NESFATIN-1 TREATMENT  ALLEVIATES INDOMETHACIN-INDUCED GASTRIC ULCER IN RATS

By

Dalia I. Abd Al-Aleem and Dalia M. Abd- Al-Mottaleb

Departments of Physiology and Pharmacology,  Faculty of Medicine, Zagazig University

ABSTRACT

Background: Indomethacin is a non-steroidal anti-inflammatory drug which is known to produce serious adverse effects including ulcerative lesions. Nesfatin-1, is an identified anorexigenic peptide to have antioxidant effects.

Objective: This study aimed to investigate the possible ameliorative effect of nesfatin-1 against indomethacin induced gastric ulcer in rats, with exploration of the possible mechanisms underlying this effect.

Materials and methods: Twenty four  adult male albino rats were the animal model of this study. Ulcers were induced using oral indomethacin (20mg/kg) daily for 7 days. The rats were randomly assigned to vehicle-treated control group (I), indomethacin-treated (II), and indomethacin-treated for 7 days followed by nesfatin-1 (1ug/kg i.p) daily for 10 days. Assessment of gastric  juice parameters (total acid output, pepsin activity and mucin content),gastric mucosal levels of malondialdehyde (MDA) , tumor necrosis factor –alpha (TNF-alpha) , nitrite and prostaglandin E2 (PGE2) levels were also determined. This in addition to assessment of gastric lesions and histopathologic examination of the gastric mucosa in each group.

Results: Nesfatin-1 displayed a significant  ameliorative effect in  gastric lesions induced by indomethacin as indicated by a significant decrease in ulcer index  and improved histopathology, along with a significant  reduction in measured gastric juice parameters .It also reduced both  gastric mucosal MDA and TNF-alpha significantly and increased nitrite and PGE2 levels in this ulcer model.

Conclusion: Nesfatin-1 attenuates indomethacin- induced gastric ulcer and potentiates  ulcer healing in the stomach of rats exposed  to chronic administration of indomethacin. This  effect  depended upon decrease in gastric secretion, increase in  nitric oxide and PGE2, besides an antioxidant anti-inflammatory role.

Keywords: Nesfatin-1, indomethacin, gastric ulcer, MDA, TNF-alpha, nitric oxide, rats.

INTRODUCTION

     Gastric ulcer is an illness that affects a considerable number of people world-wide. The etiological factors of this disorder include stress, smoking, nutritional deficiencies, frequent and indiscriminate use of non steroidal anti-inflammatory drugs (NSAIDs) (Khazaei and Salehi , 2006).

    The pathophysiology of gastric ulcer has generally focused on imbalance between aggressive and protective factors in the stomach (Lima et al., 2006). The gastric ulcerogenic action of NSAIDs is believed to occur mainly due to their local inhibitory effect on gastric prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) that are the main inhibitors of gastric acid secretion (Ribeiro-Rama et al., 2009), in addition to oxidative gastric damage and the generation of pro-inflammatory mediators (Kolgazi et al., 2014).

     Nesfatin-1 was identified as an anorexigenic factor associated with leptin and melanocortin signaling in the hypothalamus (Maejima et al., 2009 and Darambazar  et al., 2015).

     This hypothalamic  neuropeptide is composed of 82 amino acids with a molecular weight of 9.8 kDa, which is derived from the larger protein nucleobindin-2  (NUCB2) (Oh-I et al., 2006).

    As a brain-gut peptide, nesfatin/ NUCB2 is expressed in appetite-control hypothalamic nuclei, including the para-ventricular nucleus of the hypothalamus (PVN), supraoptic nucleus (SON), arcuate nucleus (ARC) and lateral hypothalamic area (LHA) (Kohno et al., 2007). Additionally, peripheral tissues, such as the stomach, adipose tissue, the heart and pancreas were considered as sources for this peptide (Stengel et al., 2009, García-Galiano et al., 2010, Foo et al., 2010, Zhang et al., 2010, Angelone et al., 2013 and Stengel et al., 2013).

     Interestingly, NUCB2 mRNA expres-sion is 10-times higher in the rat gastric mucosa than in the brain giving rise to a predominant production of nesfatin-1 in the stomach (Stengel et al., 2009). Nesfatin-1 regulates gastrointestinal motility  and, in particular, it inhibits gastric contractions in the fasted state (Watanabe et al., 2015).

It is worth mentioning that nesfatin-1 has neuroprotective function in rats with subarachnoid hemorrhage through anti-inflammatory and antiapoptotic properties (Ozsavci et al., 2011 andTang et al., 2012).

     The present study aimed to investigate the possible ameliorative effect of nesfatin-1 against indomethacin induced gastric ulcer in rats, with exploration of the possible mechanisms underlying this effect.

MATERIALS AND METHODS

     All the experimental procedures were conducted in accordance with the guiding principles for the care and use of research animals and were approved by the Institutional Review Board of Faculty of Medicine, Zagazig University.

Drugs and chemicals: Nesfatin-1 was obtained from Sigma chemical Co. (St. Louis, MO. USA) as a powder and was dissolved in normal saline. Indomethacin was obtained from Sigma chemical Co. (St. Louis, MO. USA), and was suspended in 0.5 % carboxymethylcellulose (Merck, Darmstadt, Germany).

Animals: Twenty four adult male albino rats of a local strain weighing 130-160 grams were purchased from the animal house of Faculty of Veterinary Medicine-Zagazig University. Animals were housed in plastic cages (40x28x18cm -4/cage) and given tap water ad libitum, fed with standard commercial rat chow and were left to accommodate for one week before dosing , normal  light ⁄  dark regular cycles  in partially humid and well aerated room.

Experimental design: The animals were randomly divided into three equal groups : (I) Vehicle-treated control group: in which the animals received  carboxymethyl- cellulose (0.5%) orally daily for 7 days then saline was administered intraperito-neally (i.p.) for 10 days   (II) indometha-cin-  treated group, chronic gastric ulcer induction where  the rats received oral indomethacin in a dose of 20 mg⁄ kg  suspended in 0.5% of carboxymethyl-cellulose daily for 7 days (Tariq et al., 2007), then received i.p saline for 10 days. (III) indomethacin-  treated group followed by nesfatin-1 : in which animals were given oral indomethacin in a dose of 20 mg⁄ kg  suspended in 0.5% of carboxymethylcellulose daily for 7 days , then given Nesfatin-1 (1ug/Kg) intraperitoneal for 10 days (Kolgazi et al., 2014).     At 17^th day, rats were fasted for 16 hours prior to sample collection (Xia et al., 2012)

Pyloric ligation: It was carried out at the end of the study period in order to collect gastric secretion (Alumets et al., 1982). This was done under ether anesthesia, a midline abdominal incision was performed, the pyloric portion of the stomach was gently mobilized and carefully ligated with a silk ligature around the pyloric sphincter taking care not to interfere with gastric blood supply. Abdominal incision was sutured and the animals were allowed to recover from anesthesia, the stomach was excised,

Analysis of gastric juice: Three hours after the ligation, rats of all groups were euthanized with an overdose of ether, their stomachs were rapidly removed, opened by an incision along the greater curvature and the gastric juice were collected. Gastric juice from each animal was centrifuged at 1000 g for 10 minutes to remove any solid debris and the volume of the supernatant was measured. The supernatant was then assayed for total acid concentration (Hara et al., 1991). The total acid output was calculated by multiplying the volume of gastric juice by the total acid concentration. Also, pepsin activity (Sanyal et al., 1971) and mucin content  (Winzler, 1955) were determined.

Quantification of ulceration: Degrees of ulceration in the indomethacin-treated animals were quantified using the procedure outlined by Szabo and Hollander, (1985). Briefly, cleaned stomachs were pinned on a corkboard and ulcers were scored using dissecting microscope with square-grid eyepiece based on grading on a 0–5 scale (depicting severity of vascular congestions and lesions/hemorrhagic erosions) as follows:0= Almost normal mucosa,1= Vascular congestions, 2= One or two lesions, 3= Severe lesions, 4= Very severe lesions, 5= Mucosa full of lesions. Mean ulcer score for each animal was expressed as ulcer index.

Biochemical analysis of gastric mucosa: The stomach was rinsed with saline and divided into three parts. Gastric mucosa of one part was scrapped and  homogenized in 2 ml normal saline containing 0.1 M dithiothreitol and centrifuged at 2000 r.p.m for 10 minutes at room temperature. The supernatant was used for determination of prostaglandin E2 (PGE2) level by ELISA using PGE2 immunoassay kit (R&D systems, USA) according to Granstrom et al. (1980).

    The mucosa of another  part of the stomach was also scrapped, homogenized in cold potassium phosphate buffer (0.05 M, pH7.4) and centrifuged at 2000 r.p.m for10 minutes at 4^oC; the supernatant was then kept at – 80^oC for measurement of malonaldehyde (MDA) using Oxis kits (MDA586, Oxis International, Foster City, CA, USA) (Niehaus  and  Samuelsson, 1968)  , and  TNF-alpha  using  enzyme linked immunosorbent assay kit (Abcam, Catalog No. ab100785, USA)(He and Ting, 2002). Also the total nitrate⁄nitrite in the gastric mucosal homogenate was assayed after reduction of nitrate to nitrite using the cadmium reduction method (Sastry et al., 2002).

Histological studies: These were performed according to the method previously described (Ogihara and Okabe, 1993). At autopsy, small pieces of tissue, including ulcers, were embedded in paraffin and sectioned at 5µm in an automated microtome. Haematoxylin and eosin staining was done. Tissue contraction, regeneration of the ulcerated mucosa, formation of granulation tissue, glands arrangement and inflammatory exudates were observed under the microscope.To get the histopathological picture of the ulcer before healing, a pilot study was done on the stomach mucosa on day seven of treatment with indomethacin.

Statistical analysis: Data were presented as mean ± S.D and statistical significance was determined by one way analysis of variance (ANOVA) followed by LSD test, P values less than 0.05 were considered to be significant. In statistical analysis, SPSS version 18 program for Windows (SPSS Inc. Chicago, IL, USA) was used.

RESULTS

     Indomethacin significantly increased gastric juice total acid output as compared to control group .In contrast, Nesfatin-1 treatment significantly reduced total acid output in group III. Pepsin activity significantly increased by indomethacin as compared with the control group. Rats treated with Nesfatin-1 showed significant reduction in pepsin activity when compared with that of group II. Also, indomethacin significantly reduced gastric juice mucin content in group II compared to the control group. In Nesfatin-1 treated rats, this value significantly raised when compared with that of group II (Table 1).

Table (1): Total acid output, pepsin activity and mucin content of gastric juice in all groups (Mean ± SD).

*=significant, a (versus group I), b (versus group II)

Indomethacin significantly increased gastric  mucosal MDA levels as compared to control group. In contrast, Nesfatin-1 treatment significantly reduced MDA in group III. Gastric mucosal TNF-alpha levels significantly increased by indomethacin as compared with  the control group. Rats treated with Nesfatin-1 showed significant reduction in TNF-alpha levels when compared with that of group II. Also, indomethacin significantly reduced both gastric  mucosal content of PGE2 and nitrite  in group II compared to the control group . In nesfatin-1 treated rats, those values significantly raised when compared with that of group II (Table 2).

Table (2): Gastric mucosal MDA,TNF-alpha,PGE2 and nitrite  in all groups (Mean ± SD).

*=significant, a (versus group I),   b (versus group II)

     Indomethacin significantly increased gastric ulcer index as compared to control group. In contrast, Nesfatin-1 treatment significantly reduced this index in group III (p<0.001) (Fig.1).

Figure (1): Ulcer index of all groups.

Histopathology of the stomachs

      On 7 th day of indomethacin adminis-tration, treated rats in the pilot study showed sharply defined mucosal ulcer in stomach. Damaged mucosal epithelium, glands, inflammatory exudates, prolifera-ted fibroblast and cellular debris were found in the ulcerated wall of stomach (Fig. 2b). No sign of healing was seen.

 On 17th day of the study, group II rats showed reduced inflammatory exudates along with some extent of mucosal regeneration, glandular organization and reduced size of ulcer (Fig. 2c). Group III, showed minimal inflammatory exudates and proper organization of glands (Fig.2d).

Figure 2: Histopathological examination of the stomach mucosa.

DISCUSSION

     The diversity of etiological factors underlying gastric ulcers and the complex nature of pathways participating in healing always make peptic ulcers treatment a complicated challenge (El-Moselhy et al., 2009).

     The current study highlighted the alleviating effect of Nesfatin-1 on indomethacin- induced gastric ulcer as it significantly improved all the deleterious effects induced by indomethacin on both gastric mucosa and gastric juice contents.  This effect was associated with down- regulation of inflammatory and oxidative stress markers together with increased in gastric mucosal levels of both PGE2 and nitric oxide. Szlachcic et al. (2013), found that peripheral and central administration of Nesfatin-1 inhibited stress induced increase in gastric acid and pepsin secretion in rats which is in keeping with the observations by Xia et al. (2012) who observed the inhibition of gastric secretion after a central administration of Nesfatin-1 that involved the vagal mechanism.

     In this regard, it is worthy to mention that peripheral Nesfatin-1 administrated by intravenous injection has been shown to be able to cross the blood-brain-barrier by a non-saturable mechanism (Pan et al., 2007 and Price et al., 2007).

     The result of the present work showed significant elevation in gastric MDA level in group II, an indicator for lipid peroxidation which is a well established mechanism for cellular injury (Kwiecien et al., 2002).  Nesfatin-1 treatment caused significant reduction in MDA level as compared to those of group II. It was reported that this peptide possesses free radicals scavenger activity (Kolgazi et al., 2014). This suggests that nesfatin-1 accelerated ulcer healing via antioxidant activity.

      The result of the current study showed significant elevations in gastric TNF-alpha in indomethacin treated group as compared to control group. This observation is in accordance with previous studies (Zhang et al., 2008 and Moustafa et al., 2013). Nesfatin-1 administration produced a significant   reduction of TNF-alpha in the gastric mucosa, adding also anti-inflammatory role to its beneficial effects. This is in accordance with the results of other investigators (Szlachcic et al., 2013 , Kolgazi et al., 2014 and Ozturk et al., 2015).

     Nitric oxide (NO) plays a crucial role in the gastroprotection and ulcer healing mechanisms (Laine et al., 2008). The result of the present work showed marked reduction in mucosal nitrite level (as indicator of NO) in indomethacin- treated group. This is attributed to the ability of indomethacin to upregulate the endothelin-1 leading to decrease production of gastric mucosal NO (Slomiany and Slomiany, 2000). Nesfatin-1 was able to increase the nitrite levels in this ulcer model in group III.

      In the present study, rats treated with nesfatin-1 showed a significant increase in PGE2 level in gastric mucosa. PGE2 influences virtually every component of the mucosal defense; stimulates mucus, bicarbonate; maintains mucosal blood flow; enhances the resistance of the epithelial cells to injury induced by cytokines (Brzozowski  et al., 2005).In addition, our results showed that , oral administration of indomethacin resulted in a significant reduction in gastric mucosal PGE2 level, which may be through  inhibition of cyclooxygenase (Bandyo-padhyay et al., 1999) .

     NO was reported to increase PGE2 biosynthesis in vivo through CGMP dependent mechanism (Abi-Gerges et al., 2001) and it is possible to assume that NO might regulate the release and or the biosynthesis of PGE2 in the stomach after damage  during ulcer healing. Gastropro-tective effects of Nesfatin-1 may involve endogenous PG and NO production. Nesfatin-1 exerts  hyperemic effect on the gastric mucosa and that the selective or non-selective cyclooxygenase (COX) blockers reduced this effect (Szlachcic et al., 2013).

CONCLUSION

     Nesfatin-1 attenuated indomethacin- induced gastric ulcer and potentiated  ulcer healing  in the stomach of rats exposed to chronic administration of indomethacin, and this effect depended upon decrease in gastric secretion, increase in nitric oxide and prostaglandin E2, besides an antioxidant anti-inflammatory role.

ACKNOWLEDGMENT

     The authors like to acknowledge and appreciate the effort of Prof. Dr. Ola A. Megahed; Professor of Pathology, Faculty of Medicine, Zagazig University, for per-forming the ulcer index and histopatho-logical studies.

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