Diminazene aceturate, an angiotensin-converting enzyme II activator,
prevents gastric mucosal damage in mice: Role of the angiotensin-(1–7)/
Mas receptor axis
Luan Kelves M. Souza
a, Lucas A.D. Nicolau
b, Nayara A. Sousa
c, Thiago S.L. Araújo
c,
Francisca Beatriz M. Sousa
c, Douglas S. Costa
d, Fabiana M. Souza
d, Dvison M. Pacífico
e,
Conceição S. Martins
e, Renan O. Silva
b, Marcellus H.L.P. Souza
b, Gilberto S. Cerqueira
a,
Jand Venes R. Medeiros
a,c,d,⇑aPostgraduate Program in Biomedical Sciences, Federal University of Piauí, Parnaíba, Piauí, Brazil
bDepartment of Physiology and Pharmacology, Laboratory of Pharmacology of Inflammation and Cancer (LAFICA), Federal University of Ceará, Fortaleza-CE, Brazil cBiotechnology and Biodiversity Center Research, BIOTEC, Postgraduate Program in Biotechnology, Federal University of Piauí, Parnaíba, Piauí, Brazil
dMedicinal Plant Research Center, NPPM, Postgraduate Program in Pharmacology, Federal University of Piauí, Teresina, Piauí, Brazil
ePostgraduate Program in Morphofunctional Sciences, Department of Morphology, Faculty of Medicine, Federal University Ceará, Fortaleza-CE, Brazil
a r t i c l e
i n f o
Article history:
Received 12 April 2016 Accepted 24 May 2016 Available online 27 May 2016
Keywords:
Gastroprotection Antioxidant Anti-inflammatory Healing Collagen
a b s t r a c t
The angiotensin (Ang) II converting enzyme (ACE II) pathway has recently been shown to be associated with several beneficial effects in various organisms, including gastroprotection. ACE II is responsible for converting Ang II into an active peptide, Ang-(1–7), which in turn binds the Mas receptor. Recent studies have shown that diminazene aceturate (Dize) a trypanocidal used in animals, activates ACE II. Thus, in this study, we aimed to evaluate the gastroprotective effects of Dize via the ACE II/Ang-(1–7)/Mas recep-tor pathway against gastric lesions induced by ethanol and acetic acid in mice. The results showed that Dize could promote gastric protection via several mechanisms, including increased levels of antioxidants and anti-inflammatory factors (e.g., decreasing tumor necrosis factor and interleukin-6 expression and reducing myeloperoxidase activity), maturation of collagen fibers, and promotion of re-epithelialization and regeneration of gastric tissue in different injury models. Thus, Dize represents a novel potential gastroprotective agent.
Ó2016 Elsevier Inc. All rights reserved.
1. Introduction
Gastric ulcer is a disorder associated with the gastrointestinal (GI) tract and affects many people worldwide[1]. They are charac-terized by necrosis, reduced blood flow, oxidative stress, and inflammatory processes[2–4]. Gastric damage occurs when there is an imbalance between offensive factors, such as gastric acid, pepsin,Helicobacter pyloriinfection, psychological stress, excessive intake of alcohol, free radicals, and nonsteroidal anti-inflammatory drugs (NSAIDs), and defensive factors, such as mucous secretion, bicarbonate secretion, prostaglandins, gastric blood flow, regener-ation of the mucosal epithelium, and enzymatic or nonenzymatic antioxidants [5–8]. Currently, the most common drugs used in the treatment of gastric ulcer are proton pump inhibitors and
histamine H2 receptor antagonists; however, these drugs are asso-ciated with recurrence of gastric ulcers after the cessation of treat-ment and undesirable side effects[9,10]. Thus, the treatment of gastric ulcers is still a major challenge, and new effective therapeu-tic agents that are less toxic and inexpensive are needed to over-come these drawbacks associated with conventional treatment options[11].
The renin–angiotensin system (RAS) is a classic endocrine sys-tem involved in the physiologic regulation of blood pressure and water-mineral balance[12,13]. The components of the RAS appear to be functionally active in numerous organs, including the kid-neys, heart, brain, reproductive organs, and skin. Angiotensin (Ang) I and Ang II also play important roles in the control of GI functions, such as electrolyte homeostasis, maintenance of regio-nal blood flow, mucosal absorption of glucose, GI motility, mucosal secretion, gastric inflammation, and carcinogenesis [13–15]. Recently, Ang I and II metabolites have been found to be present throughout the GI tract, including the stomach, colon, pancreatic
http://dx.doi.org/10.1016/j.bcp.2016.05.010
0006-2952/Ó2016 Elsevier Inc. All rights reserved.
⇑Corresponding author at: BIOTEC/LAFFEX/UFPI, Av. São Sebastião, n°2819, CEP 64202-020 Parnaíba, PI, Brazil.
E-mail address:jandvenes@ufpi.edu.br(J.V.R. Medeiros).
Contents lists available atScienceDirect
Biochemical Pharmacology
islets, and liver[16–18]. Moreover, recent advances have improved our understanding of the RAS[19]. For example, Ang-(1–7), a bio-logically active product of the RAS cascade, functions as a major vasoactive metabolite of Ang I and is an important component of the RAS. Recent studies have shown that Ang-(1–7) is the endoge-nous ligand for the G protein-coupled receptor Mas, which is asso-ciated with protective responses against heart, kidney, and brain damage[20].
Diminazene aceturate (Dize: C14H15N72C4H7NO3) is an aro-matic diamidine that was developed more than six decades ago and has been marketed for the control of trypanosomiasis[21]. In recent years, however, this drug has been extensively studied with respect to its therapeutic potential and pleiotropic effects and has consequently attracted much interest [21,22]. Indeed, several studies have shown that Dize may influence other physio-logical conditions. Dize has been shown to activate ACE II and stim-ulate the protective axis of the RAS, leading to the cleavage of Ang II[23]. ACE II metabolizes Ang II to Ang-(1–7) and thus counter-regulates the deleterious effects of Ang II[23]. Few studies have examined the effects of Dize on epithelial and connective tissue and the role of the ACE II/Ang-(1–7)/Mas receptor axis in healing in acute and chronic gastric ulcer models[13,15,16]; therefore, fur-ther in-depth studies of the mechanisms involved in re-epithelialization and healing of gastric lesions are needed.
In this study, we aimed to evaluate the gastroprotective effects of Dize through the Ang-(1–7)/Mas receptor axis in gastric lesions induced by ethanol and acetic acid in mice.
2. Materials and methods
2.1. Reagents
Dize, ethanol, and omeprazole were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-Ang-(1–7) Mas receptor and A-779 were obtained from Alomone Labs (Jerusalem, Israel). An enzyme-linked immunosorbent assay (ELISA) kit for Ang-(1–7) was purchased from R&D Systems (Minneapolis, MN, USA). Ethanol and acetic acid were obtained from Dinâmica (Diadema, SP, Brazil). All other chemicals were of analytical grade and were obtained from standard commercial suppliers. When appropriate, these chemicals were dissolved in saline before use.
2.2. Animals
Mice (Swiss strain, 25–30 g) of both sexes were obtained from the Vivarium Sector of the Center for Research on Medicinal Plants, Federal University do Piauí. The animals were maintained in cages and kept at the laboratory at a temperature of 22 ± 1°C under a
12-h lig12-ht/12-12-h dark cycle. T12-he animals 12-had free access to drinking water and a standard pellet diet. The experimental groups con-sisted of 5–6 animals per group. The animals were deprived of food for 18–24 h before the experiments, 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 local ethics committee (Protocol N°. 068/14).
2.3. Effects of Dize on ethanol-induced gastric damage
The animals were initially pretreated with vehicle (0.9% saline), Dize (0.7, 2, 7, or 20 mg/kg) or omeprazole (10 mg/kg) orally [24–27]. After 1 h, ethanol 50% (0.5 mL/25 g) was administered by gavage. One hour after ethanol ingestion, mice were euthanized, and stomach samples were collected for biochemical analysis and macroscopic and histopathological evaluations of gastric mucosa
[28]. Gastric damage was measured using a computer planimetry program (ImageJ software; National Institutes of Health, Bethesda, MD, USA). Samples of the stomach were fixed in 10% formalin immediately after their removal for subsequent histopathological assessment. Unfixed samples were then weighed, frozen, and stored at 80°C until determination of glutathione (GSH) [29]
and malondialdehyde (MDA) concentrations[30].
2.4. Evaluation of the Ang-(1–7)/Mas receptor pathway in mediating the protective effects of Dize on ethanol-induced gastric damage
To evaluate the role of the Ang-(1–7)/Mas receptor axis in the protective effects of Dize, the animals were treated with a selective antagonist for the Mas receptor (A-779; 5 mg/kg, intraperitoneally [i.p.]). After 30 min, mice received Dize (7 mg/kg, by gavage). One hour later, gastric damage was induced by intragastric instillation of ethanol 50% (0.5 mL/25 g). One hour later, gastric damage was determined as described above. Finally, a sample of the corpus region of each stomach was fixed in 10% formalin for subsequent histopathological assessment, and other full-thickness pieces of the gastric corpus were weighed, frozen, and stored at 70°C until
determination of GSH and MDA concentrations.
2.5. Histological evaluation of gastric damage
For histological evaluation, the glandular stomach was fixed in 10% neutral-buffered formalin solution for 24 h. After fixation, the samples were transferred to a solution of 70% alcohol, sec-tioned, and embedded in paraffin. Four-micrometer-thick sections were deparaffinized, stained with hematoxylin and eosin, and then examined under a light microscope by an experienced pathologist who was blinded to the treatment. The specimens were assessed according to previously described criteria[31]and were 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), hem-orrhagic damage (a score of 0–4), and presence of inflammatory cells (a score of 0–3), yielding a maximum possible score of 14.
2.6. Glutathione assay
Reduced GSH content in stomach tissues as non-protein sulfhy-dryls was estimated according to the method described by Sedlak and Lindsay [29]. A glandular segment from each stomach was homogenized in 5 mL of ice-cold 0.02 M EDTA solution (1 mL/100 mg tissue). Aliquots (400
l
L) of tissue homogenate were mixed with 320l
L distilled water and 80l
L of 50% (w/v) trichlor-oacetic acid in glass tubes. The samples were then centrifuged at 3000gfor 15 min, and supernatants (400l
L) were mixed with 800l
L Tris buffer (0.4 M, pH 8.9), followed by addition of 20l
L 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB; 0.01 M). After shaking the reaction mixture, the absorbance was measured at 412 nm within 5 min of DTNB addition against a blank with no homoge-nate. The GSH concentration was determined using a standard curve and expressed asl
g GSH/g wet tissue.2.7. Malondialdehyde assay
layer was determined at 535 and 520 nm, and the optical density difference between the two determinations was calculated (as the tert-butyl alcohol value). The MDA concentration was expressed as nmol MDA/g tissue.
2.8. Effects of Dize on acetic acid-induced gastric damage
Ulcers were induced using a previously described model[32,33] with some modifications. Initially, the animals were anesthetized with a mixture of xylazine hydrochloride (5 mg/kg) and ketamine (60 mg/kg), injected i.p. Then, a midline laparotomy was made to gently exteriorize the stomach. Acetic acid 40% (100
l
L/1 min) was administered in the serosa of the stomach, and a 4-mm2 holder was then used to deposit acetic acid; all animals were exposed to the same injury. Afterward, the fluid was aspirated off carefully, and the area that remained in contact with acid was gently rinsed with saline. All mice were allowed to recover from anesthesia and returned to their cages. Animals were treated from the second to seventh day after injury. The groups were treated with saline; Dize (7 mg/kg); A-779 (5 mg/kg) + Dize (7 mg/kg); or omeprazole (10 mg/kg). At the end of the seventh day, the animals were euthanized, and the stomach was removed and immediately opened to perform macroscopic analysis. Furthermore, tissues were removed for histological and immunohistochemistry analy-ses and evaluation of myeloperoxidase (MPO) activity, collagen levels, and cytokine levels.2.9. MPO activity
MPO activity, an index of polymorphonuclear cell accumulation into various tissues, including the gastrointestinal tract, was mea-sured according to the method previously described [34]. The results were reported as MPO units (UMPO)/mg tissue. A unit of MPO activity was defined as that sufficient for converting 1
l
mol H2O2to water in 1 min at 22°C.2.10. Immunohistochemistry of the Mas receptor
The samples from the stomachs of mice undergoing acetic acid-induced gastric damage were assessed for the expression of the Mas receptor by employing an immunohistochemical technique. The slides mounted from the paraffin blocks were deparaffinized and then hydrated. Endogenous peroxidase activity was blocked with 1% H2O2diluted in methanol. Then, the slides were washed in phosphate-buffered saline (PBS). Next, the slides were incubated with the primary antibody (rabbit anti-Ang-1–7; 1:100 dilution) overnight at 4°C. After washing, the slides were incubated with
the biotinylated secondary antibody, diluted in PBS containing bovine serum albumin (BSA). Negative control sections were pro-cessed simultaneously as described above, but without the addi-tion of an antibody. Finally, the tissue was stained for antigen– antibody complexes using a peroxidase detection system and then viewed under a microscope[35].
2.11. Concentration of Ang-(1–7) in the gastric mucosa
The concentrations of Ang-(1–7) in gastric tissue were mea-sured using ELISA kits (R&D Systems Inc., MN, USA) according to the manufacturer’s instructions[36]. Briefly, the plate was homog-enized mechanically, and the samples were kept under refrigera-tion. The homogenized samples were then centrifuged at 10,000gfor 10 min at 4°C. The supernatant was then removed,
and the level of Ang-(1–7) was measured using an ELISA plate reader. Samples of both types of gastric lesions were selected.
2.12. Determination of collagen levels
Collagen levels in the segmented stomach tissue were mea-sured as previously described[37]. Samples of glandular stomach were fixed in 10% neutral-buffered formalin solution for 24 h. After fixation, the samples were transferred to a solution of 70% alcohol, sectioned, and embedded in paraffin. Five-micrometer-thick sec-tions were placed on slides and incubated for 1 h for adequate adhesion of the cutting sections. The sections were then deparaf-finized in xylene, hydrated in water, and incubated in 0.1% Picrosir-ius Red colorant for 1 h at ambient temperature. Samples were then washed with distilled water and stained with hematoxylin. Finally, the sections were examined using polarized light microscopy.
2.13. Cytokine measurements
The levels of tumor necrosis factor (TNF)-
a
, interleukin (IL)-6, and (IL)-10 were determined using ELISAs, as previously described [38]. ELISA results were expressed as pg cytokine/mL reaction mixture.2.14. Statistical analysis
All values are expressed as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) and Student–Newman–Keuls tests were used to determine the significance of differences between groups. For histological assessment, the Kruskal–Wallis nonparametric test was used, followed by Dunn’s test for multiple comparisons. Differences were considered to be significant when P< 0.05.
3. Results
3.1. Effects of Dize on ethanol-induced gastric damage
In the present study, we confirmed that treatment of mice with ethanol led to severe macroscopic gastric damage (29.02 ± 6.11 mm2). Interestingly, Dize pretreatment significantly prevented the ethanol-induced macroscopic gastropathy in a dose-dependent manner (Fig. 1A;P< 0.005). The maximal effective dose of Dize was 7 mg/kg, which led to 94% lesion inhibition in the stomach. Because this dose of Dize afforded the highest protection against gastric lesions induced by ethanol, this dose was selected to study the possible mechanisms of action involved in Dize-mediated gastroprotection and the role of the ACE II/Ang-(1–7)/ Mas receptor axis in this mechanism.
3.2. Evaluation of the role of Ang-(1–7)/Mas receptor axis in the gastroprotective effects of Dize
To assess the contribution of the Ang-(1–7)/Mas receptor axis in mediating the protective effects of Dize, other groups of mice were pretreated with A-779, a selective antagonist for the Mas receptor (Fig. 1C). As shown in Figs. 1C and 2, A-779 reversed the gastroprotective effects of Dize against ethanol-induced macroscopic and microscopic gastric damage. This result showed that the Ang-(1–7)/Mas receptor axis may be associated with the gastroprotective effects of Dize.
3.3. Histological evaluation of ethanol-induced gastric damage
The gastroprotective effects of 7 mg/kg Dize were confirmed by histological analysis (Fig. 2). Microscopic analysis revealed that ethanol administration disrupted the integrity of the gastric mucosa, resulting in excessive loss of epithelial cells, rupture of mucosa surface, and enhanced hemorrhage (Fig. 2C and D). How-ever, administration of Dize maintained the integrity of the mucosa
Fig. 1.Diminazene aceturate reduced ethanol-induced gastric damage. (A) Macroscopic analysis of the gastric lesions induced by ethanol and pretreatment with different doses of Dize. (B) Effects of Dize (7 mg/kg) on levels of the peptide Ang-(1–7) in gastric damage. (C) Effects of Dize (7 mg/kg) and the role of ACE II/Ang-(1–7)/Mas on macroscopic gastric lesions were analyzed by ANOVA followed by Newman–Keuls post hoc test. Significant differences:WP< 0.005 versus the saline group;*P< 0.005 versus
the ethanol group;#P< 0.005 versus the Dize-treated group.
Fig. 2.Photomicrographs of the gastric mucosa (magnifications: 100and 400for the first and second lines, respectively). (A and B) Control (saline). (C and D) Mice
administered 50% ethanol (0.5 mL/25 g). (E and F) Mice pretreated with Dize (7 mg/kg). (G and H) Mice pretreated with A-779 and Dize (7 mg/kg) and treated with 50% ethanol. (I and J) Mice treated with omeprazole (standard drug) and 50% ethanol. (G–J) Quantitative results (scores) of microscopic analysis of the lesions induced by ethanol (scores for hemorrhage, edema, loss of epithelium, and infiltration, respectively). Scale bar = 50lm. Data are presented as mean ± SEM of 5–6 mice per group. Results were analyzed by Kruskal–Wallis nonparametric tests, followed by Dunn’s test for multiple comparisons. Significant differences:*P< 0.05 versus the ethanol group;#P< 0.05
versus the Dize-treated group.
(Fig. 2E and F). Administration of A-779 reversed the gastroprotec-tive effects promoted by Dize (Fig. 2G and H). Furthermore, analy-sis of microscopic scores showed that Dize decreased hemorrhagic damage, edema, and epithelial cell loss induced by ethanol (Fig. 2K–M). Thus, analysis of macroscopic and microscopic find-ings revealed an excellent correlation, confirming the efficacy of the compound.
3.4. GSH and MDA levels
Treatment with 50% ethanol significantly decreased GSH con-centrations (187.8 ± 13.3
l
g/g tissue) and increased MDA levels (327.0 ± 44.8 mmol/g tissue) when compared with the control group (P< 0.05), as shown in Fig. 3. Pretreatment with Dize (7 mg/kg) significantly increased the levels of the endogenous antioxidant GSH (403.3 ± 23.9l
g/g tissue), restoring the effects of ethanol (Fig. 3A). The administration of Dize (72.2 ± 1 0.7 mmol/g tissue) also significantly reduced the concentration of gastric mucosal MDA induced by ethanol (Fig. 3B). Moreover, A-779 reversed the gastroprotective effects of Dize (Fig. 3), signifi-cantly decreasing the levels of GSH (279.9 ± 9.1l
g/g tissue) and increasing those of MDA (481.1 ± 39.0 mmol/g tissue).3.5. Effects of Dize on acetic acid-induced gastric damage
As shown inFig. 4B and F, serosal application of acetic acid in the animals resulted in extensive gastric lesions on the seventh day of ulcer induction (31.9 ± 4.9 mm2). In contrast, administration of Dize (Fig. 4C and F) significantly reduced the ulceration area
(11.5 ± 1.9 mm2; P< 0.0001). Moreover, pretreatment with A-779 reversed the beneficial effects of Dize and caused a significant increase in the ulceration area (89.5 ± 2.2 mm2; Fig. 4D and F). The group treated with omeprazole also exhibited a significantly reduced ulceration area (16.1 ± 2.8 mm2), as shown inFig. 4E and F. Next, we evaluated the levels of Ang-(1–7) in acetic acid-induced gastric lesions (Fig. 4G). On the seventh day after acetic acid administration, there were no changes in the levels of Ang-(1–7) when compared to those in the saline group. However, Dize administration resulted in obviously increased Ang-(1–7) levels.
3.6. Analysis of regeneration and re-epithelialization in acetic acid-induced gastric damage
The gastric damage induced by acetic acid produced an average regeneration area of 78.93 ± 16.65
l
m after 7 days (Fig. 5 and Table 1). On the other hand, daily treatment with Dize resulted in a significant acceleration of ulcer healing (156.08 ± 25.32l
m; curative ratio of 97.74%) because ulcer areas (Table 1), which were confirmed histologically (Fig. 5C and D), significantly decreased compared with those in the acetic acid group (P< 0.0005). The administration of A-779 inhibited the protective effects of Dize on accelerated ulcer healing (65.24 ± 10.73l
m). Omeprazole also reduced the area of injury to 142.16 ± 10.23l
m compared to that in the acetic acid group (P< 0.0005). This result showed that Dize could accelerate ulcer healing (Fig. 5).3.7. Determination of collagen levels
Picrosirius red is a well-known stain that is used to detect col-lagen in histology. The results obtained for the determination of collagen in acetic acid-induced gastric lesions showed that saline-treated mice exhibited an extensive layer of collagen type I immediately below the basal lamina, as shown inFig. 6A and B. However, administration of acetic acid reduced the levels of colla-gen significantly when compared with the saline group (Fig. 6C and D). The groups pretreated with Dize (7 mg/kg) exhib-ited significantly increased collagen type I levels (Fig. 6E and F). In contrast, treatment with A-779 significantly decreased collagen type I levels when compared with the levels in Dize-treated mice. Furthermore, A-779 treatment increased the levels of collagen type III, a type of immature collagen that has less rigid fibers compared with collagen type I (Fig. 6G and H). These results showed that Dize could promote the maturation of type III collagen into type I colla-gen more rapidly, thus promoting gastric healing.
3.8. Immunohistochemistry of the Mas receptor
The gastric tissues obtained from the acetic acid-induced gastric ulcer model were used for immunohistochemical localization of the Mas receptor. In the control group (saline alone), the expres-sion of the Mas receptor (Fig. 7A) was evident in the gastric mucosa, and immunostaining was observed primarily in mucosal cells as well as some parietal cells. However, acetic acid treatment reduced the levels of Mas receptor, as observed by immunostaining (Fig. 7B). On the other hand, in animals treated with Dize, the Mas receptor was obviously expressed in mucosal cells (Fig. 7C).
3.9. MPO activity following acetic acid-induced gastric damage
In this study, we observed significantly increased levels of MPO in the stomach (30.9 ± 4.8 U/mg tissue) of acetic acid-treated mice compared to those in the control group (Fig. 8). However, pretreat-ment with 7 mg/kg Dize significantly attenuated the acetic acid-induced increase in MPO activity in the stomach (2.3 ± 1.7 U/mg of tissue). Therefore, Dize may protect the gastric mucosa by
Fig. 3.Antioxidant effects of Dize in ethanol-induced gastric damage. (A) Effects of Dize (7 mg/kg) on GSH levels in gastric damage. (B) Effects of Dize (7 mg/kg) on MDA concentrations in gastric damage. Data are presented as mean ± SEM of 5–6 mice per group. Results were analyzed by ANOVA followed by Newman–Keuls post hoc test. Significant differences:W
P< 0.05 versus the saline group;*P< 0.05 versus
reducing the recruitment of leukocytes, thereby hindering super-oxide anion production. However, blocking of the Mas receptor by A-779 significantly decreased the protective effects of Dize on MPO activity (12.9 ± 12.8 U/mg tissue).
3.10. Levels of TNF-
a
, IL-6, and IL-10As shown inTable 2, both IL-6 and TNF-
a
levels decreased fol-lowing treatment with Dize (7 mg/kg). In saline-treated mice, TNF-a
and IL-6 levels were 851.4 ± 68.0 and 6.3 ± 1.6 pg/mL, respectively. In contrast, the levels of TNF-a
and IL-6 in the acetic acid group significantly increased (1911.0 ± 314.4 and 14.8 ± 3.1 pg/mL, respectively) compared to those in the salineFig. 4.Effects of Dize on gastric lesions induced by acetic acid and the role of the ACE II/Ang-(1–7)/Mas pathway. (A) Control group (saline). (B) Positive control (40% acetic acid), which presents a severe ulcer, with minimal tissue healing and regeneration. (C) Mice treated with Dize (7 mg/kg). (D) Mice pretreated with A-779 and Dize (7 mg/kg), followed by treatment with 40% acetic acid. (E) Mice treated with omeprazole and 40% acetic acid. (G) Effects of Dize on the macroscopic features of gastric lesions induced by acetic acid. (G) Effects of Dize (7 mg/kg) on the levels of Ang-(1–7) in acetic acid-induced gastric damage. Results were analyzed by ANOVA, followed by Newman–Keuls post hoc test. Significant differences:W
P< 0.0001 versus the saline group;*P< 0.0001 versus the ethanol group;#P< 0.0001 versus the Dize-treated group.
Fig. 5.Histopathological analysis of lesions induced by acetic acid (serosal side and mucosal side, first and second lines respectively). (A and B) Mice administered 40% acetic acid. (C and D) Mice pretreated with Dize (7 mg/kg). (E and F) Mice pretreated with A-779 and Dize (7 mg/kg), followed by treatment with 40% acetic acid. (G and H) Mice treated with omeprazole.*Reorganization area and re-epithelialization at the edge of the lesion (100
). Scale bar = 50lm.#Glandular disarray area with ulcer on the surface
of the mucosa and neutrophilic infiltrate (100).
Table 1
Histopathological analysis of the gastric lesions induced by acetic acid. Treatments Histological analyses
Regeneration area Curative ratio (lm) (%) Acetic acid 78.93 ± 16.65 – Dize + acetic acid 156.08 ± 25.32⁄ 97.74
A-779 + Dize + acetic acid 65.24 ± 10.73# –
OMP + acetic acid 142.16 ± 10.23⁄ 80.10
* P< 0.0005 compared to harmful group (acetic acid).
# P< 0.0005 compared to the group that was treated with Dize (no antagonist).
group (P< 0.05). Moreover, treatment with Dize resulted in a significant reduction (P< 0.05) in the levels of the two cytokines (TNF-
a
: 971.5 ± 47.9 pg/mL; IL-6: 7.2 ± 0.5 pg/mL). Acetic acid administration caused a concomitant decrease in IL-10 level, which was unaffected by Dize. These results showed that Dize could decrease the levels of TNF-a
and IL-6 in acetic acid-induced gas-tropathy (Table 2), similar to the effects of omeprazole.4. Discussion
In the present study, we investigated the potential application of Dize as a new pharmacotherapeutic target for the treatment of gastric ulcers using an ethanol-induced acute gastric ulcer model [28,39] and an acetic acid-induced chronic gastric ulcer model [31]. In addition, we examined the role of the Ang-(1–7)/Mas receptor axis in gastroprotection, which may provide important insights into pharmacological therapies since studies on the action of Ang-derived metabolites in the gastrointestinal tract are lacking. Recently, several studies have shown that Dize influences a variety of physiological conditions[40]. For example, Dize has been shown to activate ACE II and thus stimulate the protective pathway of the RAS, leading to the cleavage of the octapeptide Ang II[23].
Fig. 6.Dize increased the levels of collagen type I in gastric lesions induced by acetic acid. In the first line, the samples are shown in natural light, and in the second line, samples are shown in polarized light. (A and B) Saline group (negative control). A wide layer of collagen type I was observed (full arrow). (C and D) Mice administered 40% acetic acid (positive control). (E and F) Mice treated with Dize (7 mg/kg). Restoration of collagen type I levels was observed (running arrow). (G and H) Mice administered A-779. Lower levels of collagen type I and high levels of collagen type III (dotted arrow) were observed.
Fig. 7.Immunohistochemical analysis of the Mas receptor on the gastric mucosa (magnification: 400). (A) Mice treated with saline. (B) Mice treated with acetic acid. (C)
Mice treated with Dize (7 mg/kg). The arrows indicate the gastric mucosa.
Fig. 8.Effects of Dize on MPO activity in gastric lesions induced by acetic acid. Results were analyzed by ANOVA followed by Newman–Keuls post hoc test. Significant differences:WP< 0.0005 versus the saline group;*P< 0.0005 versus the
ethanol group;#P< 0.05 versus the Dize-treated group.
Table 2
Measurements of inflammatory cytokines.
Treatments Analysis of inflammatory cytokines TNF-a IL-6 IL-10 Saline 851.4 ± 68.02 6.3 ± 1.6 226.8 ± 33.6 Acetic acid 1911.0 ± 314.4⁄ 14.8 ± 3.1⁄ 94.5 ± 19.3⁄
Dize + acetic acid 971.5 ± 47.97# 7.2 ± 0.5# 95.8 ± 9.2
OMP + acetic acid 869.4 ± 84.94# 6.1 ± 2.1# 114.7 ± 36.4
Results expressed as mean ± SEM. Statistical differences:*when compared to the
saline group;#compared to the positive control group (acetic acid). ANOVA was
ACE II metabolizes Ang II to Ang-(1–7), which in turn will bind to the G protein-coupled receptor, Mas receptor[41], thereby regulat-ing the deleterious effects of Ang II. Consistent with this, chronic administration of Dize has been shown to prevent and arrest pulmonary hypertension in experimental models of lung injury [42]. Furthermore, increased expression ofACE IImRNA has been observed in rats with bleomycin and monocrotaline (MCT)-induced pulmonary hypertension treated with Dize[43]. Addition-ally, Dize decreases ACE activity, with consequent decrease of Ang II, contributing to their beneficial effects[44]. Thus, Dize has been shown to have protective effects under various physiological con-ditions, including acute myocardial infarction [45], uveitis [27], ischemic stroke [43], hyperglycemia [46], erectile dysfunction [47], metabolic disorders [48], and abdominal aortic aneurysm [49]. Thus, Dize may have potential for treatment of several dis-eases or conditions, including gastric ulcers[41]by activation of the ACE II/Ang-(1–7)/Mas receptor pathway. There are many mechanisms through which gastric protection occurs, and activa-tion of ACEII/Ang-(1–7)/Mas axis is one of them.
Consistent with the previous findings [28,50], our results showed that administration of 50% ethanol caused macroscopic and microscopic gastric damage characterized by submucosal edema, intense hemorrhage, and loss of epithelial cells. However, pretreatment with Dize prevented gastric damage and maintained the integrity of the gastric mucosa against ethanol-induced gas-tropathy. When animals were treated with A-779, the protective effects of Dize were reversed, as shown in other studies[51]. The pathogenesis of ethanol-induced gastric mucosal damage is a mul-tifactorial process that depends on the imbalance between the aggressive and protective factors induced by the direct or indirect action of mediators, such as lipoxygenase and oxygen-derived free radicals, with increased lipid peroxidation and reduced non-protein sulfhydryl groups [50,52,53]. Ethanol rapidly penetrates the gastric mucosa; promotes injury characterized by membrane damage, erosive hemorrhage, and ulcer formation via destruction of the mucus barrier; and increases vascular permeability [54,55]. Moreover, in our study, ethanol increased the levels of Ang-(1–7); however, pretreatment with Dize caused a greater increase in Ang-(1–7) levels than that in the ethanol group. This increase of Ang-(1–7) by ethanol alone may be a mechanism of defense against the ethanol-induced gastric damage to improve resistance to gastric mucosa injury.
Recent studies have identified Ang I and II metabolites in the GI tract[17], and Ang-(1–7) has been recognized as the main product of Ang I conversion during endogenous synthesis in the rat stomach [13]. Ang-(1–7) has profound physiological effects on the vasculature and therefore could be considered as an important component of the GI defense system. Since the injured gastric mucosa produces an abundant amount of Ang-(1–7), Ang-(1–7) may protect the gastric mucosa and help restore the mucosa after damage [51]. Thus, drugs that enhance the levels of Ang-(1–7), such as Dize, may be excellent candidates for therapy of gastric ulcers.
GI diseases, including damage induced by ethanol, involve oxidative stress produced via the generation of free radicals and lipid peroxidation[56,57]. Therefore, it is possible that the effects of Dize on the Ang-(1–7)/Mas receptor pathway decrease the redox state in ethanol-induced gastropathy. Our results showed that administration of Dize reversed the decrease in gastric GSH levels after ethanol administration, whereas A-779 partially reversed these protective effects, suggesting the involvement of other path-ways in reducing the effectiveness of A-779. GSH is a tripeptide (L-c-glutamyl-L-cysteinyl-glycine) that acts mainly as an endoge-nous antioxidant [58]. GSH administration also decreases ethanol-induced gastric damage by inactivating reactive oxygen species and products of lipid peroxidation[47]. Alternatively, the
increase in GSH levels could be secondary to a decrease in free rad-ical production. Our results demonstrated that Dize treatment resulted in a significant decrease in MDA concentrations in ethanol-induced gastropathy. Recent studies have demonstrated that Ang-(1–7) can effectively regulate lipid metabolism and can help in the formation of cellular lipid bilayer[59,60]. In our study, the inhibition of Ang-(1–7) by A-779 also increased MDA levels induced by ethanol, since it inhibits an endogenous protection favoring the effect of ethanol on lipid peroxidation. Thus, the mechanism through which Dize exerts its gastroprotective effects could involve the reduction of ethanol-induced lipid peroxidation in the gastric mucosa.
In this study, we also investigated the effects of Dize via the Ang-(1–7)/Mas receptor pathway in inflammation and the repara-tive action against acetic acid-induced chronic gastric mucosal damage. Acetic acid-induced ulcers in rodents have been reported to be similar to clinical ulcers in terms of location, chronicity, and severity and serve as the most reliable model to study the ulcer healing process[61]. Our results showed that, in addition to pro-tecting the gastric mucosa against acute gastric lesions, treatment with 7 mg/kg Dize also accelerated the healing of chronic ulcers in a manner similar to that of omeprazole. Moreover, this effect was dependent on activation of the Mas receptor. Additionally, histo-logical analysis revealed that mice treated with Dize had the ability to regenerate the gastric mucosa.
The process of healing involves a coordinated cascade of events that result in tissue reconstitution. The healing process is common to all wounds and is independent of the agent that caused the wound. Didactically, healing is divided into three phases: inflam-mation, proliferation or granulation, and remodeling or matura-tion. In the final phase, collagen plays a key role in tissue reorganization in the mucosa. Collagen is the most abundant pro-tein involved in the healing phase of connective tissues and is orga-nized in a thick, dynamic net owing to its constant deposition and reabsorption[62,63]. The wound scar is a result of the interactions among tissue synthesis, degradation, and remodeling. The results obtained in this study showed marked deposition of collagen type I, also called mature collagen, in the control group. However, acetic acid administration resulted in a significant loss of collagen depo-sition, demonstrating an absence of epithelial regeneration and reorganization. In contrast, Dize pretreatment reversed the effects of acetic acid and caused significant deposition of collagen type I, whereas administration of A-779 resulted in accumulation of colla-gen type III, also called immature collacolla-gen, indicating a delay in the healing process. This result showed that Dize increased the deposi-tion and maturadeposi-tion of collagen fibers via Mas activadeposi-tion. Indeed, Ang II and its metabolites have been shown to stimulate collagen production, increase collagen I and III contents, and upregulate levels of collagen types I and III transcripts in cardiac fibroblasts [64]and vascular smooth muscle cells[65]. Collagen type III has little stiffness and low sustainability; therefore, maturation into collagen type I is necessary[36]. Our results are consistent with previous studies[66]showing that normal tissues consist of about 90% collagen type I and 10% collagen type III, but that collagen type III is predominant following injury in the granulation phase and undergoes remodeling over time during the tissue repair process [66].
This study is the first report describing the localization of Mas in the stomach. However, other studies have demonstrated Mas expression and localization in several organs, including the kidney, vascular endothelium, retina, ciliary body, and epithelial and suprabasal cells[67]. In the heart, for example, Mas expression is regulated by various stimuli, and the stage of diseases and the ACE II/Ang-(1–7)/Mas axis can be modulated at the receptor level by altering Mas expression in response to different pathophysio-logical conditions.
Acetic acid-induced gastric lesions are associated with inflammation in the mucosa and involve changes in the levels of prostaglandins, growth factors, nitric oxide, cytokines, and mucus [68]. Similarly, acetic acid-induced neutrophil accumulation and subepithelial edema in the gastric mucosa have been shown to play major roles in the development of ulcers[32,33,69]. In fact, in the present study, we observed increases in neutrophil migra-tion and in the levels of the pro-inflammatory cytokines TNF-
a
and IL-6 in the gastric mucosa of mice treated with acetic acid. Additionally, we found that Dize inhibited the acetic acid-induced elevation of MPO activity and increase of TNF-
a
and IL-6 levels, suggesting that the gastroprotective effects of Dize may be dependent on its inhibitory effects on neutrophil infiltration and the neutrophil-associated cytokine response. In fact, several stud-ies have shown that Dize interferes with cell migration at the site of inflammation; this protective effect is associated with decreased pro-inflammatory cytokines, such as TNF-a
, IL-6, and IL-12 [41]. Other studies have confirmed that Ang-(1–7) decreases the expres-sion and release of the pro-inflammatory cytokines IL-1bandTNF-a
, which are known to impair gastric defense[70], suggesting that the anti-inflammatory properties of Ang-(1–7) could contribute to the protective effects of this Ang I metabolite in the rat stomach.IL-10 is one of the most important anti-inflammatory and immunosuppressive cytokines produced by various cells including monocytes/macrophages and T lymphocyte[71]. In our study, after the induction of gastric ulcer by acetic acid, IL-10 level decreased and Dize did not alter this harmful effect. This could be because when the gastric mucosa was damaged by acetic acid, T and B lymphocytes in the submucosa beneath the damaged area that typically produce basal level of IL-10, were also damaged. The macrophages were actually located beyond the damaged area; therefore, the macrophages survived. The survived macrophages could stimulate the release of additional TNF-
a
in response to acetic acid-induced injury.In conclusion, our findings showed that Dize prevented ethanol-and acetic acid-induced gastric damage in mice. Although there are many mechanisms through which this effect can occur, our data supported the hypothesis that the activation of the Ang-(1–7)/ Mas receptor axis was of primary importance in this pathway. Moreover, our results indicated that the gastroprotective effects of Dize were mediated, at least in part, by its antioxidant and anti-inflammatory activities and involved improvement of wound healing and re-epithelialization of the gastric mucosa. These obser-vations also suggested that Dize and the Ang-(1–7)/Mas receptor axis may be involved in enhancing the resistance of the gastric mucosa to damage. Further studies are needed to confirm this hypothesis.
Conflict of interest
The authors declare that there are no conflicts of interest.
Ethical approval
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
Acknowledgments
The authors gratefully acknowledge the financial support from the National Counsil of Technological and Scientific Development – CNPq (Brazil) and Research Foundation for the State of Piauí – FAPEPI. Dr. Medeiros is recipient of CNPq fellowships.
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