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The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

Abstract

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg,ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

Alendronate; Lawesson's reagent; H2S donors; Gastric damage


Introduction

The discovery and development of bisphosphonates have been of great clinical importance for the prevention and treatment of bone diseases. Among the various bisphosphonates used clinically, those with primary amino side chains, such as alendronate (ALD) and pamidronate, may also have increased potential for causing gastric damage (11. Adami S, Mian M, Gatti P, Rossini M, Zamberlan N, Bertoldo F, et al. Effects of two oral doses of alendronate in the treatment of Paget's disease of bone. Bone 1994; 15: 415-417, doi: 10.1016/8756-3282(94)90818-4.
https://doi.org/10.1016/8756-3282(94)908...

2. Francis RM. Oral bisphosphonates in the treatment of osteoporosis: a review. Curr Ther Res 1995; 56: 831-851, doi: 10.1016/0011-393X(95)85087-2.
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3. Abdelmalek MF, Douglas DD. Alendronate-induced ulcerative esophagitis. Am J Gastroenterol 1996; 91: 1282-1283.
- 44. Blank MA, Ems BL, Gibson GW, Myers WR, Berman SK, Phipps RJ, et al. Nonclinical model for assessing gastric effects of bisphosphonates. Dig Dis Sci 1997; 42: 281-288, doi: 10.1023/A:1018849415297.
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).

The most common adverse effects related to ALD use are acute upper gastrointestinal bleeding, abdominal pain, and discomfort (55. Graham DY, Malaty HM. Alendronate gastric ulcers. Aliment Pharmacol Ther 1999; 13: 515-519, doi: 10.1046/j.1365-2036.1999.00488.x.
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). ALD use is also associated with irritant effects on the esophagus, stomach, and duodenum (66. Lanza F, Schwartz H, Sahba B, Malaty HM, Musliner T, Reyes R, et al. An endoscopic comparison of the effects of alendronate and risedronate on upper gastrointestinal mucosae. Am J Gastroenterol 2000; 95: 3112-3117, doi: 10.1111/j.1572-0241.2000.03258.x.
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, 77. Graham DY. Excess gastric ulcers are associated with alendronate therapy. Am J Gastroenterol 1998; 93: 1395-1396, doi: 10.1111/j.1572-0241.1998.01395.x.
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). However, the mechanism underlying ALD-mediated toxicity is unknown.

Hydrogen sulfide (H2S) is a well-known toxic gas. Toxicity of H2S is observed at concentrations well above those produced endogenously and is usually associated with the presence of high concentrations in the lung and blood (88. Milby TH, Baselt RC. Hydrogen sulfide poisoning: clarification of some controversial issues. Am J Ind Med 1999; 35: 192-195, doi: 10.1002/(SICI)1097-0274(199902)35:2<192::AID-AJIM11>3.0.CO;2-C.
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, 99. Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator. J Neurosci 1996; 16: 1066-1071.). Given the potential toxicity of this gas, efficient systems exist to metabolize and scavenge H2S in vivo. H2S is metabolized by oxidation in mitochondria or by methylation in cytosol (1010. Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 2002; 16: 1792-1798, doi: 10.1096/fj.02-0211hyp.
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).

H2S is synthesized endogenously from l-cysteine by two enzymes: cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) (1010. Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 2002; 16: 1792-1798, doi: 10.1096/fj.02-0211hyp.
https://doi.org/10.1096/fj.02-0211hyp...
). CSE is the main enzymatic source of H2S in the vasculature and heart, and CBS predominates in the central nervous system (1010. Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 2002; 16: 1792-1798, doi: 10.1096/fj.02-0211hyp.
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, 1111. Boehning D, Snyder SH. Novel neural modulators. Annu Rev Neurosci 2003; 26: 105-131, doi: 10.1146/annurev.neuro.26.041002.131047.
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). Both enzymes are expressed in the gastric mucosa (1212. Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, et al. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology 2005; 129: 1210-1224, doi: 10.1053/j.gastro.2005.07.060.
https://doi.org/10.1053/j.gastro.2005.07...
, 1313. Medeiros JV, Bezerra VH, Gomes AS, Barbosa AL, Lima-Junior RC, Soares PM, et al. Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther 2009; 330: 764-770, doi: 10.1124/jpet.109.152801.
https://doi.org/10.1124/jpet.109.152801...
).

Results from recent reports suggest that H2S protects against mucosal injury. NaHS and Lawesson's reagent, both H2S donors, reduce the gastric damage induced by ethanol (1313. Medeiros JV, Bezerra VH, Gomes AS, Barbosa AL, Lima-Junior RC, Soares PM, et al. Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther 2009; 330: 764-770, doi: 10.1124/jpet.109.152801.
https://doi.org/10.1124/jpet.109.152801...
) and nonsteroidal anti-inflammatory drugs (NSAIDs) in rats (1414. Wallace JL, Dicay M, McKnight W, Martin GR. Hydrogen sulfide enhances ulcer healing in rats. FASEB J 2007; 21: 4070-4076, doi: 10.1096/fj.07-8669com.
https://doi.org/10.1096/fj.07-8669com...
). Furthermore, H2S donors participate in ulcer repair, regulate gastric mucosal blood flow, and contribute to the maintenance of gastric mucosal integrity (1212. Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, et al. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology 2005; 129: 1210-1224, doi: 10.1053/j.gastro.2005.07.060.
https://doi.org/10.1053/j.gastro.2005.07...
, 1515. Kanatsu K, Aihara E, Okayama M, Kato S, Takeuchi K. Mucosal irritative and healing impairment action of risedronate in rat stomachs: comparison with alendronate. J Gastroenterol Hepatol 2004; 19: 512-520, doi: 10.1111/j.1440-1746.2003.03314.x.
https://doi.org/10.1111/j.1440-1746.2003...
, 1616. Costa NR, Silva RO, Nicolau LA, Lucetti LT, Santana AP, Aragão KS, et al. Role of soluble guanylate cyclase activation in the gastroprotective effect of the HO-1/CO pathway against alendronate-induced gastric damage in rats. Eur J Pharmacol 2013; 700: 51-59, doi: 10.1016/j.ejphar.2012.12.007.
https://doi.org/10.1016/j.ejphar.2012.12...
). However, the role of H2S in ALD-induced gastric damage is unknown.

Because there is still no curative therapy for gastropathy caused by ALD and there are few studies on the mechanisms involved in its toxicity, the aim of this study was to evaluate the gastroprotective effect of Lawesson's reagent, an H2S donor, against ALD-induced gastric damage in rats, and the possible mechanisms.

Material and Methods

Animals

Female Wistar rats, weighing 180-200 g, were obtained from Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil. The animals were housed in cages in a temperature-controlled environment under a 12:12-h light-dark cycle. The animals had free access to drinking water and a standard pellet diet (Purina chow, Brazil). The animals were deprived of food for 18-24 h before the experiment, but had free access to water. All animal treatments and surgical procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and were approved by the Ethics Committee of Universidade Federal de Piauí (Protocol No. 0067/10).

Drugs And Solutions

Lawesson's reagent, glibenclamide, and ALD were purchased from Sigma (USA). Vehicle solutions consisted of saline. ALD was dissolved in saline and adjusted to pH 7.0 by adding NaOH or HCl (1515. Kanatsu K, Aihara E, Okayama M, Kato S, Takeuchi K. Mucosal irritative and healing impairment action of risedronate in rat stomachs: comparison with alendronate. J Gastroenterol Hepatol 2004; 19: 512-520, doi: 10.1111/j.1440-1746.2003.03314.x.
https://doi.org/10.1111/j.1440-1746.2003...
). Glibenclamide was dissolved in 0.01 N NaOH containing 4% glucose.

H2s Donor And Ald-induced Gastric Damage

The animals (n=5) were initially treated with Lawesson's reagent (H2S donor: 3, 9, or 27 µmol/kg, po) or an equivalent volume of their respective vehicle. After 30 min, the rats received ALD (30 mg/kg, pH 7.0) by gavage. All drugs were administered once daily for 4 days (1616. Costa NR, Silva RO, Nicolau LA, Lucetti LT, Santana AP, Aragão KS, et al. Role of soluble guanylate cyclase activation in the gastroprotective effect of the HO-1/CO pathway against alendronate-induced gastric damage in rats. Eur J Pharmacol 2013; 700: 51-59, doi: 10.1016/j.ejphar.2012.12.007.
https://doi.org/10.1016/j.ejphar.2012.12...
). On the last day of treatment, 4 h after ALD administration, the animals were killed and their stomachs removed. Gastric damage was measured using a computer planimetry program (Image J¯, USA). A sample of the stomach was fixed in 10% formalin immediately after its removal for subsequent histopathological assessment. Other samples of the stomach were then weighed, frozen, and stored at -70°C until assayed for glutathione (GSH) (1717. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968; 25: 192-205, doi: 10.1016/0003-2697(68)90092-4.
https://doi.org/10.1016/0003-2697(68)900...
), malondialdehyde (MDA) (1818. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test.Anal Biochem 1978; 86: 271-278, doi: 10.1016/0003-2697(78)90342-1.
https://doi.org/10.1016/0003-2697(78)903...
), myeloperoxidase (MPO) activity (1919. Bradley PP, Christensen RD, Rothstein G. Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 1982; 60: 618-622.), and cytokine concentrations (2020. Cunha FQ, Boukili MA, da Motta JI, Vargaftig BB, Ferreira SH. Blockade by fenspiride of endotoxin-induced neutrophil migration in the rat. Eur J Pharmacol 1993; 238: 47-52, doi: 10.1016/0014-2999(93)90503-A.
https://doi.org/10.1016/0014-2999(93)905...
).

Histological Evaluation Of Gastric Lesions

For histological evaluation, stomach samples were fixed in 10% formalin solution, where they remained for 24 h. After this procedure, the samples were transferred to a solution of 70% alcohol. Then, the samples were embedded in paraffin and sectioned. Four-micrometer-thick sections were deparaffinized, stained with hematoxylin and eosin, and then examined under a microscope. The specimens were assessed according to the criteria of Laine and Weinstein (2121. Laine L, Weinstein WM. Histology of alcoholic hemorrhagic “gastritis”: a prospective evaluation. Gastroenterology 1988; 94: 1254-1262.), who assigned scores according to the following parameters: epithelial cell loss (a score of 0-3), edema in the upper mucosa (a score of 0-4), hemorrhagic damage (a score of 0-4), and presence of inflammatory cells (a score of 0-3), yielding a maximum total score of 14. Afterward, the sections were assessed by an experienced pathologist who was blinded to the treatment.

Role Of Atp-sensitive Potassium (kAtp) Channels In Lawesson's Reagent-mediated Gastric Protection

To study the role of KATP in Lawesson's reagent-mediated gastric protection, animals (n=5) were pretreated with glibenclamide (5 mg/kg,ip), a drug that blocks KATP-dependent channels, or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/k,ip). After 1 h, the rats received Lawesson's reagent (27 µmol/kg). After 30 min, the rats received ALD (30 mg/kg, pH 7.0) by gavage. All drugs were administered once daily for 4 days. On the last day of treatment, 4 h after ALD administration, gastric damage was determined as described earlier.

Glutathione Analysis

The reduced GSH content of stomach tissues was estimated according to the method described by Sedlak and Lindsay (1717. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968; 25: 192-205, doi: 10.1016/0003-2697(68)90092-4.
https://doi.org/10.1016/0003-2697(68)900...
). Briefly, 50-100 mg frozen gastric tissue was homogenized in 1 mL 0.02 M EDTA for each 100 mg tissue. Aliquots (400 µL) of the homogenate were mixed with 320 µL distilled water and 80 µL 50% (w/v) trichloroacetic acid to precipitate proteins. The tubes were centrifuged at 3000 g for 15 min at 4°C. Supernatants (400 µL) were mixed with 800 µL Tris buffer (0.4 M, pH 8.9) and 20 µL 5,5′-dithiobis(2-nitrobenzoic acid) (0.01 M). The mixture was then stirred for 3 min and the absorbance was read at 412 nm using a spectrophotometer. The results are reported as micrograms of GSH per gram of tissue (µg/g).

Mda Analysis

The level of MDA in the homogenates from each group was measured using the method of Mihara and Uchiyama (1818. Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test.Anal Biochem 1978; 86: 271-278, doi: 10.1016/0003-2697(78)90342-1.
https://doi.org/10.1016/0003-2697(78)903...
), which is based on the reaction with thiobarbituric acid. Fragments of gastric mucosa weighing between 100 and 150 mg were homogenized in cold KCl (1.15%) to prepare a 10% solution of homogenate. Briefly, 250 µL of this homogenate was added to 1.5 mL 1% H3PO4 and 0.5 mL 0.6% thiobarbituric acid (aqueous solution). Then, the mixture was stirred and heated in a boiling water bath for 45 min. Next, the reaction mixture was cooled immediately in an ice water bath, followed by addition of 4 mL n-butanol. This mixture was shaken for 1 min, and the butanol layer was separated by centrifugation at 1,200g for 10 min. Absorbance was determined at 535 and 520 nm, and the absorbance difference between the two determinations was calculated and considered as the thiobarbituric acid value. MDA concentrations are reported as nanomoles per gram of tissue (nmol/g).

Mpo Activity

MPO is an enzyme found primarily in neutrophil azurophilic granules. It has been used extensively as a biochemical marker for granulocyte infiltration into various tissues, including the gastrointestinal tract. The extent of neutrophil accumulation in the gastric mucosa was measured by MPO activity evaluation as previously described (1919. Bradley PP, Christensen RD, Rothstein G. Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 1982; 60: 618-622.). Briefly, 50-100 mg tissue was homogenized in 1 mL potassium phosphate buffer (50 mM, pH 6.0) with 0.5% hexadecyltrimethylammonium bromide for each 50 mg tissue. Then, homogenates were centrifuged at 40,000 g for 7 min at 4°C. MPO activity in the resuspended pellet was assayed by measuring the change in absorbance at 450 nm using o-dianisidine dihydrochloride and 1% hydrogen peroxide. The results are reported as MPO U/mg tissue. A unit of MPO activity is defined as that converting 1 µmol of H2O2to water in 1 min at 22°C.

Cytokine Measurements

The animals had a sample of their stomach removed on day 4 for analysis of cytokines. The specimens were stored at -70°C until required for assay. The collected tissues were homogenized and processed as described by Safieh-Garabedian et al. (2222. Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ. Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 1995; 115: 1265-1275, doi: 10.1111/j.1476-5381.1995.tb15035.x.
https://doi.org/10.1111/j.1476-5381.1995...
). The concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were determined by using an enzyme-linked immunosorbent assay (ELISA), as described previously (2020. Cunha FQ, Boukili MA, da Motta JI, Vargaftig BB, Ferreira SH. Blockade by fenspiride of endotoxin-induced neutrophil migration in the rat. Eur J Pharmacol 1993; 238: 47-52, doi: 10.1016/0014-2999(93)90503-A.
https://doi.org/10.1016/0014-2999(93)905...
). Briefly, microtiter plates were coated overnight at 4°C with an antibody against rat TNF-α or IL-1β (4 μg/mL, DuoSet ELISA Development kit, R&D Systems, USA; Catalog Nos. DY501 and DY510, respectively). After blocking the plates, the samples and standards were added at various dilutions in duplicate and incubated at 4°C for 24 h. The plates were washed three times with buffer. After the plates were washed, biotinylated sheep polyclonal anti-TNF-α or anti-IL-1β (diluted 1:1000 with assay buffer containing 1% bovine serum albumin) was added to the wells. After further incubation at room temperature for 1 h, the plates were washed and 50 µL avidin-conjugated horseradish peroxidase diluted 1:5000 was added to the wells. The color reagent o-phenylenediamine (50 µL) was added 15 min later and the plates were incubated in the dark at 37°C for 15-20 min. The enzyme reaction was stopped with H2SO4 and absorbance was measured at 490 nm. Values are reported as picograms of cytokines per milliliter (pg/mL).

Statistical Analysis

Data are reported as means±SE. One-way ANOVA and the Student-Newman-Keuls test were used to determine statistical significance of differences between groups. For histological assessment, the Kruskal-Wallis nonparametric test was used, followed by the Dunn test for multiple comparisons. Differences were considered to be significant when P<0.05.

Results

Ald-induced Gastric Damage

Oral administration of ALD once daily for 4 days caused damage to the epithelium of the corporal and antral mucosa of the stomach, leading to severe ulcers with white caps (Figure 1A). Histopathological evaluation confirmed the presence of hemorrhagic lesions and inflammation in the mucosa of the glandular stomach, reflecting true ulcer formation (Table 1 and Figure 2). Furthermore, ALD-treated rats showed decreased GSH levels compared with controls. Conversely, MDA levels were higher in the ALD-treated rats (Table 2). The ALD-treated rats also showed elevated MPO activity (see Figure 3), as well as elevated levels of TNF-α and IL-1β (seeFigure 4).

Figure 1
Lawesson's reagent reduces alendronate-induced gastric damage (A and B). Rats were treated by gavage with saline (Sal) or Lawesson's reagent (Law) for 4 days, followed by the administration of alendronate (ALD). Treatment with ALD produced widespread damage to the epithelium and the luminal surface was covered with cellular debris (A) and Law decreased alendronate-induced gastropathy (A andB). The results are reported as means±SE for 5-7 animals per group. *P<0.05 vs saline group;+P<0.05 vs alendronate group (one-way ANOVA and Student-Newman-Keuls test).
Figure 2
Histopathological alterations in the gastric mucosa of control and treated rats after 4 days of treatment. A, Saline control group showing gastric mucosal integrity (40X).B, Effect of alendronate on the gastric mucosa showing lesion in the superficial gastric glandular region with bleeding and loss of epithelial cells (arrows) (40X). C, Pretreatment with Lawesson's reagent (27 µmol/kg,po) showing a reduction in microscopic lesions caused by alendronate (40X).
Figure 3
Effect of the Lawesson's reagent (27 µmol/kg) on gastric myeloperoxidase (MPO) activity in a rat model of alendronate-induced gastric damage. Rats were treated by gavage with saline (Sal) or Lawesson's reagent (Law) for 4 days, followed by the administration of alendronate. Results are reported as means±SE for 5-7 animals per group. *P<0.05 vs control group;+P<0.05 vs alendronate group (one-way ANOVA and Student-Newman-Keuls test).
Figure 4
Treatment with Lawesson's reagent (27 µmol/kg) decreased concentration of TNF-α and IL-1β (A andB, respectively) in alendronate-induced gastric damage. Rats were treated by gavage with Lawesson's reagent (Law) 30 min before alendronate (30 mg/kg) administration. The control group was treated with saline (Sal) only. All drugs were administered once daily for 4 days. Results are reported as means±SE for at least 5 rats per group. *P<0.05, compared to control group.+P<0.05, compared to alendronate group (one-way ANOVA and Student-Newman-Keuls test).

Gastroprotective Effect Of Lawesson's Reagent Against Ald-induced Gastric Damage

Lawesson's reagent (27 µmol/kg) protected against ALD-induced macroscopic (Figure 1B) and microscopic gastric damage (Table 1 and Figure 2). It also decreased hemorrhagic damage, inflammatory cell infiltration, and epithelial cell loss induced by ALD (Table 1 and Figure 2). Furthermore, Lawesson's reagent prevented the ALD-mediated reduction in GSH levels (Table 2) and increase in MDA expression in the gastric mucosa. It also significantly attenuated the ALD-induced increase in MPO activity (Figure 3), and TNF-α and IL-1β concentrations in gastric tissue (Figure 4).

Role Of KAtp Channels In The Gastroprotective Effects Of Lawesson's Reagent

To assess the contribution of KATP channels to the protective effects of Lawesson's reagent, other groups of rats were pretreated with glibenclamide alone or with diazoxide. In Figure 5, we demonstrated that glibenclamide (1 mg/kg) alone, without diazoxide, reversed the gastroprotective effect of Lawesson's reagent against ALD-induced macroscopic gastric damage.

Figure 5
Effect of glibenclamide and diazoxide pretreatment on the protective effect of Lawesson's reagent on alendronate-induced gastric lesions in rats. Glibenclamide (Glib, 1 mg/kg, ip) was injected 30 min before Lawesson's reagent (27 µmol/kg) or saline (Sal). Another group was treated in the same manner but diazoxide (3 mg/kg,ip)+Glib (1 mg/kg) was administered. Rats were treated by gavage with Lawesson's reagent 30 min before alendronate (30 mg/kg) administration. The control group was treated with alendronate only. All drugs were administered once daily for 4 days. Results are reported as means±SE for at least 5 rats per group. *P<0.05, compared to control group. +P<0.05, compared to Lawesson's reagent+alendronate group.#P<0.05, compared to Glib+Lawesson's reagent+alendronate group (one-way ANOVA and Student-Newman-Keuls test).

Discussion

H2S has been identified as a modulator of many physiological processes, including neurotransmission (1010. Wang R. Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter? FASEB J 2002; 16: 1792-1798, doi: 10.1096/fj.02-0211hyp.
https://doi.org/10.1096/fj.02-0211hyp...
), acute inflammation (2323. Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G, Wallace JL. Hydrogen sulfide is an endogenous modulator of leukocyte-mediated inflammation. FASEB J 2006; 20: 2118-2120, doi: 10.1096/fj.06-6270fje.
https://doi.org/10.1096/fj.06-6270fje...
, 2424. Dal-Secco D, Cunha TM, Freitas A, Alves-Filho JC, Souto FO, Fukada SY, et al. Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels. J Immunol 2008; 181: 4287-4298.), and pain (2525. Distrutti E, Sediari L, Mencarelli A, Renga B, Orlandi S, Russo G, et al. 5-Amino-2-hydroxybenzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl ester (ATB-429), a hydrogen sulfide-releasing derivative of mesalamine, exerts antinociceptive effects in a model of postinflammatory hypersensitivity. J Pharmacol Exp Ther 2006; 319: 447-458, doi: 10.1124/jpet.106.106435.
https://doi.org/10.1124/jpet.106.106435...
, 2626. Cunha TM, Dal-Secco D, Verri WA Jr, Guerrero AT, Souza GR, Vieira SM, et al. Dual role of hydrogen sulfide in mechanical inflammatory hypernociception. Eur J Pharmacol 2008; 590: 127-135, doi: 10.1016/j.ejphar.2008.05.048.
https://doi.org/10.1016/j.ejphar.2008.05...
). Of particular relevance to the present study is that H2S is known to contribute to the maintenance of gastric mucosal integrity against damage caused by NSAIDs (1212. Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, et al. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology 2005; 129: 1210-1224, doi: 10.1053/j.gastro.2005.07.060.
https://doi.org/10.1053/j.gastro.2005.07...
) and ethanol (1313. Medeiros JV, Bezerra VH, Gomes AS, Barbosa AL, Lima-Junior RC, Soares PM, et al. Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther 2009; 330: 764-770, doi: 10.1124/jpet.109.152801.
https://doi.org/10.1124/jpet.109.152801...
). Therefore, we evaluated the protective effect of Lawesson's reagent, an H2S donor, against ALD-induced gastric damage in rats.

We found that ALD causes severe gastric mucosal damage, accompanied by hemorrhage, infiltration of inflammatory cells, and loss of epithelial cells. Our finding that Lawesson's reagent decreased ALD-induced gastropathy is consistent with studies showing that the H2S precursor, l-cysteine, or H2S donors attenuate NSAID- or ethanol-induced gastropathy (1212. Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, et al. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology 2005; 129: 1210-1224, doi: 10.1053/j.gastro.2005.07.060.
https://doi.org/10.1053/j.gastro.2005.07...
, 1313. Medeiros JV, Bezerra VH, Gomes AS, Barbosa AL, Lima-Junior RC, Soares PM, et al. Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther 2009; 330: 764-770, doi: 10.1124/jpet.109.152801.
https://doi.org/10.1124/jpet.109.152801...
). Together, these findings highlight an important role for H2S in gastric protection.

The mechanism by which ALD causes mucosal injury has not been fully elucidated. However, ALD-induced neutrophil accumulation and subepithelial edema in the gastric mucosa have been shown to play major roles in the development of ulcers (2727. Wallace JL, Dicay M, McKnight W, Bastaki S, Blank MA. N-bisphosphonates cause gastric epithelial injury independent of effects on the microcirculation. Aliment Pharmacol Ther 1999; 13: 1675-1682, doi: 10.1046/j.1365-2036.1999.00658.x.
https://doi.org/10.1046/j.1365-2036.1999...
). Neutrophils are a potential source of oxygen-free radicals (2828. Parks DA, Granger DN. Ischemia-reperfusion injury: a radical view. Hepatology 1988; 8: 680-682, doi: 10.1002/hep.1840080341.
https://doi.org/10.1002/hep.1840080341...
, 2929. Sullivan GW, Sarembock IJ, Linden J. The role of inflammation in vascular diseases. J Leukoc Biol 2000; 67: 591-602.) and are considered major effector cells in the tissue damage that occurs in several inflammatory diseases (3030. Kettle AJ, Winterbourn CC. Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Report 1997; 3: 3-15.). When neutrophils are stimulated, MPO and proinflammatory cytokines are released, and granulocytes are activated, which promote cell death and gastric damage (3131. Yamaguchi K, Motegi K, Iwakura Y, Endo Y. Involvement of interleukin-1 in the inflammatory actions of aminobisphosphonates in mice. Br J Pharmacol 2000; 130: 1646-1654, doi: 10.1038/sj.bjp.0703460.
https://doi.org/10.1038/sj.bjp.0703460...
,3232. Thiebaud D, Sauty A, Burckhardt P, Leuenberger P, Sitzler L, Green JR, et al. An in vitro and in vivo study of cytokines in the acute-phase response associated with bisphosphonates. Calcif Tissue Int 1997; 61: 386-392, doi: 10.1007/s002239900353.
https://doi.org/10.1007/s002239900353...
). In the present study, the increases in TNF-α and IL1-β levels, and in MPO activity in the gastric mucosa, reflect the impact of proinflammatory cytokines and neutrophil infiltration, respectively, in ALD-mediated gastric damage. There is evidence to suggest that H2S inhibits leukocyte-endothelial cell adhesion (2323. Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G, Wallace JL. Hydrogen sulfide is an endogenous modulator of leukocyte-mediated inflammation. FASEB J 2006; 20: 2118-2120, doi: 10.1096/fj.06-6270fje.
https://doi.org/10.1096/fj.06-6270fje...
, 3333. Fiorucci S, Distrutti E, Cirino G, Wallace JL. The emerging roles of hydrogen sulfide in the gastrointestinal tract and liver. Gastroenterology 2006; 131: 259-271, doi: 10.1053/j.gastro.2006.02.033.
https://doi.org/10.1053/j.gastro.2006.02...
).

Because Lawesson's reagent inhibited the ALD-induced elevation in MPO activity, and TNF-α and IL-1β levels, our results suggest that the gastroprotective effect of an H2S donor may be dependent on its inhibitory effect on neutrophil infiltration and the neutrophil-associated TNF-α and IL-1β response.

Results from several studies suggest that reactive oxygen species may contribute to ALD-induced gastric mucosal lesions (1616. Costa NR, Silva RO, Nicolau LA, Lucetti LT, Santana AP, Aragão KS, et al. Role of soluble guanylate cyclase activation in the gastroprotective effect of the HO-1/CO pathway against alendronate-induced gastric damage in rats. Eur J Pharmacol 2013; 700: 51-59, doi: 10.1016/j.ejphar.2012.12.007.
https://doi.org/10.1016/j.ejphar.2012.12...
,3434. Sener G, Paskaloglu K, Kapucu C, Cetinel S, Contuk G, Ayanoglu-Dulger G. Octreotide ameliorates alendronate-induced gastric injury. Peptides 2004; 25: 115-121, doi: 10.1016/j.peptides.2003.11.017.
https://doi.org/10.1016/j.peptides.2003....
). This observation is consistent with our finding that ALD decreased GSH levels and increased MDA levels. The rise in the MDA level, an index of lipid peroxidation, may also be partly due to free radicals generated by neutrophils (3535. Szabo S, Trier JS, Frankel PW. Sulfhydryl compounds may mediate gastric cytoprotection. Science 1981; 214: 200-202, doi: 10.1126/science.7280691.
https://doi.org/10.1126/science.7280691...
). The importance of GSH in gastric defense was described by Szabo et al. (3636. Szabo S, Nagy L, Plebani M. Glutathione, protein sulfhydryls and cysteine proteases in gastric mucosal injury and protection. Clin Chim Acta 1992; 206: 95-105, doi: 10.1016/0009-8981(92)90010-N.
https://doi.org/10.1016/0009-8981(92)900...
), who showed that substances containing sulfhydryl radicals protect the gastric mucosa in a manner similar to that of prostaglandins (PGs) and that sulfhydryl group blockers, such as diethylmaleate and iodoacetamide, reverse the gastroprotective effect of PGF2α. They also demonstrated that GSH administration decreases ethanol-induced gastric damage by inactivating reactive oxygen species and products of lipid peroxidation (3636. Szabo S, Nagy L, Plebani M. Glutathione, protein sulfhydryls and cysteine proteases in gastric mucosal injury and protection. Clin Chim Acta 1992; 206: 95-105, doi: 10.1016/0009-8981(92)90010-N.
https://doi.org/10.1016/0009-8981(92)900...
). Thus, the ability of Lawesson's reagent to inhibit the effects of ALD on the GSH and MDA levels in the present study suggests that an H2S donor may attenuate the redox state during ALD-induced gastric injury.

We have shown here that blockade of KATP channels with glibenclamide alone, without diazoxide, reversed Lawesson's reagent's protective effect against ALD-induced gastric damage. The participation of KATP channels in several models of gastric protection was previously described (3737. Medeiros JVR, Gadelha GG, Lima SJ, Garcia JA, Soares PMG, Santos AA, et al. Role of the NO/cGMP/K(ATP) pathway in the protective effects of sildenafil against ethanol-induced gastric damage in rats. Br J Pharmacol 2008; 153: 721-727, doi: 10.1038/sj.bjp.0707605.
https://doi.org/10.1038/sj.bjp.0707605...
, 3838. Gomes AS, Gadelha GG, Lima SJ, Garcia JA, Medeiros JVR, Havt A, et al. Gastroprotective effect of heme-oxygenase 1/biliverdin/CO pathway in ethanol-induced gastric damage in mice. Eur J Pharmacol 2010; 642: 140-145, doi: 10.1016/j.ejphar.2010.05.023.
https://doi.org/10.1016/j.ejphar.2010.05...
). Other studies reported that glibenclamide pretreatment prevented the protective effects of NaHS and Lawesson's reagent against ethanol (1313. Medeiros JV, Bezerra VH, Gomes AS, Barbosa AL, Lima-Junior RC, Soares PM, et al. Hydrogen sulfide prevents ethanol-induced gastric damage in mice: role of ATP-sensitive potassium channels and capsaicin-sensitive primary afferent neurons. J Pharmacol Exp Ther 2009; 330: 764-770, doi: 10.1124/jpet.109.152801.
https://doi.org/10.1124/jpet.109.152801...
) and NSAIDs (1212. Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, et al. Inhibition of hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology 2005; 129: 1210-1224, doi: 10.1053/j.gastro.2005.07.060.
https://doi.org/10.1053/j.gastro.2005.07...
) induced macroscopic gastric damage, suggesting an involvement of KATP channels in H2S gastroprotective effects.

We showed that Lawesson's reagent, an H2S donor, prevents ALD-induced gastric damage. We propose that Lawesson's reagent inhibits neutrophil infiltration and decreases damage secondary to the release of proinflammatory cytokines and elevations in oxidative stress. Our data support the hypothesis that the activation of KATP channels is of primary importance.

The authors gratefully acknowledge the financial support from Research Foundation for the Estado do Piauí (FAPEPI) and CNPq.

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    Szabo S, Nagy L, Plebani M. Glutathione, protein sulfhydryls and cysteine proteases in gastric mucosal injury and protection. Clin Chim Acta 1992; 206: 95-105, doi: 10.1016/0009-8981(92)90010-N.
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    » https://doi.org/10.1016/j.ejphar.2010.05.023
  • First published online August 19, 2013.

Publication Dates

  • Publication in this collection
    16 Aug 2013
  • Date of issue
    Aug 2013

History

  • Received
    14 Feb 2013
  • Accepted
    21 June 2013
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