LASSBio-596 protects gastric mucosa against the development of ethanol-induced gastric lesions in mice

European Journal of Pharmacology 863 (2019) 172662

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LASSBio-596 protects gastric mucosa against the development of ethanolinduced gastric lesions in mice

T

Carlos Eduardo S. Monteiroa,e, Johnatan Alisson Oliveira Sousaa,e, Lídia Moreira Limab, Eliezer J. Barreirob, Kaira Emanuella Sales da Silva-Leitec,e, Cecília Mendes Morais de Carvalhoe, Deysen Kerlla Fernandes Bezerra Girãoe, André Luiz Reis Barbosad, Marcellus Henrique Loiola Ponte de Souzae, Gomes Soares Pedro Marcosa,e,∗ a

Department of Morphology, Federal University of Ceara, Coronel Nunes de Melo Street, 1315 Rodolfo Teófilo, 60416-030, Fortaleza, Ceará, Brazil Institute of Biomedical Sciences, Federal University of Rio de Janeiro, 373 Carlos Chagas Filho Avenue, Cidade Universitária, 21941-590, Rio de Janeiro, Rio de Janeiro, Brazil c Superior Institute of Biomedical Sciences, State University of Ceara, Av. Dr. Silas Munguba, 1700, Itaperi, 60714-903, Fortaleza, CE, Brazil d Biotechnology and Biodiversity Center Research, Campus of Parnaíba, São Sebastiao Avenue, 64202-020, Federal University of Piauí, Parnaíba, Piauí, Brazil e LEFFAG- Laboratory of Physiopharmacology Study of Gastrointestinal Tract, Federal University of Ceará, Coronel Nunes de Melo Street, 1315 Rodolfo Teófilo, 60416030, Fortaleza, Ceará, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: LASSBio-596 Acute gastritis Ethanol Nitric oxide

LASSBio-596 (2-[4-(1,4-thiazinan-4-ylsulfonyl) phenylcarbamoyl] benzoic acid) is a molecular hybrid of antitumor necrosis factor α (TNF-α) and phosphodiesterase 5 inhibitors, and its anti-inflammatory effects have been demonstrated in experimental models of inflammation. The aim of this study was to evaluate the gastroprotective effect of LASSBio-596 in an ethanol-induced acute gastritis model. Before induction of gastric damage, mice were pretreated with LASSBio-596 (20 mg per os (p.o.), Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME; 3 mg/kg, intraperitoneally [i.p.]) or with 1400W (10 mg/kg, i.p.) given alone or in their combinations. Thirty minutes later, gastric damage was induced by intragastric instillation of 50% ethanol (0.5 ml/25 g, by gavage). After 1 h, gastric damage (hemorrhagic or ulcerative lesions) was measured by planimetry. Samples of the stomach were also taken for histopathological assessment and for assays of tissue myeloperoxidase (MPO), glutathione (GSH), malondialdehyde (MDA), and inflammatory cytokines. Ethanol administration induced the development of gastric lesions in mice. LASSBio-596 reduced gastric damage, epithelial cell loss and hemorrhage, and restored the antioxidant defense system by decreasing the levels of MDA and the consumption of GSH in gastric mucosa. LASSBio-596 also decreased gastric TNF-α and interleukin-1β (IL-1β) protein levels, MPO enzymatic activity, and hemoglobin levels. Treatment with the nitric oxide synthase inhibitors L-NAME and 1400W reversed the effects of LASSBio-596 on ethanol-induced gastric lesions. LASSBio-596 did not alter mucus content and pH of gastric secretions. In summary, LASSBio-596 exerts protective effects against ethanol-induced gastric injury. The gastroprotective effects of LASSBio seem to be NO-dependent.

1. Introduction Symbiotic drugs developed by molecular hybridization have been used for the treatment of multifactorial diseases. The 2-[4-(1,4thiazinan-4-ylsulfonyl) phenylcarbamoyl] benzoic acid (LASSBio596) is a new symbiotic drug, structurally designed as a hybrid drug originating from thalidomide, arylsulfonamide, and sildenafil (Lima et al., 2002; Rocco et al., 2010). The compound is considered safer

than thalidomide due the absence of phthalimide and glutarimide subunits, present in thalidomide and responsible for its teratogenic profile. Thalidomide is an anti-inflammatory drug that functions by modulating the expression of tumor necrosis factor-alpha (TNF-α), while aryl sulfonamides and sildenafil are described as selective phosphodiesterases 4 (PDE-4) and 5 (PDE-5) inhibitors. LASSBio-596 has been reported as a novel PDE-4 and PDE-5 inhibitor (Campos et al., 2006). However, there is no hard experimental evidence

∗ Corresponding author. Department of Morphology, Federal University of Ceara, Coronel Nunes de Melo Street, 1315 Rodolfo Teófilo, 60, 430-270, Fortaleza, Ceará, Brazil. E-mail address: [email protected] (P.M. Gomes Soares).

https://doi.org/10.1016/j.ejphar.2019.172662 Received 7 August 2019; Received in revised form 3 September 2019; Accepted 16 September 2019 Available online 17 September 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

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showing that LASSBio-596 inhibits PDE. On the other hand, antiinflammatory properties of LASSBio-596 have been demonstrated in acute respiratory distress syndrome (Silva et al., 2016). Despite this, it remains to established whether LASSBio-596 would protect the gastric mucosa against inflammatory damage. The integrity of the gastric mucosa is maintained by intrinsic defense mechanisms that include pre-epithelial factors (muco-bicarbonate barrier) and epithelial barriers (continuous layer of cells with narrow junctions); and by proliferation of progenitor cells (regulated by growth factors, prostaglandins, and survival of epithelial cells). The role of prostaglandins in the health of the gastric mucosa has been demonstrated by Ahluwalia et al. (2014) and Baatar et al. (2002), where the use of the cyclooxygenase 2 inhibitor significantly delayed the healing of esophageal ulcer. Excessive consumption of alcohol is one of the major triggers of gastric damage (Feinstein et al., 2010; Thorsen et al., 2013). The loss of epithelial cells accompanied by diffuse leukocyte infiltration is observed in the mucosa and submucosa in ethanol-induced gastric injury (Medeiros et al., 2009; Pan et al., 2008). In addition, the inflammatory response to excess alcohol can generate reactive oxygen species that trigger intracellular protein damage and disrupt the gastrointestinal (GI) tract barrier, leading to ulcerogenesis (Kwiecien et al., 2014; Bhattacharyya et al., 2014). NO is a key signaling molecule in the pathogenesis of inflammation and GI mucosal defense (Sharma et al., 2007). Physiologically, it is mainly released by enteric inhibitory nitrergic neurons and endothelial cells (Groneberg et al., 2016; Magierowski et al., 2015). NO plays a crucial role in smooth muscle relaxation and the regulation of GI motility (Groneberg et al., 2016). NO also leads to vasodilatation with increase in gastric blood flow (Magierowski et al., 2015). Moreover, NO is involved in the maintenance of mucosal integrity (Magierowski et al., 2015). On the other hand, an excess of NO can lead to pathology, inflammation and tissue damage (Sharma et al., 2007). The aim of the present study was to determine whether LASSBio596 exhibits any gastroprotective effect in a mouse model of ethanolinduced gastric lesions. In addition, we tried to determine the mechanisms involved in this effect and to compare the effectiveness of gastroprotective effect of LASSBio-596 with gastroprotective effect of omeprazole, a well-known proton pomp inhibitor.

animals were used in the study. Mice were maintained in a temperature (22–24 °C) and humidity (50–60%)-controlled room at a 12 h light:12 h dark cycle, and received free access to water and food ad libitum. Before the experiments, the animals were deprived of food for 18–24 h, but had free access to water. All surgical procedures and animal treatments were conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and were approved by the local ethics committee (Protocol no. 99/2017). 2.3. Experimental protocol All mice were fasted with free access to water for 24 h before a final experiments. Experiments were carried out in four separate series of studies. The first series was performed to determine the optimal gastroprotective dose of LASSBio-596 for the further study, and to compare effects of LASSBio-596 and omeprazole on the development of ethanol-induced gastric lesions, on mucosal oxidative stress, tissue hemoglobin content and severity of mucosal inflammation. DMSO (4%; diluted in saline) was used as a vehicle for omeprazole and LASSBio-596. The second series was to determine the influence of pretreatment with optimal dose of LASSBio-596 on gastric mucus secretion in animals with ethanol-induced gastric lesions. The third series was to evaluate the effect of LASSBio-596 on gastric secretion and pH of gastric content. Euthanasia was performed after 1 h (gastric mucus experiments) or 4 h (for gastric secretion and pH experiments) after the administration of 50% ethanol (volume of 0.5 ml/25 g of weight). The fourth series was to determine the influence of NO synthase on gastroprotective effect of LASSBio-596 in ethanol-induced gastric lesions. Mice were intraperitoneally pretreated with L-NAME (3 mg/kg, i.p.) or with 1400W (10 mg/kg, i.p.) 30 min before treatments with DMSO, LASSBio-596 (20 mg/kg, p.o.) (Fig. 1). 2.4. Macroscopic analyses

2. Materials and methods

The stomachs were removed and opened along the greater curvature in order to photograph the gastric mucosa using a digital camera (Nikon L315). The gastric lesion area was quantified using ImageJ software (National Institute of Mental Health: Bethesda, MD, USA) and the results expressed as a percentage of lesion area in relation to total tissue area.

2.1. Chemicals

2.5. Histopathological analysis

Ethanol, omeprazole, dimethyl sulfoxide (DMSO), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a non-selective inhibitor of nitric oxide synthase, and N-[[3-(Aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), a selective inhibitor of inducible nitric oxide synthase (iNOS), were purchased from Millipore Sigma (St Louis, MO, USA). LASSBio-596 was synthesized and provided by the Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio) at the Federal University of Rio de Janeiro. The remaining drugs and reagents were of analytical grade.

Stomach segments (1 cm) were fixed with a 10% formaldehyde solution, dehydrated with an alcoholic solution, and embedded in paraffin. Sections were cut at 6 μm thickness for staining. The sections were stained with hematoxylin/eosin (H&E) and analyzed at random by an experienced pathologist. Histopathological parameters were evaluated using the following criteria: Haemorrhagic damage was scored from 0 to 4 (0= no hemorrhage; 1= if up to one-fourth of the horizontal span of the specimen contained areas of hemorrhage with a minimum of two adjacent areas; 2= if than more one-fourth and less three-fourth of the horizontal span of the specimen contained areas of hemorrhage; 3= if up to three-fourths of the horizontal span of the specimen contained areas of hemorrhage and 4= more than three-fourths of the span was occupied by hemorrhage), Subephitelial edema was scored from 0 to 4 (0= no edema; 1= if up to one-fourth of the specimen contained areas of edema; 2= if more than one-fourth and less than three-fourths of the specimen contained areas of edema; 3= if up to three-fourths of the specimen

2.2. Animals The studies were performed in accordance with the protocol approved on 09/21/2017 by the Animal Care and Use Committee at the Federal University of Ceará. Male Swiss mice (20–25 g) were obtained from our facilities at the Department of Physiology and Pharmacology, Federal University of Ceará. A total number of 203

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Fig. 1. Experimental protocol for each series of studies.

contained areas of edema and 4= more than three-fourths of the areas was occupied by edema in the surrounding mucosa), Epithelial cell loss was scored from 0 to 3 (0= no lesion; 1= damage to the surface epithelium only; 2= damage extending down to the mucus neck cell region; 3= damage penetrating the parietal cell area and involving tissue necrosis with cytoplasmic basophilia, disturbed nuclear polarity, enlarged nuclei, and prominent nucleoli); Inflammatory infiltration was scored from 0 to 3 (0= no infiltration; 1= one or two extravascular foci of polymorphonuclear cells in the lamina propria or in the epithelium, 2= indicated three or four foci and 3= represented more than four foci). Each section was evaluated on a cumulative basis to give the mean histological score, the maximum score thus being 14 (Lacy and Ito, 1982; Laine and Weinstein, 1988).

preparation was then cooled immediately, followed by addition of 2 ml of n-butanol. The mixture was stirred and centrifuged at 132 g for 10 min, and absorbance measured at 520 and 535 nm. MDA level was calculated by ratio difference of absorbance at 520 and 535 nm. Linear regression data of the MDA standard curve (1,1,3,3-tetraethoxypropane) were used for to calculate nanomoles of MDA per gram tissue. 2.8. Hemoglobin assay The presence of hemorrhage in the gastric mucosa was determined by the hemoglobin assay, using the cyanmethemoglobin method (Bioclin, Belo Horizonte, MG and Brazil). This standard hemoglobin kit (Bioclin) contains the color reagent for hemoglobin detection (Drabkin's reagent). A glandular segment of the stomach was homogenized in Drabkin's reagent (100 mg of tissue per milliliter reagent). Shortly afterward, the samples were centrifuged at 10,000×g for 10 min. The supernatants were then removed, filtered using a 0.22 μm filter, and centrifuged at 10,000×g for 10 min. Absorbance was measured at 540 nm, and hemoglobin concentration was read off a standard curve and expressed as Hb (milligrams per gram of tissue).

2.6. Evaluation of GSH level Stomach samples were homogenized in 0.02 M EDTA (1 ml/ 100 mg of tissue). Thereafter, 400 μl of homogenate was mixed with 320 μl of distilled water and 80 μl of trichloroacetic acid (50%, w/v), and centrifuged at 826 g for 15 min. Next, 400 μl of supernatant was mixed with 800 μl of Tris buffer (0.4 M, pH 8.9) and 20 μl of 0.01 M dithionitrobenzoic acid. The samples were shaken for 3 min and absorbance measured at 412 nm using a spectrophotometer. Linear regression data of the reduced gluthatione standard curve were used for to calculate micrograms of GSH per gram tissue (Silva et al., 2011).

2.9. Myeloperoxidase activity (MPO) Neutrophil infiltration in the gastric mucosa was measured using the MPO activity method described previously by Bradley et al. (1982). Briefly, stomach tissue (50 mg/ml) was homogenized in HTAB buffer (Sigma-Aldrich, St Louis, MO, USA). Next, the homogenate was centrifuged (1.859 g, 4 °C, 20 min). MPO activity was measured in the resuspended pellet at 450 nm using o-dianisidine dihydrochloride (SigmaAldrich, St Louis, MO, USA) and 1% hydrogen peroxide (Merck, Whitehouse Station, NJ, USA). The results are reported as MPO activity units per milligram of tissue (UMPO/mg of tissue). A unit of MPO activity was defined as conversion of 1 μmol of hydrogen peroxide to water in 1 min at 22 °C.

2.7. Levels of malondialdehyde (MDA) Lipid peroxidation was determined by quantification of MDA levels using the thiobarbituric acid reactive substances method adapted from Lee et al. (2012). For malondialdehyde analysis, stomach segment (100 mg of tissue) was homogenized in 1.15% KCl (1 ml). Briefly, 250 μl of homogenate was added to 1.5 ml of 1% H3PO4 and 500 μl of 0.6% thiobarbituric acid. Afterward, the mixture was stirred and heated in a boiling water bath for 45 min. The

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Fig. 2. Effect of oral pretreatment with LASSBio-596 on the gastric mucosa in a mouse model of ethanol-induced gastric mucosal injury by macroscopic examination. (A) Saline (Sal, Control group); (B) dimethyl sulfoxide (4% DMSO) + ethanol (0.5 ml/25 g); (C) omeprazole (OME, 30 mg/kg) + ethanol; (D) LASSBio-596 (L-596, 0.1 mg/kg) + ethanol; (E) LASSBio-596 (L-596, 1 mg/kg) + ethanol; (F) LASSBio-596 (L-596, 10 mg/kg) + ethanol; (G) LASSBio-596 (L-596, 20 mg/kg) + ethanol. The results are presented as mean ± S.E.M (n = 6–8). (*P < 0.05) versus saline; (#P < 0.05) versus dimethyl sulfoxide + ethanol.

2.10. Cytokine measurements

50 μl of avidin-conjugated horseradish peroxidase diluted 1:5000 was added to the wells. The color reagent o-phenylenediamine (OPD; 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 expressed as picograms of cytokines per gram of tissue.

Stomach samples (100 mg of tissue) were homogenized in 1 ml of 1 × PBS and centrifuged at 826 g for 10 min. The concentration of TNF-α and interleukin (IL)-1β was determined by using an enzymelinked immunosorbent assay (ELISA). 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; Catalog DY501 or 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 3 times with buffer. After washing the plates, biotinylated sheep polyclonal antiTNF-α or anti-IL-1β (diluted 1:1000 with assay buffer containing 1% bovine serum albumin [BSA]) was added to the wells. After further incubation at room temperature for 1 h, the plates were washed and

2.11. Gastric wall mucus determination The procedure described by Corne et al. (1974) was used to quantify the mucus on the gastric wall. The glandular segments of each stomach were weighed and added immediately to 1% Alcian blue solution (sucrose solution buffered with sodium acetate pH 5), and the excess dye was removed with a sucrose solution. The dye, which adhered to the mucus on the gastric wall, was extracted with a 4 ml solution of

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2.12. Measurements of pH and gastric secretions The pylorus ligature method was used to evaluate gastric acid secretion. After 24 h of starving with free access to water, the animals had pyloric ligation and were intraduodenally administered omeprazole (30 mg/kg) or LASSBio-596 (20 mg/kg) followed intraperitoneal administration of histamine (5 mg/kg). Group false-operated (sham) were used for comparison (Kinoshita et al., 1997; Carvalho et al., 2015). Four h after pyloric ligation, the animals were euthanized using a combination of xylazine (5 mg/kg) and ketamine (60 mg/kg), and the stomach contents were collected and centrifuged at 1.124 g for 10 min. The pH of the supernatant containing the gastric juice was analyzed in a beaker, using 0.1 ml of gastric secretion with 2 drops of phenolphthalein 1% (indicator), and 0.9 ml of distilled water added. The pH of the supernatant for each sample was measured by titration with 0.1 N NaOH. 2.13. Statistical analysis Data are expressed as the mean ± standard error of the mean (S.E.M) and analyzed using GraphPad Prism ver. 5.0 Software. Groups were compared using one-way ANOVA followed by Bonferroni and Dunnett's multiple comparisons. The non-parametric Kruskal-Wallis test was performed for histological analyses followed by Dunn's test. A P < 0.05 was considered statistically significant. 3. Results 3.1. Gastroprotective effect LASSBio-596 and omeprazole on macroscopic gastric damage Fig. 3. Histological analysis of gastric tissue sections. Mice were treated with saline (Sal, control group) (A), dimethyl sulfoxide (4% DMSO) + ethanol (B), omeprazole (OME, 30 mg/kg) + ethanol (C), and LASSBio-596 (L-596, 20 mg/kg) + ethanol (D). Arrowheads indicate the presence of edema, hemorrhagic damage, and loss of epithelial cells. Gastric tissue sections were analyzed by H&E staining (magnification × 400).

Fig. 2 shows that pretreatment with LASSBio-596 (1.0, 10.0, and 20.0 mg/kg) and omeprazole (30 mg/kg) reduced the percentage of gastric lesions to 50.42%, 21.96%, 11.10% and 11.10% respectively, as compared to DMSO plus ethanol group. As a result, the concentration of LASSBio 20 mg/kg was selected as the effective dose for subsequent studies. 3.2. Histopathological analysis

magnesium chloride mixed with an equal volume of diethyl ether. The resulting emulsion was centrifuged, and the absorbance measured using a spectrophotometer at a wavelength of 580 nm. The quantity of Alcian blue extracted per gram of glandular tissue was calculated. The quantity of Alcian blue was determined by linear regression analysis, and a calibration curve generated with different concentrations of Alcian blue. The results are expressed as micrograms of Alcian blue extracted per gram of gastric tissue.

Fig. 3 and Table 1 show that ethanol led to induction of hemorrhages (average score: 3), edemas (2), epithelial cell loss (3), neutrophilic infiltration (1), and total score (9) compared to the salinetreated group (total score 0). In contrast, treatment with LASSBio-596 was able to reduce hemorrhagic lesions and epithelial cell loss. 3.3. Effect of LASSBio-596 on GSH, MDA, and Hb Fig. 4A shows that ethanol administration (183.4 ± 21.3 μg/g of tissue) reduced gastric GSH levels compared to those in the saline-

Table 1 Effect of LASSBio-596 on the pathology of the gastric mucosa in response to ethanol treatment in mice. Data are presented as median values with minimum and maximum values in parentheses. Statistical significance was determined using the non-parametric Kruskal-Wallis test followed by the Dunn's test. (ap < 0.05) vs saline group; (bp < 0.05) vs Ethanol. Experimental groups (n=8)

Haemorrhagic damage (Score 0–4)

Edema (Score 0–4)

Epithelial cell loss (Score 0–3)

Inflammatory infiltration (Score 0–3)

Total (Score 0–14)

Saline Ethanol Ethanol + Omeprazole Ethanol + LASSBio-596

0 (0–0) 3(2–4)a 1(0–2)a 1(0–1)b

1(0–1) 2(2–3)a 1.5(0–2) 1.5(1–2)

0.5(0–1) 3(2–3)a 1(0–2)a 1(0–1)b

0(0–1) 1(1–2)a 1(0–1) 1(1–2)a

0(0–3) 9(8–10)a 4.5(0–7)a 4.5(3–7)b

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Fig. 4. Effect of LASSBio-596 on reduced glutathione (GSH) (A), malondialdehyde (MDA) (B) and hemoglobin (Hb) (C) levels in the gastric mucosa. Mean ± S.E.M (n = 6–8). (*P < 0.05) versus saline group; (#P < 0.05) versus dimethyl sulfoxide + ethanol group.

Fig. 5. Effect of LASSBio-596 on myeloperoxidase (MPO) (A) activity, and interleukin-1β (IL-1β) (B) and tumor necrosis factor-alpha (TNF-α) (C) levels in the gastric mucosa. Each value represents the mean ± S.E.M (n = 6–8). (*P < 0.05) versus saline group; (#P < 0.05) versus dimethyl sulfoxide (DMSO) + ethanol group.

treated group (409.5 ± 49.1 μg/g of tissue). Conversely, LASSBio596 treatment significantly increased (314.7 ± 29.7 μg/g of tissue) gastric GSH levels compared to those in the DMSO plus ethanol group. Omeprazole administration (285.1 ± 15.7 μg/g of tissue) significantly increased gastric GSH levels compared to DMSO plus ethanol group. As shown in Fig. 4B, the levels of gastric MDA increased significantly after ethanol administration (1411.0 ± 143.5 nmol/g of tissue) relative to saline-treated group (253.9 ± 17.9 nmol/g of tissue). Treatment with LASSBio-596 significantly reduced (P < 0.05) MDA (115.1 ± 12.3 nmol/g of tissue) levels. However, mice treated with omeprazole (165.8 ± 19.1 nmol/g of tissue) had a significant reduction of gastric MDA levels compared to DMSO plus ethanol group. Acute administration of ethanol increased (44.7 ± 3.7 mg/g of tissue) tissue hemoglobin content in gastritis (DMSO-treated) mice compared to that in the saline-treated group (15.4 ± 0.8 mg/g of tissue) (Fig. 4C). Conversely, LASSBio-596 (30.1 ± 2.7 mg/g of tissue) treatment reduced the Hb content relative to the group treated with DMSO (vehicle) and exposed to ethanol (Fig. 4C). Omeprazole administration (26.1 ± 1.1 mg/g of tissue) significantly reduced gastric hemoglobin content compared to DMSO plus ethanol group.

Fig. 6. Effect of LASSBio-596 on the content of mucus in the stomach after ethanol treatment. Saline (Sal, control group), dimethyl sulfoxide (4% DMSO), omeprazole (OME, 30 mg/kg), and LASSBio-596 (L-596, 20 mg/kg) were orally administered. The results are presented as mean ± S.E.M. (n = 6–8). (*P < 0.05) vs saline group; (#P < 0.05) vs dimethyl sulfoxide + ethanol group.

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Fig. 7. Effects of LASSBio-596 on changes in gastric pH and secretion. The animals after pyloric ligation were intraduodenally administered saline, omeprazole (30 mg/kg) or LASSBio596 (20 mg/kg) followed intraperitoneal administration of histamine (5 mg/kg). Group false-operated (Sham) were used for comparison. The evaluation of secretion volume (A) and pH (B) was performed 4 h after pylorus ligation. Data are presented as mean ± S.E.M. (n = 6–8). (*P < 0.05) vs Sham group; (#P < 0.05) vs histamine group.

3.4. Effect of LASSBio-596 on MPO activity

(155.0 ± 26.9 μl) and omeprazole (116.3 ± 18.0 μl) was not significantly different from sham group. As shown in Fig. 7B, histamine administration after pyloric ligation resulted in a significant decrease in the pH of the gastric juice (1.7 ± 0.0) compared to that in the sham group (3.2 ± 0.01). However, the LASSBio-596 (3.1 ± 0.05) and omeprazole (3.2 ± 0.07) groups are different statisticantly.

MPO activity was significantly increased in response to ethanol treatment in the gastritis group (19.1 ± 0.8 Units/mg tissue) compared to that in the control group (3.7 ± 0.3 Units/mg tissue) (Fig. 5A). In contrast, LASSBio-596 treatment significantly attenuated MPO activity (9.8 ± 0.6 Units/mg tissue) (Fig. 5A). Mice treated with omeprazole (8.27 ± 1.04 Units/mg tissue) had a significant reduction of the MPO activity compared to DMSO plus ethanol group.

3.8. L-NAME and 1400W affect the gastroprotective effects of LASSBio

3.5. Effect of LASSBio-596 on cytokines levels

In Fig. 8, L-NAME and 1400W reduced the gastroprotective effect of LASSBio-596. In addition, pretreatment with these inhibitors increased the percentage of microscopic and macroscopic lesions, MDA levels, and the hemoglobin concentration. L-NAME and 1400W were able to reduce the GSH levels and thus reduce the antioxidant effect of LASSBio-596. L-NAME and 1400W inhibitors administered alone did not change the biochemical parameters evaluated.

Ethanol treatment increased the levels of TNF-α (270.1 ± 57.6 pg/ ml) and IL-1β (317.1 ± 67.1 pg/ml) compared to those in the salinetreated group (TNF-α: 73.0 ± 11.6; IL-1β: 80.9 ± 15.9 pg/ml) (Fig. 5B and C). Treatment with LASSBio-596 reduced gastric TNF-α (113.1 ± 8.7 pg/ml; Fig. 5C) and IL-1β (82.9 ± 13.3 pg/ml; Fig. 5B) levels compared to those in the DMSO plus ethanol group. Omeprazole administration significantly reduced gastric IL-1β (136.6 ± 36.5 pg/ ml; Fig. 5B) and TNF-α (135.3 ± 6.9 pg/ml; Fig. 5C) levels compared to DMSO plus ethanol group.

4. Discussion Excessive alcohol consumption, particularly by young individuals can result in a range of physical, academic, and social problems (Adger and Saha, 2013). Ethanol-induced gastric lesions in mice is an experimental model commonly utilized for the preclinical evaluation of new drugs developed for the protection and healing of gastric mucosa. This model mimics some human conditions. Ethanol, among others, leads to the dissolution of mucus and lipoproteins on the cell membrane, and inhibits the protective capacity of the mucosa. This results in the restriction of gastric blood flow, leading to hemorrhage, and the development of lesions (Halabi et al., 2014; Rehman et al., 2013; Selmi et al., 2017). Ethanol may also induce mucosal edema, epithelial cell loss, and inflammatory cell infiltration (Medeiros et al., 2008). The development of gastric lesions affects NO synthesis and release. NO plays a key role in the regulation of gastric mucosal defense, maintenance of local mucosal blood flow (Pique et al., 1989), and mucus secretion (Brown et al., 1993). Excess of NO may be involved in neutrophil adhesion (Lai et al., 2017). Previous studies have shown exposure of gastric mucosa to damaging factors leads to little or no damage if adequate mucosal blood

3.6. Effect of LASSBio-596 on mucus content Mice with gastric lesions had a significant reduction in mucus content (153.5 ± 6.1 μg Alcian blue/g wet stomach) compared to the negative control group (264.9 ± 12.3 μg Alcian blue/g stomach wet weight) (Fig. 6). Pretreatment with LASSBio-596 (228.1 ± 13.5 μg Alcian blue/g stomach wet weight) and omeprazole (240 ± 16.6 μg Alcian blue/g stomach wet weight) increased the mucus content in gastric lesions of mice compared to the DMSO plus ethanol group (Fig. 6). 3.7. Effect of LASSBio-596 on pH and gastric secretion Fig. 7A shows that intraperitoneal histamine (286.7 ± 49.5 μl) after pyloric ligation increased the volume of gastric juice compared to that in the sham group (45.2 ± 10.8 μl). However, the volume of gastric juice produced in response to pretreatment with LASSBio-596

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Fig. 8. Effects of LASSBio-596 in response to the inhibition of nitric oxide synthase by L-NAME and 1400W. L-NAME and 1400W inhibitors were intraperitoneally administered alone or along with orally administered LASSBio-596. The mice received ethanol by oral administration. We evaluated the percentage of gastric lesions (A), malondialdehyde (MDA) (B), reduced glutathione (GSH) (C) and hemoglobin (Hb) levels (D). Data are presented as mean ± S.E.M. (n = 6–8). (*P < 0.05) versus saline group; (#P < 0.05) versus dimethyl sulfoxide (DMSO) + ethanol group; (&P < 0.05) versus LASSBio-596 (20 mg/kg).

flow is ensured (Sørbye and Svanes, 1994). On the other hand, the reduction in blood flow through the gastric mucosa results in increased sensitivity of the mucous membrane to damaging factors and leads to a significant increase in gastric damage (Sørbye and Svanes, 1994). The protective and healing-promoting effect of appropriate organ blood flow has been found in the stomach (Warzecha et al., 2000, 2001), as well as in other organs of digestive tract such as the oral mucosa (Cieszkowski et al., 2017), duodenum (Ceranowicz et al., 2009; Warzecha et al., 2012), colon (Konarska et al., 2018; Matuszyk et al., 2016) or pancreas (Warzecha et al., 2017;

Bukowczan et al., 2016). Several drugs of varying categories have been used in the treatment of gastric ulcers, including proton pump inhibitors, antacids, prostaglandin analogs, and H2-receptor antagonists (den Hollander and Kuipers, 2012). However, there are various side effects associated with these drugs, including arrhythmia, impotence, gynecomastia, and hematopoietic changes. Therefore, more effective treatment with fewer health-associated side effects are needed. We investigated the gastroprotective effect of LASSBio-596, a symbiotic compound designed by structural modification of thalidomide (an

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anti-TNF-α drug) – by optimizing its pharmacodynamic properties and eliminating its teratogenic effect (phthalimide ring removal) – and molecular hybridization with sildenafil (a phosphodiesterase 5 inhibitor) and arylsulfonamide (a selective phosphodiesterase 4 prototype) (Lima et al., 2002), on an alcohol-induced gastritis model. LASSBio-596 showed no genotoxic and/or mutagenic effect on different cell lines in vitro, and no deaths were observed in acute toxicity murine trials (at doses 200 times greater than that required for anti-inflammatory effect), or during chronic toxicity trials (at doses up to 25 times that required for anti-asthmatic effect) (Rocco et al., 2010). However, there are no studies on teratogenic effects of LASSBio-596 in pregnant animals. In agreement with previous studies, we have found that intragastric administration of 50% ethanol induces macroscopic and microscopic mucosal damage in the stomach. Histological examination of gastric damage has shown foci of hemorrhage, edema, neutrophil infiltration, and epithelial loss. Similar effects were observed by Arab et al. (2015). Moreover, Warzecha et al. (2014) have reported that the induction of gastric lesions by ethanol is associated with a reduction in gastric blood flow and mucosal DNA synthesis. The investigators also found that induction of ethanol-evoked gastric ulcers significantly increases the mRNA expression of COX-2, IL-1β, and TNF-α in gastric mucosa (Warzecha et al., 2014). The novelty of the findings of our study was the observation that LASSBio-596 attenuates hemorrhagic damage, hemoglobin levels, and restores gastric GSH and MDA levels. GSH acts as a scavenger of free radicals, inhibiting lipid peroxidation and other free radical-mediated processes (Gazzieri et al., 2007; Kurutas, 2016). Another phenotype of ethanol-induced gastric mucosal injury is lipid peroxidation, which is quantified by measuring MDA, an important factor involved in the progression of alcohol-induced oxidative gastrointestinal mucosal injury (Medeiros et al., 2009). Ethanol increases the secretion/release of inflammatory mediators and induces neutrophil migration to the gastric mucosa (Shin et al., 2013). Therefore, we investigated the activity of MPO, a biochemical marker of neutrophil infiltration. LASSBio-596 reduced MPO release, and TNF-α and IL-1β secretion because it comprises thalidomide. TNF-α and IL-1β cytokines are significantly increased in the gastric tissue after ethanol treatment (Amirshahrokhi and Khalili, 2016). TNF-α is secreted by macrophages during inflammation and plays a key role in the induction of gastric mucosal damage (Dang et al., 2015). Several factors are known to control the development of gastric lesions, such as mucus production and a bicarbonate layer that attempt to shield newly generated cells from the acidic environment and peptic injury (Tarnawski et al., 2001). Mucus production, the formation of a protective physical barrier that inhibits the activity of toxic agents on the surface of the epithelium, is one of the main gastric mucosal defense mechanisms (Laine et al., 2008). Herein, we found that LASSBio-596 prevents the loss of mucous content in contact with gastric mucosa in mice exposed to ethanol. The presence of NO synthase and cGMP stimulates mucus secretion in gastric epithelial cells; suggesting an effector role of NO in the modulation of gastric mucus release (Brown et al., 1993). As mentioned above, our data suggest that gastroprotective effects of LASSBio may be NO-dependent. This concept is additionally supported by our current observation that L-NAME (a nonselective NOS inhibitor) and 1400W (a selective iNOS inhibitor) inhibit the gastroprotective effect of LASSBio-596. The present study, in accordance with others, demonstrates that inhibition of the constitutive and inducible isoforms of NOS exacerbates damage caused by ethanol in animals. These enzymes facilitate the gastric mucosa to resist damage (Masuda et al., 1995; Forstermann and Sessa, 2012; Morini et al.,

2001). Additional studies are required to further elucidate these findings. 5. Conclusions LASSBio-596 attenuates ethanol-induced gastric mucosal damage. This gastroprotective effect may be associated with reduced oxidative stress and inflammation. Moreover, gastroprotective effects of LASSBio seem to be NO-dependent. Conflicts of interest The authors declare that there are no conflicts of interest. Author contributions 1) Conception and Design: Carlos Eduardo S Monteiro, Johnatan Alisson Oliveira Sousa and Pedro Marcos Gomes Soares 2) Provision of Study materials and funding acquisition: Lídia Moreira Lima, Eliezer J Barreiro, Pedro Marcos Gomes Soares and Marcellus Henrique Loiola Ponte de Souza 3) Manuscript writing: Carlos Eduardo S Monteiro, André Luiz Reis Barbosa, Kaira Emanuella Sales da Silva-Leite, Cecília Mendes Morais de Carvalho and Pedro Marcos Gomes Soares 4) Formal analysis and Investigation: Deysen Kerlla Fernandes Bezerra Girão, Carlos Eduardo S Monteiro, Johnatan Alisson Oliveira Sousa Acknowledgements This research was supported by National Council for Scientific and Technological Development (CNPq) (nº6379341/2015) and Cearense Foundation for Scientific and Technological Development Support (FUNCAP) (nº 12535684-6/2012). References Adger Jr., H., Saha, S., 2013. Alcohol use disorders in adolescents. Pediatr. Rev. 34, 103–114. https://doi.org/10.1542/pir.34-3-103. Ahluwalia, A., Baatar, D., Jones, M.K., Tarnawski, A.S., 2014. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB. Am. J. Physiol. Gastrointest. Liver Physiol. 307, G602–G610. https://doi.org/10.1152/ajpgi.00177.2014. Amirshahrokhi, K., Khalili, A.R., 2016. Gastroprotective effect of 2-mercaptoethane sulfonate against acute gastric mucosal damage induced by ethanol. Int. Immunopharmacol. 34, 183–188. https://doi.org/10.1016/j.intimp.2016.03.006. Arab, H., Salama A., S., Omar A., H., Arafa A., el-S., Maghrabi A., I., 2015. Diosmin Protects against Ethanol-Induced Gastric Injury in Rats: Novel Anti-Ulcer Actions. PLoS One 10(3), e0122417. https://doi.org/10.1371/journal.pone.0122417. Baatar, D., Jones, M.K., Pai, R., Kawanaka, H., Szabo, I.L., Moon, W.S., Kitano, S., Tarnawski, A.S., 2002. Selective cyclooxygenase-2 blocker delays healing of esophageal ulcers in rats and inhibits ulceration-triggered c-Met/hepatocyte growth factor receptor induction and extracellular signal-regulated kinase 2 activation. Am. J. Pathol. 160, 963–972. https://doi.org/10.1016/S0002-9440(10)64918-8. Bhattacharyya, A., Chattopadhyay, R., Mitra, S., Crowe, S.E., 2014. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol. Rev. 94, 329–354. https://doi.org/10.1152/physrev.00040.2012. Bradley, P.P., Christensen, R.D., Rothstein, G., 1982. Cellular and extracellular myeloperoxidase in pyogenic inflammation. Blood 60, 618–622. Brown, J.F., Keates, A.C., Hanson, P.J., Whittle, B.J., 1993. Nitric oxide generators and cGMP stimulate mucus secretion by rat gastric mucosal cells. Am. J. Physiol. Liver Physiol. 265, G418–G422. https://doi.org/10.1152/ajpgi.1993.265.3.G418. Bukowczan, J., Cieszkowski, J., Warzecha, Z., Ceranowicz, P., Kusnierz-Cabala, B., Tomaszewska, R., Dembinski, A., 2016. Therapeutic effect of obestatin in the course of cerulein-induced acute pancreatitis. Pancreas 45, 700–706. Campos, H.S., Xisto, D.G., Oliveira, M.B.G., Teixeira, I., Negri, E.M., Mauad, T., Carnielli, D., Lima, L.M., Barreiro, E.J., Faffe, D.S., Zin, W.A., Lapa e Silva, J.R., Rocco, P.R.M., 2006. Protective effects of phosphodiesterase inhibitors on lung function and remodeling in a murine model of chronic asthma. Braz. J. Med. Biol. Res. 39, 283–287. https://doi.org/10.1590/S0100-879X2006000200016.

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