epithelial cells, (C) treatment only with esculin (25 mg/kg, p.o.), showing histological aspects similar to group D, (D) pre-treatment with NAC (300 mg/kg, p.o.) before administration of ethanol, showing preservation of the gastric mucosa.
The capsazepine was able to antagonize the gastroprotection promoted by capsaicin (12.00 ± 3.135%; p < 0.01) compared to capsaicin alone. 3.8. Nitrite content in the injured stomach after ethanol-induced lesions To increase understanding of the role of nitric oxide in the gastroprotective action of esculin, the concentration of nitrite in the tissue was measured as an indirect way of assessing the amount of tissue NO. In corroboration with previous results, the group pretreated with esculin (25 mg/kg, p.o.) showed a signifi-
cantly higher concentration of nitrite (24.47 ± 4.15 mM; p < 0.001) than the group pretreated with vehicle (3% Tween 80 in distilled water, p.o; 7.50 ± 0.32 mM). No statistical difference was observed between the vehicle-treated group (11.79 ± 0.98 mM) and the ulcerated group. As standards, two groups were pre-treated with Nacetylcysteine (NAC; 300 mg/kg) or l-arginine (l-ARG; 600 mg/kg). The group pre-treated with NAC did not show a statistically significant difference (8.16 ± 0.40 mM) compared to the lesioned group, whereas the group treated with l-ARG (19.61 ± 2.55 mM; p < 0.001) showed a higher nitrite concentration compared to the injured control and was similar to the group pre-treated with esculin (Fig. 4).
Table 2 Scores of histopathological changes in the gastric mucosa of mice subjected to gastric lesions induced by ethanol pretreated or not with esculin. Animals were treated orally with vehicle (saline 0.9–3% Tween 80), esculin (ESC 25 mg/kg), or N-acetylcysteine (NAC 300 mg/kg), 60 min before administration of ethanol (0.2 mL, p.o.). Histopathological changes of glandular portion of stomach were assessed as described by Laine and Weinstein [12]: 1 – edema (score 0–4), 2 – hemorrhagic damage (score 0–4), 3 – Inflammatory infiltration (0–3), and 4 – loss epithelial cells (score 0–3). The results are presented as median with minimum and maximum values shown in parentheses (n = 4). * p < 0.05, ** p < 0.01 vs. vehicle + ethanol. We used the nonparametric Kruskal–Wallis followed by Dunn’s test as post hoc. Treatment
Vehicle Vehicle + ethanol ESC (25 mg/kg) + ethanol NAC (300 mg/kg) + ethanol
Microscopic scores Edema (score 0–4)
Hemorrhagic damage (score 0–4)
Inflammatory infiltration (score 0–3)
Loss epithelial cell (score 0–3)
0 3 (2–4) 0* 0,5 (0–2)
0 3,5 (2–4) 0** 0**
0 1 0** 0**
0 3 (2–3) 0 (0–1)* 0 (0–1)*
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Fig. 3. Effects of pre-treatment with l-NAME, glibenclamide, indomethacin or capsazepine on the gastroprotective effect of esculin in mice (n = 8/group). The results are presented as mean ± SEM. ** p < 0.01, *** p < 0.001, significant difference compared with saline group, ## p < 0.01, ### p < 0.001, significant difference compared with ESC 25 group and ++ p < 0.01, +++ p < 0.001, significant difference compared with l-ARG, DIAZ or MISO group. ANOVA followed by Newman–Keuls as the post hoc test was used.
3.9. Evaluation of the involvement of the antioxidant effect in the gastroprotection promoted by esculin 3.9.1. Evaluation of the involvement of glutathione (GSH) metabolism in the gastroprotective effects of esculin Fig. 5 shows the influence of pre-treatment with esculin or NAC on the gastric content of GSH in ethanol-induced ulcers in healthy animals. Animals treated with NAC (300 mg/kg) before ethanol administration showed a significant increase in the GSH levels (649.4 ± 16.170 g/g of protein; p < 0.001) when compared with the saline plus ethanol group (285.4 ± 22.130 g/g of protein). However, in the group treated with esculin (263.6 ± 14.510 g/g of protein), the GSH content showed no change when compared with the saline plus ethanol group. Healthy animals (399.5 ± 55.29 g/g of protein; p < 0.01) showed a higher GSH content than the control.
Fig. 4. Effect of esculin 25 mg/kg on nitrite content of gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to stressor agent. The results are presented as mean ± SEM. *** p < 0.001, significant difference compared with vehicle + absolute ethanol group. ANOVA followed by Newman–Keuls as the post hoc test was used.
3.9.2. Measurement of the superoxide dismutase (SOD) activity Ethanol reduced the SOD activity in ulcerated stomachs pretreated with vehicle (332.90 ± 42.86 U/g of protein; p < 0.05) when compared with controls without lesions (534.30 ± 30.69 U/g of protein). Pre-treatment with esculin (25 mg/kg) increased the activity of the enzyme (642.90 ± 57.39 U/g of protein; p < 0.01 and p < 0.001) compared with the lesioned group and the group without lesions. Similarly, N-acetylcysteine increased SOD activity (748.60 ± 76.86 U/g of protein; p < 0.001) compared with lesioned and non-lesioned controls (Fig. 5). 3.9.3. Measurement of the catalase (CAT) activity Ethanol-induced gastric lesions increased the CAT activity in the animals pre-treated only with the vehicle (3% Tween 80 in distilled water) (129.90 ± 8.53 g/min/g of protein; p < 0.01) when compared with non-ulcerated healthy controls that received vehicle alone (93.08 ± 7.58 g/min/g of protein). Pre-treatment
Fig. 5. Effect of esculin 25 mg/kg on enzymes activity (SOD and CAT) and nonprotein sulfhydryl (GSH) content of gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to stressor agent. The results are presented as mean ± SEM. ** p < 0.01 and * p < 0.05, significant difference compared with vehicle (non-lesioned) group and ### p < 0.001 significant difference with vehicle + absolute ethanol group. ANOVA followed by Newman–Keuls as the post hoc test was used.
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Fig. 6. Effect of esculin 25 mg/kg on lipoperoxidation level (MDA account) and MPO activity in the gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to stressor agent. The results are presented as mean ± SEM. *** p < 0.001, significant difference compared with vehicle (non-lesioned) group and ### p < 0.001 significant difference with vehicle + absolute ethanol group. ANOVA followed by Newman–Keuls as the post hoc test was used.
with esculin (25 mg/kg) did not alter the activity of the enzyme (79.19 ± 5.06 g/min/g of protein) compared with the healthy group, but CAT activity diminished significantly (p < 0.001) when compared with the ulcerated group. Similarly, the group treated with N-acetylcysteine showed diminished CAT activity (81.97 ± 7.00 g/min/g of protein; p < 0.001) when compared with the ulcerated controls (Fig. 5). 3.9.4. Measurement of the myeloperoxidase (MPO) activity MPO activity in ulcerated stomachs pre-treated only with the vehicle (3% Tween 80 in distilled water) (20.29 ± 2.318 U of MPO/min/mg of tissue, p < 0.001) was higher when compared with the healthy group without lesions (7.39 ± 0.98 U of MPO/min/mg of tissue). In the stomachs of the animals pre-treated with esculin (25 mg/kg), the activity of the enzyme was decreased (12.08 ± 0.89 U of MPO/min/mg of tissue, p < 0.001) compared with the ulcerated group. Similarly, treatment with N-acetylcysteine (300 mg/kg) decreased activity (10.16 ± 1.22 U of MPO/min/mg of tissue; p < 0.001) compared with ulcerated controls (Fig. 6). 3.9.5. Measurement of membrane lipid peroxidation Lipoperoxidation in the lesioned stomachs pretreated only with vehicle (3% Tween 80 in distilled water) (86.15 ± 10.76 mol of MDA/mg of tissue; p < 0.001) was higher when compared with the non-lesioned group (26.77 ± 4.13 mol of MDA/mg of tissue). In the stomachs of the animals pretreated with esculin (25 mg/kg) (42.03 ± 5.28 mol of MDA/mg of tissue; p < 0.001) or N-acetylcysteine (300 mg/kg) (44.54 ± 7.39 mol of MDA/mg of tissue; p < 0.001), a decrease in lipoperoxidation was observed compared with the lesioned group, and this was a similar result to that seen in the healthy group (Fig. 6). 4. Discussion The present work describes the gastroprotective action of esculin against both indomethacin- and ethanol-induced lesions in mice and demonstrates the possible mechanisms involved. Several mechanisms are involved in the production of gastric lesions and the precise gastroprotective mechanism of esculin has not been identified. To investigate the gastroprotective effects of esculin, different pharmacological tools were used. Although the mechanism(s) of ethanol-induced gastric ulcers are not fully understood, it is well documented in the literature that the pathogenesis in animals is multifactorial, involving super-
ficial aggressive cellular necrosis and the release of tissue-derived mediators, which act on the gastric microvasculature to trigger a series of events that lead to mucosal and possibly submucosal tissue damage [19]. Ethanol-induced gastric ulceration in rats and mice is considered to be a reliable tool to study the pathogenesis of acute gastric mucosal ulceration [3]. In this study, esculin (12.5, 25 and 50 mg/kg) was shown to effectively protect the gastric mucosa against lesions induced by ethanol, but the effect was not dose-dependent and was similar to that exhibited by cyproheptadine, which is frequently used as a reference in these models [16,25]. From microscopic analysis, ethanol-induced lesions were characterized by hemorrhage, edema, inflammatory infiltrate, and loss of epithelial cells; this result is consistent with other studies [25,26]. Our findings showed that esculin was able to maintain the integrity of the gastric mucosa against the damaging effects of ethanol by a reduction in all histopathological parameters that were analyzed. In the indomethacin-induced lesion model, pre-treatment with esculin at two doses (25 and 50 mg/kg) also promoted protection against gastric damage, similar to ranitidine. These results do not necessarily mean that the protective action of the drug is directly linked to increased synthesis or decreased metabolism of PGs. Protection from this type of injury depends on several factors, and thus antisecretory drugs such as ranitidine, an H2receptor antagonist, are often used as reference drugs in this model due to a reduction of gastric lesions caused by decreasing acid secretion [27].Several pathological mechanisms are implicated in ethanol-induced gastric injury. The important role of nitric oxide (NO) as a crucial mediator of gastrointestinal mucosal defense is well described [28]. NO is involved in the prevention and healing of injuries in the gastrointestinal tract via several mechanisms, including the following: by regulating the capillary flow in the gastrointestinal tract wall, by acting as a cytoprotective and an anti-inflammatory factor, and by facilitating the protective effects of prostaglandins (PGs) in the stomach. These defense mechanisms can be stimulated and/or facilitated by PGs, which seem to inhibit acid secretion, stimulate mucus, bicarbonate and phospholipid secretion, increase mucosal blood flow, and accelerate epithelial restitution and mucosal healing [16]. The ATP-dependent K+ channels are involved with NO and PGs in gastroprotection, but the mechanisms remain unclear [29]. Another important factor in the generation of the lesion is the formation of free radicals and alteration in the activity of antioxidant enzymes that are promoted by ethanol. NO modulates several elements of gastric mucosal defense, including blood flow [30], neutrophil adhesion [31] and mucus secretion [32,33]. In the present study, the evaluation of mechanisms of action of esculin was performed using the dose of 25 mg/kg. These results showed that the gastroprotection of esculin and l-arginine against ethanol-induced gastric damage was reversed after pre-treatment with a non-selective inhibitor of NOsynthase, l-NAME; furthermore, stomachs pretreated with esculin showed higher nitrite levels compared to the stomachs of animals pretreated only with vehicle or of the non-injured group. Even the group pretreated with l-NAME showed a large lesion area. These results suggest the possible involvement of NO synthesis in the gastroprotective mechanism of esculin. Nitric oxide is a crucial mediator of gastrointestinal mucosal defense; paradoxically, it also contributes to mucosal injury in several situations. In 2008, Medeiros et al. [26] showed the beneficial effects of nitric oxide in the gastric mucosal defense. However, agents that can scavenge nitric oxide or peroxynitrite are promising drugs for the prevention of nitric oxide-associated tissue injury. Indeed, the coupling of a nitric oxide source with non-steroidal anti-inflammatory drugs is considered to be a valid means of reduc-
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ing the gastrointestinal toxicity of these drugs without decreasing their efficacy [26]. Cytoprotection in the stomach, consisting of the secretion and circulation of mucus and bicarbonate secretion to the gastric lumen, is highly dependent on prostaglandins [34]. To verify the role of prostaglandins in the gastroprotective role of esculin, mice were pretreated with indomethacin, a non-selective cyclooxygenase inhibitor. The results reveal that gastroprotection by esculin against ethanol-induced mucosal injury is reduced by indomethacin, suggesting a role for endogenous prostaglandins in gastroprotection. Recently, the involvement of KATP channels in several models of gastric protection was described [14,29]. The gastroprotection of esculin and diazoxide (3 mg/kg) against ethanol-induced gastric damage was reduced after the KATP channels were blocked with glibenclamide, showing the possible involvement of KATP channels in the gastroprotective mechanism of esculin. Capsaicin acts on the sensory neurons stimulating the receptors of membrane TRPV-1, and in small doses, it has a gastroprotective effect by stimulating gastric microcirculation. In the stomach, the afferent sensory nerves sensitive to capsaicin are involved in the local defense mechanism against the formation of gastric ulcers, and the oral administration of capsaicin exercises a protective effect on the gastric mucosa against injury caused by ethanol [35]. Based on these data, we investigated the role of the capsaicinsensitive afferent nerves in the gastroprotective effect of esculin in the model of gastric injury prompted by ethanol. For this, mice were pretreated with capsazepine, an antagonist of TRPV-1. The results reveal that the gastroprotection by esculin against ethanolinduced mucosa injury was similar when the TRPV-1 was blocked, suggesting a limited role for the capsaicin-sensitive afferent nerves in gastroprotection. On the other hand, the oxygen-derived free radicals have received increasing attention as a possible pathogenic factor in gastric mucosal injury associated with anti-inflammatory drugs and ethanol-induced ulcers. It is known that ethanol is able to deplete the levels of reduced glutathione in gastric tissue and alter the activity of antioxidant enzymes [16]. The results obtained in our experiments were consistent with those described in the literature for injured stomachs with ethanol. A decrease in the amount of GSH and an increase in both SOD activity and lipid peroxidation was observed for the group exposed to the deleterious action of ethanol [36]. With regard to the level of LPO, which was measured indirectly by the MDA amount, pre-treatment with esculin was able to reduce this rate, probably due to the elimination of toxic compounds, mainly by SOD. The decrease in the amount of GSH may be linked to its oxidation by toxic metabolites of ethanol, to a decrease in its synthesis caused by ethanol, or to consumption of GSH in the sequestration of free radicals or even with Glutathione Peroxidase activity (not investigated). Whereas esculin normalized the CAT activity, this may have occurred through a decrease in substrates for the enzyme [37]. The MPO is described as a marker of infiltration/aggregation of neutrophils and is often increased in ulcerogenic lesions [38]. It is present in neutrophils and catalyzes the breakdown of H2 O2 , generating ROS that is responsible for the antimicrobial activity of neutrophils. The data obtained for activity of this enzyme indicate a possible antioxidant mechanism promoted by esculin, as reduction was observed in MPO activity. The data concur with the antioxidant effects found previously; in addition, the data provide evidence of a possible anti-inflammatory mechanism that was not investigated and concur with the results of histopathological analyses. In conclusion, the results of this study indicate that the gastroprotective effect of esculin against gastric damage induced by ethanol and indomethacin is possibly mediated, in part, by endogenous prostaglandin synthesis, nitric oxide release, KATP channel
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