Dig Dis Sci (2011) 56:314–322 DOI 10.1007/s10620-010-1300-8
ORIGINAL ARTICLE
Role of Capsaicin-Sensitive Primary Afferent Neurons and Non-protein Sulphydryl Groups on Gastroprotective Effect of Amifostine Against Ethanol-Induced Gastric Damage in Rats Jeroˆnimo Junqueira-Ju´nior • Ana Fla´via Torquato Arau´jo Junqueira • Jand Venes R. Medeiros • Sergio Henrique Brito Barbosa • Ana Carolina Pereira Nogueira Jose´ Maurı´cio Segundo Correia Mota • Ana Paula Maceˆdo Santana • Gerly Anne C. Brito • Ronaldo A. Ribeiro • Roberto Ce´sar P. Lima-Ju´nior • Marcellus H. L. P. Souza
•
Received: 28 October 2009 / Accepted: 3 June 2010 / Published online: 16 June 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Background Amifostine has been widely tested as a cytoprotective agent against a number of aggressors in different organs. Recently, a gastroprotective effect was observed for this drug in a model of indomethacin-induced gastric injury. Our objective was to investigate the effect of amifostine on ethanol-induced gastric injury and the role played in this mechanism by afferent sensory neurons, nonprotein sulfhydryl groups, nitric oxide, ATP-sensitive potassium channels, and cyclooxygenase-2. Methods Rats were treated with amifostine (22.5, 45, 90, or 180 mg/kg, PO or SC). After 30 min, the rats received absolute ethanol (5 ml kg-1, PO). One hour later, gastric damage was quantified with a planimeter. Samples from the stomach were also taken for histopathological assessment and for assays of non-protein sulfhydryl groups. The other groups were pretreated with L-NAME (10 mg kg-1, IP),
glibenclamide (10 mg kg-1, PO), or celecoxib (10 mg kg-1, PO). After 30 min, the animals were given amifostine (90 mg kg-1, PO or SC), followed 30 min later by gavage with absolute ethanol (5 ml kg-1). Other rats were desensitized with capsaicin (125 mg kg-1, SC) 8 days prior to amifostine treatment. Results Amifostine administration PO and SC significantly and dose-dependently reduced ethanol-induced macroscopic and microscopic gastric damage by restoring glutathione levels in the stomach mucosa. Amifostine-promoted gastroprotection against ethanol-induced stomach injury was reversed by pretreatment with neurotoxic doses of capsaicin, but not by L-NAME, glibenclamide, or celecoxib. Conclusions Amifostine protects against ethanol-induced gastric injury by increasing glutathione levels and stimulating the afferent sensory neurons in the stomach. Keywords Amifostine Ethanol Afferent sensory neurons GSH Gastric defense
J. Junqueira-Ju´nior A. F. T. A. Junqueira J. V. R. Medeiros S. H. B. Barbosa A. C. P. Nogueira J. M. S. C. Mota A. P. M. Santana G. A. C. Brito R. A. Ribeiro R. C. P. Lima-Ju´nior M. H. L. P. Souza Brazilian Semi-Arid Institute of Biomedicine (INCT-IBISAB), Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceara´, Rua Cel. Nunes de Melo, 1315, Rodolfo Teo´filo, Fortaleza, CE 60430-270, Brazil J. V. R. Medeiros Department of Biology, Federal University of Piaui, Minister Reis Velloso campus, Av. Sa˜o Sebastia˜o, 2819, Parnaiba, PI 64202-020, Brazil M. H. L. P. Souza (&) Centro de Biomedicina, Faculdade de Medicina, Universidade Federal do Ceara´, Fortaleza, CE, Brazil e-mail:
[email protected]
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Introduction Ethanol ingestion is related to the development of gastric mucosal lesions in humans [1, 2]. The mechanisms involved are complex. Some are known to promote gastric damage, such as the release of inflammatory mediators inducing vasoconstriction/ischemia followed by cell death [3]. In addition, gastric blood flow stasis and microvascular disruption lead to hemorrhage and necrotic tissue injury [3]. Furthermore, the healing of concentrated ethanolinduced gastric lesions was reported to be delayed in animals submitted to high-dose capsaicin-induced functional ablation of the sensory neurons (reviewed by Evangelista
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[4]). In contrast, gastroprotective effects against ethanolinduced damage have been observed in rodents treated with ATP-dependent potassium channel openers [5], nitric oxide [6], or hydrogen sulphide donors [7]. Reactive oxygen species appear to contribute to the development of mucosal lesions, while the administration of scavengers promotes gastroprotection, as demonstrated in a number of gastric damage models (reviewed by Kountouras et al. [8]). In a study by Konturek et al. [9], the injection of sulfhydryl-containing cysteamine caused a dose-dependent reduction in absolute ethanol-induced gastric lesions in rodents. The fact that this effect was neutralized by the administration of the sulfhydryl blockers N-ethylmaleimide and indomethacin (a potent inhibitor of cyclooxygenase) suggests the involvement of prostaglandins and endogenous sulfhydryls in the mechanism protecting the gastric mucosa. Amifostine (AMF) (WR-2721) is a cysteamine analogue used clinically for its cytoprotective activity against sideeffects from radiotherapy and chemotherapy including post-irradiation xerostomia, cisplatin-induced neuro/nephrotoxicity, and cyclophosphamide-induced bone marrow suppression [10]. Amifostine is an inactive phosphorylated pro-drug metabolically activated via alkaline phosphatase on the cell membrane to an active dephosphorylated form, WR-1065. Alkaline phosphatase activity is seen primarily in non-tumor cells and contributes significantly to protecting healthy tissue against chemoradiotherapy [11]. In animal inflammation models, AMF beneficially reduced ifosfamide- and acrolein-induced hemorrhagic cystitis in mice [12] and indomethacin-induced gastropathy in rats by increasing gastric NP-SH levels [13]. As noted, the objective of this study was to evaluate the effects of amifostine, if any, on ethanol-induced gastric injury and to identify the possible mechanisms involved.
Methods Animals Male Wistar rats (180–220 g) from the laboratory animal facility of the Federal University of Ceara´ were kept in a temperature-controlled room with free access to water and food. Prior to the experiment, the animals were fasted for 18 h. All study procedures involving animals were in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and approved by the local ethics committee. Drugs Administered The study used absolute ethanol (5 ml kg-1) (Merck, Sa˜o Paulo, Brazil); amifostine (EthyolÒ, Schering-Plough);
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capsaicin diluted in ethanol/Tween 80/PBS solution at 1:1:8; theophylline (Fluka, India) diluted in 0.9% saline; terbutaline (Sigma Chemical Co., St. Louis, MO, USA) diluted in 0.9% saline; glibenclamide dissolved in 0.01 N NaOH containing 4% glucose. Amifostine in Ethanol-Induced Gastric Damage The animals were divided into groups of 6–8 rats each and given sterile saline or amifostine (22.5, 45, 90, or 180 mg kg-1) by gavage (PO) or subcutaneously (SC). After 30 min, absolute ethanol (5 ml kg-1) was administered by gavage, and after 1 h, the animals were euthanized by cervical dislocation. The stomach was excised and the mucosa was carefully exposed by an incision along the greater curvature, pinned out on a platform, and photographed with a planimeter. The hemorrhagic or ulcerative lesions were measured using computer software (Image J, v.1.38, NIH, USA). A sample of the corpus region of each stomach was fixed in 10% formalin for subsequent histopathological assessment. Additional full-thickness sections of the gastric corpus were weighed, frozen, and stored at -70°C until they were assayed for non-protein sulphydryl groups [14]. Histopathological Studies For histological assessment, the glandular stomach was fixed in 10% neutral buffered formalin solution, sectioned, and then embedded in paraffin. Sections 4 lm thick were deparaffinized, stained with hematoxylin-eosin (H&E), and examined under a light microscope. The specimens were then assessed according to the criteria of Laine [2]. Briefly, a 1-cm segment of each histological section was assessed for epithelial cell loss (score: 0–3), edema in the upper mucosa (score: 0–4), hemorrhagic damage (score: 0–4), and presence of inflammatory cells (score: 0–3). Finally, the sections were evaluated by a blinded experienced pathologist (GACB). Non-protein Sulfhydryl Group Assay Non-protein sulfhydryl groups (NP-SH), such as glutathione, promote cytoprotection by preventing free-radical oxidative damage in various tissues, including the gastric mucosa [15]. In this study, NP-SH levels were measured using a previously described assay [14]. Briefly, 50– 100 mg portions of frozen gastric tissue were homogenized in 1 ml 0.02 M EDTA/100 mg tissue. Then, 400-ll aliquots of the homogenate were mixed with 320 ll of distilled water and 80 ll of 50% trichloroacetic acid (TCA) in order to precipitate the proteins. The tubes were centrifuged at 3,000 9 g for 15 min at 4°C. Finally, 400 ll of
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supernatant was mixed with 800 ll 0.4 M Tris buffer (pH 8.9) and 20 ll [5,50 dithiobis-(2-nitro-benzoic acid)] (DTNB, Fluka) and the mixture was shaken for 3 min. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent blank with no homogenate. Tissue levels of NP-SH were reported as lg g-1 tissue.
analysis of variance (ANOVA) followed by the Newman– Keuls test when appropriate. Microscopy scores were expressed as median values and range and analyzed with the Kruskal–Wallis non-parametric test and Dunn’s multiple comparison test. The statistical significance was set at p \ 0.05.
Chemical Ablation of Sensory Afferent Neurons by Capsaicin
Results
Pharmacological Modulation with ATP-Sensitive Potassium Channels (KATP), Nitric Oxide Synthase, or Cycloxygenase-2 Inhibitors In another experimental setting, rats were treated with 10 mg kg-1 of either glibenclamide PO (an ATP-sensitive potassium channel blocker; KATP), L-NAME IP (a nonspecific inhibitor of nitric oxide synthase; NOS) or celecoxib PO (a cycloxygenase-2 inhibitor) 30 min prior to administration with amifostine PO or SC (90 mg kg-1). Thirty minutes after amifostine administration, the animals were given absolute ethanol (5 ml kg-1, PO). In all protocols the effect of the treatment on the gastric lesions was assessed as previously described. Statistical Analysis Findings were expressed as mean values ± SEM for each group of 6–8 rats. The statistical analysis included one-way
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Figure 1 shows that intragastric instillation of absolute ethanol induces macroscopic damage in the stomach (panel A: 74.76 ± 17.45 mm2, panel B: 111.0 ± 25.85 mm2) when compared to the saline-treated animals (panel A: 2.0 ± 1.6 mm2, panel B: 4.2 ± 2.4 mm2). It is also shown that amifostine administered either PO (panel A) or SC (panel B) protected (in a dose-dependent manner) the ethanol-induced macroscopic gastric injury (93.0 and
Macroscopic gastric lesion (mm2)
A
100 80 60 40
*
20
**
**
90
180
0 Sal
Sal
22.5
45
Amifostine p.o (mg kg-1) Absolute Ethanol (5 ml kg-1)
B Macroscopic gastric lesion (mm2)
To evaluate the involvement of the sensory afferent neurons in the gastroprotective effect of amifostine, the rats were injected subcutaneously with neurotoxic doses of capsaicin (25 mg kg-1 plus 2 9 50 mg kg-1 at 12-h intervals) for chemical ablation of sensory afferent neurons, as described by Maggi et al. [16], or Ehrlich et al. [17], and adapted to the conditions of the present experiment. To counteract any respiratory impairment, the animals were injected with terbutaline (0.2 mg kg-1, IP) and theophylline (20 mg kg-1, IM) and lightly anesthetized with tribromoethanol 5% (10 ml kg-1, IP) prior to capsaicin administration. Eight days later, one or two drops of capsaicin solution (10 lg ml-1) were instilled in the eyes and the wiping reflex was observed to make sure the animals had been desensitized. The absence of this reflex was taken as an effective ablation of the primary afferent sensory neurons. As previously described, a control group of naive rats (n = 8) were treated with vehicle or amifostine (90 mg kg-1, PO or SC) prior to gavage with absolute ethanol. Similar treatments were administered to groups of capsaicin-desensitized rats before the intragastric instillation of ethanol. In addition, a saline-treated group (normal control) was included.
160 140 120 100 80 60 40
*
*
90
180
20 0 Sal
Sal
22.5
45
Amifostine SC (mg kg-1) Absolute Ethanol (5 ml kg-1)
Fig. 1 Effect of amifostine PO (panel a) and amifostine SC (panel b) on ethanol-induced gastric injury. The rats were treated with saline (Sal) or amifostine PO or SC. After 30 min, absolute ethanol (5 ml kg-1) was administered. The control group was treated with saline only. Gastric lesions were quantified after 1 h. The results are expressed as mean values ± SEM for each group of five or more rats. * p \ 0.05 in relation to the ethanol group. ** p \ 0.01 in relation to the ethanol group. ANOVA and Newman–Keuls test
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66.7%, respectively, to the highest protection reached either in PO or SC routes). Additionally, Table 1 demonstrates ethanol’s capacity to induce a high degree of epithelial cell loss [3(2–3)], edema formation [3(3–4)], and hemorrhagic erosions [3(3–4)] on the gastric tissue when assessed microscopically. However, we did not observe an increase in inflammatory cells infiltration during ethanol-induced gastropathy. Such observations were significantly reduced (p \ 0.05) in animals given amifostine 90 mg kg-1 PO [1(0–1); 1(0–2); 1(0–2), respectively] or SC [0(0–1); 1(0–1); 1(0–1), respectively] when compared to the ethanol-treated group. Figure 2 showed that ethanol administration (panel B) induced disruption of the gastric gland superficial region with epithelial cell loss (vertical arrow) and intense hemorrhage (head of arrow). We did not observe these alterations in rats pretreated with amifostine 90 mg kg-1 PO (panel C) or SC (panel D). In Fig. 3, we observed that the amifostine protective effects were maximal after 30 min (98 and 73%, respectively, to PO and SC routes) and persisted for at least 24 h (PO: 73% and SC: 71%) independent of the route of amifostine administration, when compared to ethanoltreated animals. In addition, Fig. 4 shows that ethanol was associated with a marked reduction in gastric mucosal NP-SH levels (80.5 ± 18.4 lg g-1 tissue) when compared to salinetreated animals (234.0 ± 26.36 lg g-1 tissue), which was dose-dependently prevented (p \ 0.05) with amifostine PO (panel A) or SC (panel B) treatments (229.6 ± 19.8 and 247 ± 10.5 lg g-1 tissue, respectively, to the highest protection reached either to PO or SC routes). Chemical ablation of the sensory afferent neurons neutralized the gastroprotective effect of amifostine PO and Table 1 Protective effects of amifostine (22.5, 45, or 90 mg kg-1) on ethanolinduced microscopic damage in gastric mucosa
Groups (n = 7) Saline
SC on ethanol-induced macroscopic (Fig. 5; 51 and 36%, respectively) and microscopic gastric injury (Table 2). However, there was no change in ethanol ability to induce gastric damage in the desensitized rats (p [ 0.05). Likewise, pretreatment with neurotoxic doses of capsaicin decreased NP-SH levels in gastric tissue of amifostinetreated animals (123.1 ± 18.2 and 125.4 ± 15.0 lg g-1 tissue, respectively, to PO and SC routes) compared to nondesensitized controls receiving amifostine (PO: 248.3 ± 20.79; SC: 224.6 ± 19.68 lg g-1 tissue) (Fig. 6). Pretreatment with glibenclamide, celecoxib, or L-NAME did not reverse the gastroprotective effects of amifostine PO (Fig. 7, panel A) or SC (Fig. 7, panel B) in ethanol-induced macroscopic gastric injury when compared with ethanoltreated group (p [ 0.05).
Discussion In this study, amifostine was shown to have a dose-dependent gastroprotective effect on ethanol-induced gastric lesions both macroscopically and microscopically, whether administered by gavage or by subcutaneous injection. This protective effect lasted up to 24 h after amifostine administration. The significant reduction observed in edema, hemorrhagic scores, and the preservation of stomach epithelium in samples from animals treated with amifostine indicate a protective action of this agent. Ethanol administration is known to increase the levels of inflammatory mediators and induce neutrophil migration to the gastric mucosa as observed in several rat models of ethanol-induced gastric injury [18–20]. However, the protocol used in the present study differed from those in the time of sacrifice post-ethanol administration and the dose
Epithelial cell loss (score 0–3) 0
Inflammatory cells (score 0–3) 0
Edema (score 0–4) 0
Hemorrhagic damage (score 0–4) 0
Ethanol
3 (2–3)
0
3 (3–4)
3 (3–4)
Ethanol ? amifostine
2 (1–2)
0
3 (2–4)
2 (2–4)
1 (1–3)
0
2 (1–2)
1 (1–3)
1 (0–1)*
0
1 (0–2)*
1 (0–2)*
2 (1–3)
0
2 (1–3)
3 (3–4)
1 (0–1)
0
1 (1–3)
2 (0–2)
0 (0–1)*
0
1 (0–1)
1 (0–1)*
22.5 mg kg-1 PO Ethanol ? amifostine 45 mg kg-1 PO The table shows median values followed by minimum and maximum scores (in brackets)
Ethanol ? amifostine
* p \ 0.05 in relation to the ethanol group
22.5 mg kg-1 SC
The Kruskal–Wallis nonparametric test, followed by Dunn’s test, were used for multiple comparisons in the histological assessment
90 mg kg-1 PO Ethanol ? amifostine Ethanol ? amifostine 45 mg kg-1 SC Ethanol ? amifostine 90 mg kg-1 SC
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Fig. 2 Photomicrographs of gastric mucosa (magnifications, 9100). a Control (saline); b absolute ethanol, showing disruption of gastric gland superficial region with epithelial cell loss, intense hemorrhage; c absolute ethanol ? amifostine 90 mg kg-1, PO, d absolute ethanol ? amifostine 90 mg kg-1, SC
Macroscopic gastric lesion (mm2)
180 160 140 120 100 80
*
60 40 20
*
*
*
*
p.o 24h
s.c 30min
s.c 6h
**
0 Sal
Sal
p.o 30min
p.o 6h
s.c 24h
Amifostine (90 mg kg-1) Absolute Ethanol (5 ml kg-1)
Fig. 3 Effect of amifostine PO and amifostine SC on ethanolinduced gastric injury. The rats were treated with saline (Sal) or amifostine PO or SC. Thirty minutes, 6 h, or 24 h later, absolute ethanol (5 ml kg-1) was administered. The control group was treated
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with saline only. Gastric lesions were quantified after 1 h. The results are expressed as mean values ± SEM for each group of five or more rats. * p \ 0.05 in relation to the ethanol group. ** p \ 0.01 in relation to the ethanol group. ANOVA and Newman–Keuls test
Dig Dis Sci (2011) 56:314–322
Control group
300
# #
250
Sensory afferent neurons ablated by capsaicin
#
Macroscopic gastric lesion (mm2)
NP-SH (μg per g tissue)
A
319
200 150
*
100 50
100 80
#
60
ψ
40 20
*
0
0 Sal
Sal
22.5
45
90
Sal
180
Amifostine PO (mg kg-1)
Sal
*
Sal Amf PO (90 mg kg -1) Amf SC (90 mg kg -1) Absolute Ethanol (5 mL kg-1)
-1
Absolute Ethanol (5 ml kg )
NP-SH (μg per g tissue)
B
300
#
#
90
180
250 200 150
*
100 50
Fig. 5 Reduction of ethanol-induced gastric damage by amifostine PO or SC mediated by sensory afferent neurons. The rats were treated with neurotoxic doses of capsaicin. After 8 days, the desensitized animals were treated with saline (Sal) or amifostine (90 mg kg-1) PO or SC. Thirty minutes later, absolute ethanol (5 ml kg-1) was administered. Gastric lesions were quantified after 1 h. The gastroprotective effect of amifostine PO or SC was inhibited by capsaicininduced ablation of the sensory afferent neurons. The results are expressed as mean values ± SEM for each group of five or more rats. * p \ 0.01 in relation to the ethanol group. # p \ 0.05 in relation to the amifostine p.o. group. W p \ 0.05 in relation to the amifostine s.c. group. ANOVA and Newman–Keuls test
0 Sal
Sal
22.5
45
Amifostine SC (mg kg-1) Absolute Ethanol (5 ml kg-1) Fig. 4 Effect of amifostine PO (panel a) and amifostine SC (panel b) on ethanol-induced decrease in nonprotein sulfhydryl (NP-SH) levels. The rats were treated with saline (Sal) or amifostine PO or SC. Thirty minutes later, absolute ethanol (5 ml kg-1) was administered. The control group was treated with saline only. One hour later, the animals were euthanized and full-thickness sections of gastric corpus were collected, weighed, and stored at -70°C until assayed for glutathione (12). The results are expressed as mean values ± SEM for each group of five or more rats. * p \ 0.05 in relation to the saline group. # p \ 0.05 in relation to the ethanol group. ANOVA and Newman– Keuls test
of ethanol instilled. The animals in our study may have been euthanized too early (only 1 h after ethanol administration) to detect an increase in neutrophil infiltration based on another study published by our group [7]. Therefore, the gastroprotective effects of amifostine on ethanol-induced gastric injury could not be explained by a decrease in leukocyte adherence, as reported in a study evaluating the protective effect of amifostine against indomethacin-induced gastropathy [13]. This observation supports the hypothesis that a local rather than an inflammatory cell-dependent mechanism of ethanol-induced gastric damage is involved. Recently, amifostine was shown to inhibit indomethacin-induced gastric damage, suggesting that its protective effect is at least partially mediated by significant prevention of NP-SH depletion [13]. The role of endogenous NP-
SH in mucosal protection has already been demonstrated in a model of ethanol-induced gastric injury. As damage develops, mucosal NP-SH levels decrease. However, the administration of exogenous sulfhydryl-containing compounds can prevent damage by restoring NP-SH levels to normal [21]. In this study, amifostine was found to prevent NP-SH depletion, suggesting that in both models of gastric damage the maintenance of NP-SH levels in the gastric mucosa is a primary element in the protective action of amifostine. The contribution of several systems (prostaglandins, nitric oxide, and afferent neurons) in gastric mucosal defense is well documented [17]. In this study, chemical ablation of the sensory afferent neurons by capsaicin significantly neutralized the protective effect of amifostine on ethanol-induced gastric damage. Capsaicin-sensitive sensory neurons contain a variety of peptides, including tachykinins and CGRP. These form a dense plexus around the gastric submucosal arterioles [22, 23] and are involved in the regulation of gastric mucosal blood flow. Capsaicin at neurotoxic concentrations per se does not cause gastric mucosal damage, but rather increases the susceptibility of the gastric mucosa to the damaging effects of a number of chemical agents [24]. In our study, there was no change in ethanol’s ability to induce gastric damage in the desensitized rats, which differs from previously published data [24]. However, that study [24] utilized intragastric instillation of ethanol to pylorus-ligated rats, which considerably differs from our protocol. We hypothesize that long-term
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Table 2 Effect of afferent neuron ablation by neurotoxic doses of capsaicin on the protective action of amifostine (90 mg kg-1) on ethanolinduced microscopic damage in gastric mucosa Groups (n = 7)
Epithelial cell loss (score 0–3)
Inflammatory cells (score 0–3)
Edema (score 0–4)
Hemorrhagic damage (score 0–4)
Saline
0 (0–1)
0
0
0
Ethanol
2 (2–3)
0
3 (2–4)
4 (3–4)
Ethanol ? amifostine PO Ethanol ? amifostine SC
1 (0–1)* 2 (1–2)
0 0
0 (0–1)* 0*
0* 0*
Ethanol ? amifostine PO ? capsaicin ablation
2 (2–3)#
0
3 (2–3)#
3 (3–4)#
0
w
Ethanol ? amifostine SC ? capsaicin ablation
2.5 (2–3)
2 (1–3)
3.5 (3–4)w
The table shows median values followed by minimum and maximum scores (in brackets) * p \ 0.05 in relation to the ethanol group #
p \ 0.05 in relation to the ethanol ? amifostine PO group
w
p \ 0.05 in relation to the ethanol ? amifostine SC group
Control group Sensory aferent neurons ablated by capsaicin
NP-SH (μg per g tissue)
300
*
250
*
200
ψ
#
150
A
140
Macroscopic gastric lesion (mm 2)
The Kruskal–Wallis nonparametric test, followed by Dunn’s test, were used for multiple comparisons in the histological assessment
120 100 80 60 40
*
20 0
100
Sal
Sal
Sal
Glib
Cel
L-Name
Amifostine PO (90 mg kg -1)
50
Absolute Ethanol (5 ml kg-1)
Sal
Sal Amf PO (90 mg kg -1 )
Amf SC (90 mg kg-1 )
Absolute Ethanol (5 ml kg-1 )
Fig. 6 Gastroprotective effect of amifostine PO and SC on ethanolinduced decrease in nonprotein sulfhydryl (NP-SH) levels mediated by sensory afferent neurons. The rats were treated with neurotoxic doses of capsaicin. After 8 days, the desensitized animals were treated with saline (Sal) or amifostine (90 ml kg-1) PO or SC. Thirty minutes later, absolute ethanol (5 ml kg-1) was administered. The control group was treated with saline only. One hour later, the animals were euthanized and full-thickness segments of gastric corpus were collected, weighed and stored at -70°C until assayed for non-protein sulphydryl groups (14). The results are expressed as mean values ± SEM for each group of five or more rats. * p \ 0.05 in relation to the ethanol group. # p \ 0.05 in relation to the amifostine PO group. W p \ 0.05 in relation to the amifostine SC group. ANOVA and Newman–Keuls test
contact of this ulcerogen with the gastric mucosa could led to the aggravated injury seen in those animals. It is evident that amifostine’s protective effect on ethanol-induced gastric damage is dependent on afferent neurons primarily due to the reversal action the desensitization caused in amifostine protection. This mode of action differs from other cytoprotective agents, such as prostacyclin, that still shielding the animals from ethanol injury despite the fact
123
B
160
Macroscopic gastric lesion (mm2)
0
140 120 100 80 60 40
*
20 0 Sal
Sal
Sal
Glib
Cel
L-Name
Amifostine SC (90 mg kg-1) Absolute Ethanol (5 ml kg-1)
Fig. 7 Effect of glibenclamide, celecoxib, and L-NAME on the protective action of amifostine PO (panel a) and amifostine SC (panel b) on ethanol-induced gastric injury. Glibenclamide (10 mg kg-1), celecoxib (10 mg kg-1), or L-NAME (10 mg kg-1) were injected 30 min prior to amifostine (90 mg kg-1) PO or SC administration. Thirty minutes later, absolute ethanol (5 ml kg-1) was administered. The control group was treated with saline only. Gastric lesions were quantified after 1 h. No statistically significant differences were observed between the three groups (glibenclamide, celecoxib, and L-NAME) and the amifostine group. The results are expressed as mean values ± SEM for each group of five or more rats. * p \ 0.01 in relation to the ethanol group. ANOVA and Newman–Keuls test
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that they were desensitized [25]. It is not clear how amifostine affects the sensory afferent fibers, but the absence of primary afferent neuron activity likely potentiates ethanol-induced release of reactive oxygen species, similar to what is observed in stress-induced gastric damage models [26]. Our results support this hypothesis since the chemical ablation of the sensory afferent neurons by capsaicin prevented amifostine from maintaining normal mucosal NPSH levels during ethanol-induced gastropathy (Fig. 6). Thus, amifostine appears to have been unable to scavenge the greatly increased number of free radicals available in the gastric mucosa. Endogenous nitric oxide (NO) [7], increased COX-2 expression [27], and activation of ATP-sensitive potassium channels (KATP) [28] have all been shown to be associated with gastric defense. In order to determine the effect of each of these factors on amifostine-promoted gastroprotection, the study included groups of animals receiving L-NAME, celecoxib, or glibenclamide. However, the protective effect of amifostine was not reversed by L-NAME or celecoxib, indicating it may be due to factors other than the presence of endogenous NO and COX-2 activation. Likewise, glibenclamide administration did not inhibit the protective effect of amifostine on ethanol-induced gastric injury. The latter finding is in contrast with the literature, since the NP-SH-containing drug dimercaprol has been shown to protect against ethanol-induced gastric lesions through KATP activation [28]. Overall, our results indicate that amifostine, administered orally or subcutaneously, prevents gastric injury by increasing the level of non-protein sulfhydryl groups in the gastric mucosa. The effect seems at least in part to involve the activation of capsaicin-sensitive afferent neurons. Acknowledgments The authors would like to thank Maria Silvandira Freire Franc¸a and Jose´ Ivan Rodrigues for their technical assistance. This study was supported by grants from CNPq (Brazil). Dr. Ribeiro, Dr. Brito, and Dr. Souza are CNPq fellowship holders. This study is part of the requirements for coauthor J. J. Junqueira to obtain a Master of Science degree in Pharmacology from the Medical School at the Federal University of Ceara´.
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