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Protective effect of l -citrulline against ethanol-induced gastric ulcer in rats
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Protective effect of l-citrulline against ethanol-induced gastric ulcer in rats Yi Liu ∗,1 , Xia Tian 1 , Lingshan Gou, Xiaobin Fu, Sai Li, Nuo Lan, Xiaoxing Yin Department of Pharmacy, Xuzhou Medical College, Jiangsu, China
a r t i c l e
i n f o
Article history:
a b s t r a c t We examined the protective effect of l-citrulline on ethanol-induced gastric ulcer in rats.
Received 22 November 2011
Administration of l-citrulline at doses of 300, 600 and 900 mg/kg body weight prior to ethanol
Received in revised form
ingestion protected the stomach from ulceration. The gastric lesions were significantly
20 April 2012
attenuated by all doses of l-citrulline as compared to the ethanol group. Pre-treatment with
Accepted 24 April 2012
l-citrulline prevented the oxidative damage and the decrease of nitric oxide content as well
Available online 11 May 2012
as the increase of the myeloperoxidase activity. Consequently, significant changes observed included the attenuation in the elevation in total nitric oxide synthase activity and inducible
Keywords:
nitric oxide synthase activity as well as the decrease in constitutive nitric oxide synthase
l-Citrulline
activity in the gastric mucosa induced by ethanol. Analysis of serum cytokines of ethanol-
Ethanol
induced rats showed a moderate decrease in interleukin-10 with considerable increase of
Gastric ulcer
interleukin-6 while l-citrulline inhibited the acute alteration of cytokines. These results
Rat
suggested the gastroprotective effect of l-citrulline. © 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Gastric ulcer is a common disease with multiple etiologies, defined as a discontinuity in the gastric mucosa penetration through the muscularis mucosa (Yeomans and Naesdal, 2008). It usually results from the imbalance between the gastric mucosal protective factors, i.e., the gastric mucosal barrier and the aggressive factors, to which the mucosa is exposed. Aggressive factors which promote gastric mucosal injury include gastric hydrochloric acid (HCl), mucosal hypoperfusion (Ham and Kaunitz, 2007), free oxygen radicals, and ethanol, etc. Among them, alcohol consumption is the greatest contributor to gastric ulceration (Franke et al., 2005). Consumption of excessive alcohol usually elevates the risk
of gastric mucosal damage. Thus, the experimental model of ethanol-induced gastric mucosal damage in rats is often employed to screen the compounds for anti-ulcer activity in that it serves as the leading cause of gastric ulcer in humans (Santos and Rao, 2001). The mechanisms underlying the ethanol-induced gastric injury have not yet been fully elucidated. There is evidence that oxidative stress and lipid peroxidation have important roles to play in the pathogenesis of acute gastric lesions induced by ethanol (Pan et al., 2008; Hernandez-Munoz et al., 2000). Numerous reports have demonstrated that ethanolinduced gastric lesions are closely related to the increased reactive oxygen species (ROS), which lead to lipid peroxidation in the membranes by the oxidation of unsaturated fatty acids (Takeuchi et al., 1991; Ames et al., 1993). Therefore, antioxidant
∗ Corresponding author at: School of Pharmacy, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, Jiangsu 221002, China. Tel.: +86 13775895636. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Tian), [email protected] (L. Gou), [email protected] (X. Fu), [email protected] (S. Li), [email protected] (N. Lan), [email protected] (X. Yin). 1 Yi Liu and Xia Tian are co-first authors. 1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2012.04.009
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defence systems, including antioxidant enzymes, foods and drugs, contribute to the prevention of the toxic ROS effects (Mates et al., 1999; Bafna and Balaraman, 2004). On the other hand, neutrophil infiltration into the gastric mucosa is also a critical process in the pathogenesis of a variety of gastric ulcers (Elliot and Wallace, 1998; Nishida et al., 1998). It has been shown that ethanol-induced neutrophil infiltration in the gastric mucosa is closely related to the genesis of lesions (La Casa et al., 2000). The neutrophil infiltration into the gastric mucosal tissues can be reflected by the determination of the activities of myeloperoxidase (MPO) and nitric oxide synthase (NOS), both of which serve as key indicators of neutrophil infiltration in various experimental gastric injuries (Coskun et al., 1996; Takeuchi et al., 1998). Similarly, cytokines such as interleukin-6 (IL-6) and interleukin-10 (IL-10) are involved in the acute-phase inflammation as well as maintenance and regulation of the severity of gastric ulcer (Rogler and Andus, 1998). Much evidence suggests that l-citrulline can decrease oxidative damage induced by exhaustive exercise (Sureda et al., 2009). Moreover, l-citrulline is closely related to larginine (Bredt and Snyder, 1990). l-Citrulline can be readily converted to l-arginine in the kidney, vascular endothelium and other tissues, thereby providing a recycling pathway for the conversion of l-citrulline to nitric oxide (NO) via l-arginine (Solomonson et al., 2003). Moreover, it has been demonstrated that l-arginine elicits a gastric protective effect via the inhibition of the increased inducible nitric oxide synthase (iNOS) activity and the decreased constitutive nitric oxide synthase (cNOS) activity in the gastric mucosa (Ohta and Nishida, 2001). As an indirect precursor of l-arginine, l-citrulline may also exert particular gastroprotective effects. Thus, the aim of the present study was: (a) to evaluate the gastroprotective effect of l-citrulline on ethanol-induced gastric ulceration in rats; (b) to determine the effect of l-citrulline on pro- and antiinflammatory cytokines as well as enzyme activities, such as superoxide dismutase (SOD), glutathione peroxidise (GSH-px), MPO, NOS and the levels of NO, malondialdehyde (MDA) as well as glutathione (GSH) in the gastric tissues in each treatment group.
2.
Materials and methods
2.1.
Animals
Adult male Sprague-Dawley rats, weighing 180–220 g, were provided by the Experimental Animal Centre of Xuzhou Medical College. All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
2.2. Ethanol-induced gastric ulcer in rats and its prevention by l-citrulline The rats were deprived of food but had ad libitum access to tap water for 48 h before ulcer induction. Gastric mucosal damage was induced in conscious rats by gavage of 1.0 mL/rat of absolute ethanol (99.5%) (Robert, 1979). The test drug l-citrulline was dissolved in normal
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saline. Animals were randomised into five groups (n = 8): control, ethanol, l-citrulline (300 mg/kg) + ethanol, l-citrulline (600 mg/kg) + ethanol, and l-citrulline (900 mg/kg) + ethanol. All the rats in groups were pre-treated by gavage with lcitrulline for 7 days prior to the ethanol administration. The animals were sacrificed 60 min later, and the blood samples were obtained with no addition of anticoagulants for centrifugation at 3000 × g for 10 min for subsequent determination of clear serum. In parallel, the murine stomachs were rapidly removed, opened along the greater curvature and rinsed thoroughly with normal saline, followed by macroscopic determination of the gastric mucosal injury index. Thereafter, each stomach was dichotomised, with one moiety of stomach immersed in 4% formaldehyde for histological evaluation and gastric mucosa from the other moiety stored at −80 ◦ C for biochemical determinations.
2.3.
Determination of gastric ulcer index
An observer who was unaware of the treatment regimen performed the determination of the severity of gastric mucosal lesions. Lesion size (mm) was measured along its greatest length, and in the case of patches, five such lesions were considered the equivalent of a 1 mm ulcer. The sum of the lesion lengths in each group of animals was divided by its number and expressed as the mean gastric haemorrhagic lesion index (Qiu et al., 1991).
2.4.
Histological procedure and assessment
For pathological examinations, all samples were fixed in 4% formaldehyde buffer. The tissues were embedded in paraffin, and pathological sections were sliced along the longitudinal axis. From each sample, 5-m-thick sections were obtained and stained with haematoxylin–eosin to evaluate gastric morphology.
2.5.
Biochemical examinations of stomach tissues
Following the macroscopic analyses, determination was conducted on the activities of SOD, GSH-px, NOS and MPO as well as the levels of NO, MDA and GSH in rat stomach tissues. To prepare the tissue homogenates, stomach tissues were thawed and then treated in ice-cold normal saline at a ratio of 1:9 (w/v). The mixtures were homogenised on ice using an Ultra-Turrax homogeniser for 15 min. Homogenates was centrifuged using a refrigerated centrifuge at 4 ◦ C. Then, these supernatants were used for the determination of biochemical examinations.
2.5.1.
Determination of SOD activity
Measurements were made as described by Sun et al. (Sun et al., 1988). SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which reacted with nitro-blue tetrazolium (NTB) to form formazan dye. SOD activity was then measured at 550 nm by the degree of inhibition of this reaction. One unit of enzyme was defined as the amount of enzyme required at the inhibition
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rate of 50%. The activity of SOD was expressed as unit/mg protein.
2.5.2.
Determination of MDA level
The level of MDA in the gastric mucosa designated as an index of lipid peroxidation was measured according to the modified method of Ohkawa et al. (1989). Gastric mucosa was weighed and homogenised in 10 mL KCl (10%). The homogenate was supplemented with 8.1% sodium lauryl sulfate, 20% acetic acid and 0.8% TBA, and was thereafter boiled at 100 ◦ C for 1 h. After cooling, the reactants were supplemented with 2.5 mL n-butanol for vigourous agitation for 1 min and centrifugation for 10 min. Absorbance was measured at 532 nm and the results were expressed as nmol TBA/mg of protein.
2.5.3.
Determination of GSH level
GSH concentration was determined by the procedures of Elmann (1959). Briefly, 0.5 mL homogenate was mixed with 1.5 mL 0.15 M KCl and 3 mL deproteinisation solution. Each sample was centrifuged at 3000 rpm for 10 min and the supernatant was removed, followed by the addition of 2 mL phosphate solution and 0.5 mL DTNB into the 0.5 mL supernatant, with the absorbance read at 412 nm and compared with glutathione standards.
2.5.4.
Determination of GSH-px activity
GSH-px activity in the tissue homogenate was determined according to Paglia and Valentine (1967). In brief, the sample was prepared by the addition of an equal volume of 50 mM potassium phosphate buffer (pH 7.0, 0.25 mL) into the original tissue homogenate. Subsequent to the centrifugation of 10 min at 750 g, the supernatant was obtained for the determination of GSH-px activity. In a quartz cell, 2.8 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 2 mM EDTA–2Na was mixed with 0.1 mL of 150 mM glutathione, 0.1 mL of 84.4 mM NADPH and 0.1 mL of 66 U/mL glutathione reductase, followed by the addition of 0.1 mL of the supernatant. 5 min after preincubation at 25 ◦ C, 0.1 mL of 22 mM H2 O2 was added and mixed to commence the reaction. The variations in absorbance of the reaction mixture at 340 nm during the first 100 s of the reaction were monitored, and the rate of decrease in absorbance was calculated by the linear proportion to the observed variations. The variations in NADPH were calculated using an extinction coefficient (M) value of 6220 M−1 cm−1 . GSH-px activity was expressed as unit/g protein.
2.5.5.
Determination of NOS activity
NOS activity was measured on the basis of the formation of l-citrulline from l-arginine (Bredt and Snyder, 1989). Frozen tissues from the stomach mucosa were homogenised at 0 ◦ C in 5 vol buffer containing 320 mmol/L sucrose, 1 mmol/L dldithiothreitol, phenylmethylsulphonyl fluoride (100 g/mL), leupeptin (10 g/mL), soybean trypsin inhibitor (100 g/mL) and aprotinin (2 g/mL) in 50 mmol/L HEPES (pH 7.0). Following centrifugation of the homogenate at 100 000 × g for 1 h, the supernatants were added to the reaction mixture containing 50 mmol/L Tris (pH 7.4), l-[U-14C]arginine (specific activity 11.8 GBq/mmoL), 10 g/mL calmodulin, 1 mmol/L CaCl2 , and
50 mmol/L l-valine. The samples were incubated for 20 min at 37 ◦ C prior to the termination of the reaction by the addition of 0.1 vol of 20% (v/v) HClO4. l-[U-14C]Citrulline was isolated from l-[U-14C]arginine by passage through Dowex 50W (Na+ form, Sigma–Aldrich, St Louis, MO, USA) and quantified by liquid-scintillation counting. The level of iNOS activity was measured by the addition of 1 mmol/L EGTA and total NOS (TNOS) activity by the addition of 2 mmol/L l-NMMA. The cNOS activity was determined by subtraction of iNOS activity from TNOS activity. Protein concentration of the supernatant was determined by spectrophotometry using a commercial assay kit (BioRadLaboratories, Richmond, CA, USA).
2.5.6.
Determination of MPO activity
MPO activity, a marker of neutrophil infiltration (Krawisz et al., 1984), was assayed according to the modified method of Bradley et al. (1982). In brief, the specimens were homogenised in 50 mM potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide (Sigma–Aldrich, St Louis, MO, USA). Suspensions were then centrifuged and MPO in the resulting supernatant was assayed with a spectrophotometer (Shimadzu UV2450, Shimadzu Corporation, Tokyo, Japan). One unit of MPO activity was defined as degrading 1 mol of peroxide/min 25 ◦ C. Proteins were measured with a modified bicinchoninic acid method with a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Results were expressed as units/g of protein.
2.5.7.
Determination of NO level
The NO content in the gastric tissue was measured with the method of nitric acid reductase, and the operational processes were measured in accordance with the NO kit instructions. Results were expressed as mol/g of protein.
2.6.
Cytokines assay
Serum levels of cytokines (IL-6 and IL-10) were evaluated by eBioscience kits (San Diego, CA, USA) for rats IL-6 and IL-10 by the manufacturer’s instruction. Primary antibodies were coated on 96-well plates, followed by removal of the non-specific binding by washing. Serum samples or cytokine standards (100 L) were respectively loaded into each well. The biotin-conjugated secondary antibodies were added into each well. Avidin-HRP and substrate solution were added for colour reaction, with the absorbance measured at 450 nm with an ELISA reader (Zenyth 3100; Anthos Labtec Instruments GmbH, Salzburg, Austria). A standard curve was run on each assay plate using recombinant IL-6 and IL-10 in serial dilutions.
2.7.
Statistical analysis
All results were expressed as means ± SEM. The data were evaluated with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The statistical significance of differences for each parameter among the groups was evaluated by one-way ANOVA, followed by Dunnett’s t test. The significance level was set at P < 0.05.
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3.
Results
3.1. index
Effect of l-citrulline pre-treatment on gastric ulcer
In this experiment, only petechiae were observed in the murine gastric lining in the control group. The injection of absolute ethanol into the gastric lumen induced gross lesions in the stomach. l-Citrulline gavage prior to ethanol administration significantly reduced the ulcerogenic effect of ethanol. As shown in Fig. 1, l-citrulline at different doses significantly reduced the ethanol-induced gastric ulcer index, particularly at the dose of 900 mg/kg (P < 0.01).
3.2.
Histological results
The morphological study of the stomach of normal animals showed no damage (Fig. 2A). However, in the group treated with absolute ethanol, it was observed that there were disruption and exfoliation of the superficial gastric epithelium, disappearance of the mucous cells from the upper portion of fundic glands, vacuolisation, necrosis, and karyopyknosis in the superficial gastric epithelium as well as mucous neck cells. In some areas, there was cell damage extending into the gastric glands. Moreover, there was apparent extensive
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Fig. 1 – l-Citrulline (l-Cit) at different doses on ethanol-induced gastric ulcer index by in rats. Data are the means ± SE for 8 animals. ***P < 0.001 compared to the control group; ## P < 0.05 compared to the ethanol group; ### P < 0.01 compared to the ethanol group.
haemorrhage in the mucosa, and hyperaemia as well as inflammatory cell infiltration in the mucosa and submucosa (Fig. 2B). Following l-citrulline pre-treatment, the pathological injuries were markedly attenuated, with only slight damage observed in the superficial epithelium (Fig. 2C).
Fig. 2 – Effects of l-citrulline (l-Cit) on histological findings of gastric damage induced by ethanol in rats after haematoxylin and eosin staining: control (A), ethanol (B), l-citrulline 900 (mg/kg) (C).
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3.3.
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Biochemical assays
3.3.1. Effect of l-citrulline on SOD and GSH-px activities and GSH and MDA levels in the stomach tissue of rats administered with ethanol As shown in Table 1, the level of MDA, an index of lipid peroxidation, was significantly increased to 0.770 ± 0.021 nmol TBA/mg protein at 1 h after ethanol administration in comparison to the control group. This increase was not significant in animals with l-citrulline gavage. The activities of the antioxidant enzymes SOD and GSH-px were significantly decreased subsequent to intragastric administration of ethanol (P < 0.001), though, l-citrulline pre-treatment protected the gastric mucosal against the loss of antioxidant enzyme activity, resulting in a significant increase in enzymatic SOD and GSH-px activities approximate to the control levels. Similarly, ethanol-treated rats showed a significant decrease in the GSH content (P < 0.001), probably due to its consumption during oxidative stress. In contrast, rats pre-treated with l-citrulline showed only a modest decrease, with no significant depletion of GSH levels.
3.3.2. Effect of l-citrulline on NOS and MPO activities in the stomach tissue of rats administered with ethanol Table 2 shows the activities of MPO and NOS in all groups. There were higher MPO activity in the ethanol-treated group than in the normal control group (P < 0.01), while MPO activities were markedly weakened (P < 0.01) in the l-citrulline treatment groups. TNOS activity of the gastric mucosal tissues was found to be higher in the ethanol group compared to the normal control group (P < 0.01). Treatment with l-citrulline significantly decreased the TNOS compared with that of the ethanol group (P < 0.01). Additionally, the cNOS activity was lower in ethanol group compared to the normal control group, but was similar in both l-citrulline pre-treatment group and the control group. However, iNOS activity was significantly higher (P < 0.01) in ethanol-treated rats than in normal control rats. l-Citrulline pre-treatment significantly lowered the iNOS activity (P < 0.01).
3.3.3.
Effect of l-citrulline on gastric mucosal NO content
The contents of NO in gastric tissue are shown in Fig. 3; the NO content in the ethanol-treated group was significantly lower as compared to the normal control group (P < 0.01), while the NO contents in the l-citrulline-treated groups were significantly higher than those in the ethanol-treated group (P < 0.05), particularly at the dose of 900 mg/kg (P < 0.01).
3.4. Effect of l-citrulline on pro-inflammatory and anti-inflammatory cytokines The levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines in ulcerated rats upon pre-treatment against ethanol challenge in rats are presented in Fig. 4. Administration of ethanol significantly (P < 0.001) elevated the levels of IL-6 when compared with normal rats. In contrast, the elevation of IL-6 was prevented in rats that received l-citrulline (P < 0.01) as pre-treatment (Fig. 4A). Similarly, the level of anti-inflammatory cytokine IL-10 was moderately reduced (P < 0.001) when compared with normal control. However, the
Fig. 3 – Effects of l-citrulline (l-Cit) on levels of NO in rats gastric tissues. Data are the means ± SE for 8 animals. ***P < 0.001 compared to the control group; ## P < 0.05, ### P < 0.01 compared to the ethanol group.
rats pre-treated with l-citrulline (P < 0.01) elevated the IL-10 levels compared with the ethanol group (Fig. 4B).
4.
Discussion
Alcohol consumption can produce acute haemorrhagic gastric erosions, and excessive ingestion can results in gastritis characterised by mucosal oedema, sub-epithelial haemorrhages, cellular exfoliation, and inflammatory cell infiltration (Guslandi, 1987; Ko et al., 2004), which is essentially an acute inflammatory reaction. However, prophylactic treatment with l-citrulline attenuated ethanol-induced gastric injury in a dose-dependent manner, with significant reduction of the gastric ulcer index. This study demonstrates that l-citrulline has gastroprotective effect against ethanol-induced gastric ulcer, when administered at oral doses between 300 and 900 mg/kg, particularly at the dose of 900 mg/kg. Despite the widely accepted notion that alcohol abuse leads to detrimental consequences in the gastrointestinal tract, the mechanisms underlying the ethanol-induced gastric mucosal injury still remain obscure. There is growing evidence that ethanol-induced gastric mucosal injury is closely related to the increased ROS level and the major source of ROS is from the activated neutrophils (Pan et al., 2008). On the other hand, organisms per se have enzymatic and non-enzymatic defences, including GSH, SOD and GSH-px against the ROS-induced lipid peroxidation (Mates et al., 1999). Thus, in order to address the role of oxidative stress in our model, we assessed several oxidant-antioxidant parameters in murine gastric tissues. The experimental results showed that ethanol markedly increased MDA, an index of lipid peroxidation, accompanied by a decrease of GSH, GSH-px and SOD, all of which are endogenous antioxidants. Our data support an imperative role for oxidative stress in the pathogenesis of ethanol-induced gastric ulcer. Nevertheless, treatment with lcitrulline resulted in significant increases in the activities of SOD and GSH-px and the levels of GSH, as well as a decrease in MDA formation, reflecting its antioxidant potential. The mechanisms which underlie l-citrulline-mediated waning of
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Table 1 – Effect of l-citrulline on SOD and GSH-px activities and GSH and MDA levels in the stomach tissue of rats administered with ethanol. Group
MDA (nmol TBA/mg)
Control group Ethanol group l-Cit 300 mg/kg l-Cit 600 mg/kg l-Cit 900 mg/kg
0.365 0.770 0.632 0.524 0.446
± ± ± ± ±
SOD (unit/mg)
0.014 0.021*** 0.017* 0.022** 0.024**
224.38 112.72 135.70 174.32 203.96
± ± ± ± ±
GSH (mg/g)
5.985 4.536*** 4.061* 7.592** 5.111**
9.82 2.74 4.04 6.49 8.35
± ± ± ± ±
0.404 0.237*** 0.328** 0.341** 0.234**
GSH-px (unit/g) 241.29 116.78 136.85 166.00 202.49
± ± ± ± ±
8.872 6.157*** 6.561* 7.656** 6.384**
Data are presented as the means ± SEM for 8 animals. P < 0.05 as compared to the ethanol control group. ∗∗ P < 0.01 as compared to the ethanol control group. ∗∗∗ P < 0.001 as compared to the control group. ∗
Table 2 – Effect of l-citrulline on NOS and MPO activities in the stomach tissue of rats administered with ethanol. Group
MPO (unit/g)
Control group Ethanol group l-Cit 300 mg/kg l-Cit 600 mg/kg l-Cit 900 mg/kg
0.197 0.773 0.654 0.473 0.292
± ± ± ± ±
0.008 0.031*** 0.025* 0.015** 0.012**
TNOS (nmol/min/g) 2.707 4.657 4.030 3.580 3.106
± ± ± ± ±
0.038 0.069*** 0.053* 0.060** 0.038**
cNOS (nmol/min/g) 2.270 1.494 2.161 2.306 2.185
± ± ± ± ±
0.051 0.058*** 0.048* 0.076** 0.065**
iNOS (nmol/min/g) 0.437 3.164 1.869 1.274 0.921
± ± ± ± ±
0.047 0.056*** 0.059* 0.113** 0.036**
Data are the means ± SEM for 8 animals. P < 0.05 as compared to the ethanol control group. ∗∗ P < 0.01 as compared to the ethanol control group. ∗∗∗ P < 0.001 as compared to the control group. ∗
oxidative stress in ethanol-induced gastric ulcer could be attributed to the direct antioxidant and free radical scavenging activity of l-citrulline or indirectly due to augmentation of intracellular GSH, GSH-px and SOD in the rat gastric tissues, all of which can scavenge superoxide, hydrogen peroxide, hydroxyl and lipid peroxyl radicals, and attenuate damages to the tissue. In addition, this observed antioxidant activity of l-citrulline might be due to the recycling pathway from lcitrulline to l-arginine and NO. The recycling pathway might be crucial in sustaining the production of NO, which is the active species in reducing the oxidative stress (Hayashi et al., 2005). Thus, we might make a hypothesis that the gastroprotection of l-citrulline might rest on its antioxidant effects.
There is growing evidence that the major source of ROS is from the activated neutrophils (Pan et al., 2008) and neutrophils play an vital role in the development of gastric damage by their aggregation and release of tissue-disrupting substance, such as oxygen free radicals and proteases (Kobayashi et al., 2001). The neutrophil infiltration into the gastric mucosal tissues is assessed by MPO as well as NOS (Coskun et al., 1996; Takeuchi et al., 1998). In the present study, we observed a significant increase in MPO activity in the stomach following ethanol administration, which confirmed the infiltration and activation of neutrophils in the gastric mucosa produced by ethanol. In our experiment, prior administration of l-citrulline also exhibited an inhibitory effect on the
Fig. 4 – Effects of l-citrulline (l-Cit) on levels of pro-inflammatory cytokine: IL-6 (A) and anti-inflammatory cytokine: IL-10 (B) in the gastric mucosa of ulcerated rats. Data are the means ± SE for 8 animals. ***P < 0.001 compared to the control group; ## P < 0.05, ### P < 0.01 compared to the ethanol group.
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increased MPO activity. It is also widely accepted that, in the digestive systems, cNOS-generated NO is cytoprotective and iNOS-generated NO is cytotoxic (Nishida et al., 1998). In the present study, we observed an increase in cNOS activity and a decrease in iNOS activity when rats were pre-treated with l-citrulline. Moreover, l-citrulline pre-treatment prevented the decrease of NO level in the gastric mucosa induced by ethanol. Thus, we estimate the protective effect of l-citrulline against ethanol-induced gastric ulcer in rats is closely related to NO generated by cNOS through l-arginine converted from l-citrulline. Our previous study confirmed that l-citrulline pretreatment prevented the increase in iNOS activity and iNOS protein expression in rats subjected to ischaemia–reperfusion (IR) (Gou et al., 2011). Thus, we hypothesise that the NOS activities might be related to the NOS protein expression in the murine stomach subjected to ethanol. These results suggest that ethanol led to the neutrophil-infiltration into the gastric tissues while l-citrulline administration attenuated the injury to the gastric mucosa. NO and ROS exert multiple modulating effects on inflammation and play key roles in the regulation of immune responses. Large amounts of NO, generated primarily by iNOS can be toxic and pro-inflammatory (Guzik et al., 2003). During the course of an inflammatory response, large amounts of NO formed by iNOS surpass the physiological amounts of NO (Xie and Nathan, 1994), which is of interest to our study of the levels of inflammatory cytokines. Interleukins play a vital role in the regulation of the mucosal defence barrier. Proinflammatory cytokines such as IL-6 in the gastric mucosa significantly increased and anti-inflammatory IL-10 significantly decreased in gastric ulceration (Paglia and Valentine, 1967), which was consistent with the results observed in this model. However, l-citrulline prophylactic treatment inhibited the depletion of IL-10 and the elevation of IL-6, which showed its anti-inflammatory effect in ethanol-induced gastric ulcer. The anti-inflammatory effect of l-citrulline might be closely related to the decrease of iNOS activity. In conclusion, gastric ulceration in this model was associated with gastric lipid peroxidation, neutrophil infiltration into the stomach and inflammatory reaction may be responsible for the spontaneous occurrence of ethanol-induced ulcers in rats. Based on the results of our study, l-citrulline was confirmed to have protective effects on ethanol-induced gastric ulceration in rats. The effect of l-citrulline can be attributed to its reduction of oxidative damage, and its inhibitory effects on neutrophil infiltration as well as its anti-inflammatory effects in rat stomach tissues.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgements The authors are cordially indebted to these financial supports: “Qing-Lan” Project of Jiangsu Province, the Industrialization of Scientific Research Promotion Projects of Universities and Colleges in Jiangsu Province (2011-16), the Natural Science Fund for Universities and Colleges in Jiangsu Province (09KJB350003
and 11KJB350005), Laboratory of Biological Therapy for Cancer of Xuzhou Medical College (JSBL0803, C0903 and C0904), the Science and Technology Plan Projects of Xuzhou (XF11C037 and XF11C062), Superiority Academic Discipline Construction Project of Jiangsu Higher Education Institutions, and Xuzhou Public Service Platform Projects of Drug Discovery and Research, Innovation Project of Postgraduates in Jiangsu Province, China (CXLX11-0752).
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Krawisz, J.E., Sharon, P., Stenson, W.F., 1984. Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Gastroenterology 87 (6), 1344–1350. La Casa, C., Villegas, I., Alarcon de la Lastra, C., Motilva, V., Martin Calero, M.J., 2000. Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced Gastric lesions. J. Ethnopharmacol. 71 (1-2), 45–53. Mates, J.M., Perez-Gomez, C., Nudez de Castro, I., 1999. Antioxidant enzymes and human diseases. Clin. Biochem. 32 (8), 595–603. Nishida, K., Ohta, Y., Ishiguro, I., 1998. Contribution of NO synthases to neutrophil infiltration in the gastric mucosal lesions in rats with water immersion restraint stress. FEBS Lett. 425 (2), 243–248. Ohkawa, H., Ohishi, N., Yagi, K., 1989. Assay for lipid peroxide for animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95 (2), 351–358. Ohta, Y., Nishida, K., 2001. Protective effect of l-arginine against stress-induced gastric mucosal lesions in rats and its relation to nitric oxide-mediated inhibition of neutrophil infiltration. Pharmacol. Res. 43 (6), 535–541. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70 (1), 158–169. Pan, J.S., He, S.Z., Xu, H.Z., et al., 2008. Oxidative stress disturbs energy metabolism of mitochondria in ethanol-induced gastric mucosa injury. World J. Gastroenterol. 14 (38), 5857–5867. Qiu, B.S., Cho, C.H., Ogle, C.W., 1991. Chronic nicotine treatment intensifies gastric ulceration by cold-restraint stress in rats. Agents Actions 33 (3-4), 367–370.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Protective effect of l-citrulline against ethanol-induced gastric ulcer in rats Yi Liu ∗,1 , Xia Tian 1 , Lingshan Gou, Xiaobin Fu, Sai Li, Nuo Lan, Xiaoxing Yin Department of Pharmacy, Xuzhou Medical College, Jiangsu, China
a r t i c l e
i n f o
Article history:
a b s t r a c t We examined the protective effect of l-citrulline on ethanol-induced gastric ulcer in rats.
Received 22 November 2011
Administration of l-citrulline at doses of 300, 600 and 900 mg/kg body weight prior to ethanol
Received in revised form
ingestion protected the stomach from ulceration. The gastric lesions were significantly
20 April 2012
attenuated by all doses of l-citrulline as compared to the ethanol group. Pre-treatment with
Accepted 24 April 2012
l-citrulline prevented the oxidative damage and the decrease of nitric oxide content as well
Available online 11 May 2012
as the increase of the myeloperoxidase activity. Consequently, significant changes observed included the attenuation in the elevation in total nitric oxide synthase activity and inducible
Keywords:
nitric oxide synthase activity as well as the decrease in constitutive nitric oxide synthase
l-Citrulline
activity in the gastric mucosa induced by ethanol. Analysis of serum cytokines of ethanol-
Ethanol
induced rats showed a moderate decrease in interleukin-10 with considerable increase of
Gastric ulcer
interleukin-6 while l-citrulline inhibited the acute alteration of cytokines. These results
Rat
suggested the gastroprotective effect of l-citrulline. © 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Gastric ulcer is a common disease with multiple etiologies, defined as a discontinuity in the gastric mucosa penetration through the muscularis mucosa (Yeomans and Naesdal, 2008). It usually results from the imbalance between the gastric mucosal protective factors, i.e., the gastric mucosal barrier and the aggressive factors, to which the mucosa is exposed. Aggressive factors which promote gastric mucosal injury include gastric hydrochloric acid (HCl), mucosal hypoperfusion (Ham and Kaunitz, 2007), free oxygen radicals, and ethanol, etc. Among them, alcohol consumption is the greatest contributor to gastric ulceration (Franke et al., 2005). Consumption of excessive alcohol usually elevates the risk
of gastric mucosal damage. Thus, the experimental model of ethanol-induced gastric mucosal damage in rats is often employed to screen the compounds for anti-ulcer activity in that it serves as the leading cause of gastric ulcer in humans (Santos and Rao, 2001). The mechanisms underlying the ethanol-induced gastric injury have not yet been fully elucidated. There is evidence that oxidative stress and lipid peroxidation have important roles to play in the pathogenesis of acute gastric lesions induced by ethanol (Pan et al., 2008; Hernandez-Munoz et al., 2000). Numerous reports have demonstrated that ethanolinduced gastric lesions are closely related to the increased reactive oxygen species (ROS), which lead to lipid peroxidation in the membranes by the oxidation of unsaturated fatty acids (Takeuchi et al., 1991; Ames et al., 1993). Therefore, antioxidant
∗ Corresponding author at: School of Pharmacy, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, Jiangsu 221002, China. Tel.: +86 13775895636. E-mail addresses: [email protected] (Y. Liu), [email protected] (X. Tian), [email protected] (L. Gou), [email protected] (X. Fu), [email protected] (S. Li), [email protected] (N. Lan), [email protected] (X. Yin). 1 Yi Liu and Xia Tian are co-first authors. 1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2012.04.009
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defence systems, including antioxidant enzymes, foods and drugs, contribute to the prevention of the toxic ROS effects (Mates et al., 1999; Bafna and Balaraman, 2004). On the other hand, neutrophil infiltration into the gastric mucosa is also a critical process in the pathogenesis of a variety of gastric ulcers (Elliot and Wallace, 1998; Nishida et al., 1998). It has been shown that ethanol-induced neutrophil infiltration in the gastric mucosa is closely related to the genesis of lesions (La Casa et al., 2000). The neutrophil infiltration into the gastric mucosal tissues can be reflected by the determination of the activities of myeloperoxidase (MPO) and nitric oxide synthase (NOS), both of which serve as key indicators of neutrophil infiltration in various experimental gastric injuries (Coskun et al., 1996; Takeuchi et al., 1998). Similarly, cytokines such as interleukin-6 (IL-6) and interleukin-10 (IL-10) are involved in the acute-phase inflammation as well as maintenance and regulation of the severity of gastric ulcer (Rogler and Andus, 1998). Much evidence suggests that l-citrulline can decrease oxidative damage induced by exhaustive exercise (Sureda et al., 2009). Moreover, l-citrulline is closely related to larginine (Bredt and Snyder, 1990). l-Citrulline can be readily converted to l-arginine in the kidney, vascular endothelium and other tissues, thereby providing a recycling pathway for the conversion of l-citrulline to nitric oxide (NO) via l-arginine (Solomonson et al., 2003). Moreover, it has been demonstrated that l-arginine elicits a gastric protective effect via the inhibition of the increased inducible nitric oxide synthase (iNOS) activity and the decreased constitutive nitric oxide synthase (cNOS) activity in the gastric mucosa (Ohta and Nishida, 2001). As an indirect precursor of l-arginine, l-citrulline may also exert particular gastroprotective effects. Thus, the aim of the present study was: (a) to evaluate the gastroprotective effect of l-citrulline on ethanol-induced gastric ulceration in rats; (b) to determine the effect of l-citrulline on pro- and antiinflammatory cytokines as well as enzyme activities, such as superoxide dismutase (SOD), glutathione peroxidise (GSH-px), MPO, NOS and the levels of NO, malondialdehyde (MDA) as well as glutathione (GSH) in the gastric tissues in each treatment group.
2.
Materials and methods
2.1.
Animals
Adult male Sprague-Dawley rats, weighing 180–220 g, were provided by the Experimental Animal Centre of Xuzhou Medical College. All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
2.2. Ethanol-induced gastric ulcer in rats and its prevention by l-citrulline The rats were deprived of food but had ad libitum access to tap water for 48 h before ulcer induction. Gastric mucosal damage was induced in conscious rats by gavage of 1.0 mL/rat of absolute ethanol (99.5%) (Robert, 1979). The test drug l-citrulline was dissolved in normal
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saline. Animals were randomised into five groups (n = 8): control, ethanol, l-citrulline (300 mg/kg) + ethanol, l-citrulline (600 mg/kg) + ethanol, and l-citrulline (900 mg/kg) + ethanol. All the rats in groups were pre-treated by gavage with lcitrulline for 7 days prior to the ethanol administration. The animals were sacrificed 60 min later, and the blood samples were obtained with no addition of anticoagulants for centrifugation at 3000 × g for 10 min for subsequent determination of clear serum. In parallel, the murine stomachs were rapidly removed, opened along the greater curvature and rinsed thoroughly with normal saline, followed by macroscopic determination of the gastric mucosal injury index. Thereafter, each stomach was dichotomised, with one moiety of stomach immersed in 4% formaldehyde for histological evaluation and gastric mucosa from the other moiety stored at −80 ◦ C for biochemical determinations.
2.3.
Determination of gastric ulcer index
An observer who was unaware of the treatment regimen performed the determination of the severity of gastric mucosal lesions. Lesion size (mm) was measured along its greatest length, and in the case of patches, five such lesions were considered the equivalent of a 1 mm ulcer. The sum of the lesion lengths in each group of animals was divided by its number and expressed as the mean gastric haemorrhagic lesion index (Qiu et al., 1991).
2.4.
Histological procedure and assessment
For pathological examinations, all samples were fixed in 4% formaldehyde buffer. The tissues were embedded in paraffin, and pathological sections were sliced along the longitudinal axis. From each sample, 5-m-thick sections were obtained and stained with haematoxylin–eosin to evaluate gastric morphology.
2.5.
Biochemical examinations of stomach tissues
Following the macroscopic analyses, determination was conducted on the activities of SOD, GSH-px, NOS and MPO as well as the levels of NO, MDA and GSH in rat stomach tissues. To prepare the tissue homogenates, stomach tissues were thawed and then treated in ice-cold normal saline at a ratio of 1:9 (w/v). The mixtures were homogenised on ice using an Ultra-Turrax homogeniser for 15 min. Homogenates was centrifuged using a refrigerated centrifuge at 4 ◦ C. Then, these supernatants were used for the determination of biochemical examinations.
2.5.1.
Determination of SOD activity
Measurements were made as described by Sun et al. (Sun et al., 1988). SOD estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which reacted with nitro-blue tetrazolium (NTB) to form formazan dye. SOD activity was then measured at 550 nm by the degree of inhibition of this reaction. One unit of enzyme was defined as the amount of enzyme required at the inhibition
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rate of 50%. The activity of SOD was expressed as unit/mg protein.
2.5.2.
Determination of MDA level
The level of MDA in the gastric mucosa designated as an index of lipid peroxidation was measured according to the modified method of Ohkawa et al. (1989). Gastric mucosa was weighed and homogenised in 10 mL KCl (10%). The homogenate was supplemented with 8.1% sodium lauryl sulfate, 20% acetic acid and 0.8% TBA, and was thereafter boiled at 100 ◦ C for 1 h. After cooling, the reactants were supplemented with 2.5 mL n-butanol for vigourous agitation for 1 min and centrifugation for 10 min. Absorbance was measured at 532 nm and the results were expressed as nmol TBA/mg of protein.
2.5.3.
Determination of GSH level
GSH concentration was determined by the procedures of Elmann (1959). Briefly, 0.5 mL homogenate was mixed with 1.5 mL 0.15 M KCl and 3 mL deproteinisation solution. Each sample was centrifuged at 3000 rpm for 10 min and the supernatant was removed, followed by the addition of 2 mL phosphate solution and 0.5 mL DTNB into the 0.5 mL supernatant, with the absorbance read at 412 nm and compared with glutathione standards.
2.5.4.
Determination of GSH-px activity
GSH-px activity in the tissue homogenate was determined according to Paglia and Valentine (1967). In brief, the sample was prepared by the addition of an equal volume of 50 mM potassium phosphate buffer (pH 7.0, 0.25 mL) into the original tissue homogenate. Subsequent to the centrifugation of 10 min at 750 g, the supernatant was obtained for the determination of GSH-px activity. In a quartz cell, 2.8 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 2 mM EDTA–2Na was mixed with 0.1 mL of 150 mM glutathione, 0.1 mL of 84.4 mM NADPH and 0.1 mL of 66 U/mL glutathione reductase, followed by the addition of 0.1 mL of the supernatant. 5 min after preincubation at 25 ◦ C, 0.1 mL of 22 mM H2 O2 was added and mixed to commence the reaction. The variations in absorbance of the reaction mixture at 340 nm during the first 100 s of the reaction were monitored, and the rate of decrease in absorbance was calculated by the linear proportion to the observed variations. The variations in NADPH were calculated using an extinction coefficient (M) value of 6220 M−1 cm−1 . GSH-px activity was expressed as unit/g protein.
2.5.5.
Determination of NOS activity
NOS activity was measured on the basis of the formation of l-citrulline from l-arginine (Bredt and Snyder, 1989). Frozen tissues from the stomach mucosa were homogenised at 0 ◦ C in 5 vol buffer containing 320 mmol/L sucrose, 1 mmol/L dldithiothreitol, phenylmethylsulphonyl fluoride (100 g/mL), leupeptin (10 g/mL), soybean trypsin inhibitor (100 g/mL) and aprotinin (2 g/mL) in 50 mmol/L HEPES (pH 7.0). Following centrifugation of the homogenate at 100 000 × g for 1 h, the supernatants were added to the reaction mixture containing 50 mmol/L Tris (pH 7.4), l-[U-14C]arginine (specific activity 11.8 GBq/mmoL), 10 g/mL calmodulin, 1 mmol/L CaCl2 , and
50 mmol/L l-valine. The samples were incubated for 20 min at 37 ◦ C prior to the termination of the reaction by the addition of 0.1 vol of 20% (v/v) HClO4. l-[U-14C]Citrulline was isolated from l-[U-14C]arginine by passage through Dowex 50W (Na+ form, Sigma–Aldrich, St Louis, MO, USA) and quantified by liquid-scintillation counting. The level of iNOS activity was measured by the addition of 1 mmol/L EGTA and total NOS (TNOS) activity by the addition of 2 mmol/L l-NMMA. The cNOS activity was determined by subtraction of iNOS activity from TNOS activity. Protein concentration of the supernatant was determined by spectrophotometry using a commercial assay kit (BioRadLaboratories, Richmond, CA, USA).
2.5.6.
Determination of MPO activity
MPO activity, a marker of neutrophil infiltration (Krawisz et al., 1984), was assayed according to the modified method of Bradley et al. (1982). In brief, the specimens were homogenised in 50 mM potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide (Sigma–Aldrich, St Louis, MO, USA). Suspensions were then centrifuged and MPO in the resulting supernatant was assayed with a spectrophotometer (Shimadzu UV2450, Shimadzu Corporation, Tokyo, Japan). One unit of MPO activity was defined as degrading 1 mol of peroxide/min 25 ◦ C. Proteins were measured with a modified bicinchoninic acid method with a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Results were expressed as units/g of protein.
2.5.7.
Determination of NO level
The NO content in the gastric tissue was measured with the method of nitric acid reductase, and the operational processes were measured in accordance with the NO kit instructions. Results were expressed as mol/g of protein.
2.6.
Cytokines assay
Serum levels of cytokines (IL-6 and IL-10) were evaluated by eBioscience kits (San Diego, CA, USA) for rats IL-6 and IL-10 by the manufacturer’s instruction. Primary antibodies were coated on 96-well plates, followed by removal of the non-specific binding by washing. Serum samples or cytokine standards (100 L) were respectively loaded into each well. The biotin-conjugated secondary antibodies were added into each well. Avidin-HRP and substrate solution were added for colour reaction, with the absorbance measured at 450 nm with an ELISA reader (Zenyth 3100; Anthos Labtec Instruments GmbH, Salzburg, Austria). A standard curve was run on each assay plate using recombinant IL-6 and IL-10 in serial dilutions.
2.7.
Statistical analysis
All results were expressed as means ± SEM. The data were evaluated with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The statistical significance of differences for each parameter among the groups was evaluated by one-way ANOVA, followed by Dunnett’s t test. The significance level was set at P < 0.05.
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3.
Results
3.1. index
Effect of l-citrulline pre-treatment on gastric ulcer
In this experiment, only petechiae were observed in the murine gastric lining in the control group. The injection of absolute ethanol into the gastric lumen induced gross lesions in the stomach. l-Citrulline gavage prior to ethanol administration significantly reduced the ulcerogenic effect of ethanol. As shown in Fig. 1, l-citrulline at different doses significantly reduced the ethanol-induced gastric ulcer index, particularly at the dose of 900 mg/kg (P < 0.01).
3.2.
Histological results
The morphological study of the stomach of normal animals showed no damage (Fig. 2A). However, in the group treated with absolute ethanol, it was observed that there were disruption and exfoliation of the superficial gastric epithelium, disappearance of the mucous cells from the upper portion of fundic glands, vacuolisation, necrosis, and karyopyknosis in the superficial gastric epithelium as well as mucous neck cells. In some areas, there was cell damage extending into the gastric glands. Moreover, there was apparent extensive
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Fig. 1 – l-Citrulline (l-Cit) at different doses on ethanol-induced gastric ulcer index by in rats. Data are the means ± SE for 8 animals. ***P < 0.001 compared to the control group; ## P < 0.05 compared to the ethanol group; ### P < 0.01 compared to the ethanol group.
haemorrhage in the mucosa, and hyperaemia as well as inflammatory cell infiltration in the mucosa and submucosa (Fig. 2B). Following l-citrulline pre-treatment, the pathological injuries were markedly attenuated, with only slight damage observed in the superficial epithelium (Fig. 2C).
Fig. 2 – Effects of l-citrulline (l-Cit) on histological findings of gastric damage induced by ethanol in rats after haematoxylin and eosin staining: control (A), ethanol (B), l-citrulline 900 (mg/kg) (C).
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3.3.
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Biochemical assays
3.3.1. Effect of l-citrulline on SOD and GSH-px activities and GSH and MDA levels in the stomach tissue of rats administered with ethanol As shown in Table 1, the level of MDA, an index of lipid peroxidation, was significantly increased to 0.770 ± 0.021 nmol TBA/mg protein at 1 h after ethanol administration in comparison to the control group. This increase was not significant in animals with l-citrulline gavage. The activities of the antioxidant enzymes SOD and GSH-px were significantly decreased subsequent to intragastric administration of ethanol (P < 0.001), though, l-citrulline pre-treatment protected the gastric mucosal against the loss of antioxidant enzyme activity, resulting in a significant increase in enzymatic SOD and GSH-px activities approximate to the control levels. Similarly, ethanol-treated rats showed a significant decrease in the GSH content (P < 0.001), probably due to its consumption during oxidative stress. In contrast, rats pre-treated with l-citrulline showed only a modest decrease, with no significant depletion of GSH levels.
3.3.2. Effect of l-citrulline on NOS and MPO activities in the stomach tissue of rats administered with ethanol Table 2 shows the activities of MPO and NOS in all groups. There were higher MPO activity in the ethanol-treated group than in the normal control group (P < 0.01), while MPO activities were markedly weakened (P < 0.01) in the l-citrulline treatment groups. TNOS activity of the gastric mucosal tissues was found to be higher in the ethanol group compared to the normal control group (P < 0.01). Treatment with l-citrulline significantly decreased the TNOS compared with that of the ethanol group (P < 0.01). Additionally, the cNOS activity was lower in ethanol group compared to the normal control group, but was similar in both l-citrulline pre-treatment group and the control group. However, iNOS activity was significantly higher (P < 0.01) in ethanol-treated rats than in normal control rats. l-Citrulline pre-treatment significantly lowered the iNOS activity (P < 0.01).
3.3.3.
Effect of l-citrulline on gastric mucosal NO content
The contents of NO in gastric tissue are shown in Fig. 3; the NO content in the ethanol-treated group was significantly lower as compared to the normal control group (P < 0.01), while the NO contents in the l-citrulline-treated groups were significantly higher than those in the ethanol-treated group (P < 0.05), particularly at the dose of 900 mg/kg (P < 0.01).
3.4. Effect of l-citrulline on pro-inflammatory and anti-inflammatory cytokines The levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines in ulcerated rats upon pre-treatment against ethanol challenge in rats are presented in Fig. 4. Administration of ethanol significantly (P < 0.001) elevated the levels of IL-6 when compared with normal rats. In contrast, the elevation of IL-6 was prevented in rats that received l-citrulline (P < 0.01) as pre-treatment (Fig. 4A). Similarly, the level of anti-inflammatory cytokine IL-10 was moderately reduced (P < 0.001) when compared with normal control. However, the
Fig. 3 – Effects of l-citrulline (l-Cit) on levels of NO in rats gastric tissues. Data are the means ± SE for 8 animals. ***P < 0.001 compared to the control group; ## P < 0.05, ### P < 0.01 compared to the ethanol group.
rats pre-treated with l-citrulline (P < 0.01) elevated the IL-10 levels compared with the ethanol group (Fig. 4B).
4.
Discussion
Alcohol consumption can produce acute haemorrhagic gastric erosions, and excessive ingestion can results in gastritis characterised by mucosal oedema, sub-epithelial haemorrhages, cellular exfoliation, and inflammatory cell infiltration (Guslandi, 1987; Ko et al., 2004), which is essentially an acute inflammatory reaction. However, prophylactic treatment with l-citrulline attenuated ethanol-induced gastric injury in a dose-dependent manner, with significant reduction of the gastric ulcer index. This study demonstrates that l-citrulline has gastroprotective effect against ethanol-induced gastric ulcer, when administered at oral doses between 300 and 900 mg/kg, particularly at the dose of 900 mg/kg. Despite the widely accepted notion that alcohol abuse leads to detrimental consequences in the gastrointestinal tract, the mechanisms underlying the ethanol-induced gastric mucosal injury still remain obscure. There is growing evidence that ethanol-induced gastric mucosal injury is closely related to the increased ROS level and the major source of ROS is from the activated neutrophils (Pan et al., 2008). On the other hand, organisms per se have enzymatic and non-enzymatic defences, including GSH, SOD and GSH-px against the ROS-induced lipid peroxidation (Mates et al., 1999). Thus, in order to address the role of oxidative stress in our model, we assessed several oxidant-antioxidant parameters in murine gastric tissues. The experimental results showed that ethanol markedly increased MDA, an index of lipid peroxidation, accompanied by a decrease of GSH, GSH-px and SOD, all of which are endogenous antioxidants. Our data support an imperative role for oxidative stress in the pathogenesis of ethanol-induced gastric ulcer. Nevertheless, treatment with lcitrulline resulted in significant increases in the activities of SOD and GSH-px and the levels of GSH, as well as a decrease in MDA formation, reflecting its antioxidant potential. The mechanisms which underlie l-citrulline-mediated waning of
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Table 1 – Effect of l-citrulline on SOD and GSH-px activities and GSH and MDA levels in the stomach tissue of rats administered with ethanol. Group
MDA (nmol TBA/mg)
Control group Ethanol group l-Cit 300 mg/kg l-Cit 600 mg/kg l-Cit 900 mg/kg
0.365 0.770 0.632 0.524 0.446
± ± ± ± ±
SOD (unit/mg)
0.014 0.021*** 0.017* 0.022** 0.024**
224.38 112.72 135.70 174.32 203.96
± ± ± ± ±
GSH (mg/g)
5.985 4.536*** 4.061* 7.592** 5.111**
9.82 2.74 4.04 6.49 8.35
± ± ± ± ±
0.404 0.237*** 0.328** 0.341** 0.234**
GSH-px (unit/g) 241.29 116.78 136.85 166.00 202.49
± ± ± ± ±
8.872 6.157*** 6.561* 7.656** 6.384**
Data are presented as the means ± SEM for 8 animals. P < 0.05 as compared to the ethanol control group. ∗∗ P < 0.01 as compared to the ethanol control group. ∗∗∗ P < 0.001 as compared to the control group. ∗
Table 2 – Effect of l-citrulline on NOS and MPO activities in the stomach tissue of rats administered with ethanol. Group
MPO (unit/g)
Control group Ethanol group l-Cit 300 mg/kg l-Cit 600 mg/kg l-Cit 900 mg/kg
0.197 0.773 0.654 0.473 0.292
± ± ± ± ±
0.008 0.031*** 0.025* 0.015** 0.012**
TNOS (nmol/min/g) 2.707 4.657 4.030 3.580 3.106
± ± ± ± ±
0.038 0.069*** 0.053* 0.060** 0.038**
cNOS (nmol/min/g) 2.270 1.494 2.161 2.306 2.185
± ± ± ± ±
0.051 0.058*** 0.048* 0.076** 0.065**
iNOS (nmol/min/g) 0.437 3.164 1.869 1.274 0.921
± ± ± ± ±
0.047 0.056*** 0.059* 0.113** 0.036**
Data are the means ± SEM for 8 animals. P < 0.05 as compared to the ethanol control group. ∗∗ P < 0.01 as compared to the ethanol control group. ∗∗∗ P < 0.001 as compared to the control group. ∗
oxidative stress in ethanol-induced gastric ulcer could be attributed to the direct antioxidant and free radical scavenging activity of l-citrulline or indirectly due to augmentation of intracellular GSH, GSH-px and SOD in the rat gastric tissues, all of which can scavenge superoxide, hydrogen peroxide, hydroxyl and lipid peroxyl radicals, and attenuate damages to the tissue. In addition, this observed antioxidant activity of l-citrulline might be due to the recycling pathway from lcitrulline to l-arginine and NO. The recycling pathway might be crucial in sustaining the production of NO, which is the active species in reducing the oxidative stress (Hayashi et al., 2005). Thus, we might make a hypothesis that the gastroprotection of l-citrulline might rest on its antioxidant effects.
There is growing evidence that the major source of ROS is from the activated neutrophils (Pan et al., 2008) and neutrophils play an vital role in the development of gastric damage by their aggregation and release of tissue-disrupting substance, such as oxygen free radicals and proteases (Kobayashi et al., 2001). The neutrophil infiltration into the gastric mucosal tissues is assessed by MPO as well as NOS (Coskun et al., 1996; Takeuchi et al., 1998). In the present study, we observed a significant increase in MPO activity in the stomach following ethanol administration, which confirmed the infiltration and activation of neutrophils in the gastric mucosa produced by ethanol. In our experiment, prior administration of l-citrulline also exhibited an inhibitory effect on the
Fig. 4 – Effects of l-citrulline (l-Cit) on levels of pro-inflammatory cytokine: IL-6 (A) and anti-inflammatory cytokine: IL-10 (B) in the gastric mucosa of ulcerated rats. Data are the means ± SE for 8 animals. ***P < 0.001 compared to the control group; ## P < 0.05, ### P < 0.01 compared to the ethanol group.
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increased MPO activity. It is also widely accepted that, in the digestive systems, cNOS-generated NO is cytoprotective and iNOS-generated NO is cytotoxic (Nishida et al., 1998). In the present study, we observed an increase in cNOS activity and a decrease in iNOS activity when rats were pre-treated with l-citrulline. Moreover, l-citrulline pre-treatment prevented the decrease of NO level in the gastric mucosa induced by ethanol. Thus, we estimate the protective effect of l-citrulline against ethanol-induced gastric ulcer in rats is closely related to NO generated by cNOS through l-arginine converted from l-citrulline. Our previous study confirmed that l-citrulline pretreatment prevented the increase in iNOS activity and iNOS protein expression in rats subjected to ischaemia–reperfusion (IR) (Gou et al., 2011). Thus, we hypothesise that the NOS activities might be related to the NOS protein expression in the murine stomach subjected to ethanol. These results suggest that ethanol led to the neutrophil-infiltration into the gastric tissues while l-citrulline administration attenuated the injury to the gastric mucosa. NO and ROS exert multiple modulating effects on inflammation and play key roles in the regulation of immune responses. Large amounts of NO, generated primarily by iNOS can be toxic and pro-inflammatory (Guzik et al., 2003). During the course of an inflammatory response, large amounts of NO formed by iNOS surpass the physiological amounts of NO (Xie and Nathan, 1994), which is of interest to our study of the levels of inflammatory cytokines. Interleukins play a vital role in the regulation of the mucosal defence barrier. Proinflammatory cytokines such as IL-6 in the gastric mucosa significantly increased and anti-inflammatory IL-10 significantly decreased in gastric ulceration (Paglia and Valentine, 1967), which was consistent with the results observed in this model. However, l-citrulline prophylactic treatment inhibited the depletion of IL-10 and the elevation of IL-6, which showed its anti-inflammatory effect in ethanol-induced gastric ulcer. The anti-inflammatory effect of l-citrulline might be closely related to the decrease of iNOS activity. In conclusion, gastric ulceration in this model was associated with gastric lipid peroxidation, neutrophil infiltration into the stomach and inflammatory reaction may be responsible for the spontaneous occurrence of ethanol-induced ulcers in rats. Based on the results of our study, l-citrulline was confirmed to have protective effects on ethanol-induced gastric ulceration in rats. The effect of l-citrulline can be attributed to its reduction of oxidative damage, and its inhibitory effects on neutrophil infiltration as well as its anti-inflammatory effects in rat stomach tissues.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgements The authors are cordially indebted to these financial supports: “Qing-Lan” Project of Jiangsu Province, the Industrialization of Scientific Research Promotion Projects of Universities and Colleges in Jiangsu Province (2011-16), the Natural Science Fund for Universities and Colleges in Jiangsu Province (09KJB350003
and 11KJB350005), Laboratory of Biological Therapy for Cancer of Xuzhou Medical College (JSBL0803, C0903 and C0904), the Science and Technology Plan Projects of Xuzhou (XF11C037 and XF11C062), Superiority Academic Discipline Construction Project of Jiangsu Higher Education Institutions, and Xuzhou Public Service Platform Projects of Drug Discovery and Research, Innovation Project of Postgraduates in Jiangsu Province, China (CXLX11-0752).
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