INTRODUCTION
Ulcerative colitis (UC) is a common inflammatory bowel disease (IBD) mediated by an autoimmune process that significantly leads to extensive mucosal disruption and ulceration [42]. Despite innumerable research in the field of IBD, the exact aetiology of UC is still unclear, and their pathogenesis is complex, requiring the co-existence of environmental and genetic factors. However, research has suggested that both UC and Crohn’s disease result from an abnormal immunological response to microflora present in the digestive system [39]. Untreated UC may proceed to colorectal cancer, life-threatening respiratory and/or circulatory complications [16]. The severity of the disease is associated with the extent of colonic barrier disruption with subsequent exposure of the mucosa and submucosa to proinflammatory and environmental factors [45, 46, 49, 58]. These factors can directly or indirectly activate local mesenchymal cells, such as fibroblasts, myofibroblasts, and smooth muscle cells, for the expression of increasing amounts of extracellular matrix components (ECM) such as collagens or fibronectins. These proceedings eventually end in fibrosis, goblet cell inhibition, and disruption of the colonic wall histological building [38, 45, 52]. In addition, the associated imbalance between tissue matrix-metalloproteinases, which degrade ECM, and tissue inhibitors of metalloproteinases, which inhibit ECM degradation, adds to the alteration of the colonic wall construction [38].
The serious complications of UC and the lack of presence of proper drug therapy necessitates more scientific trials to choose and/or discover alternative drug candidates with improved tolerability, minimum side effects, and more safety for the treatment of this disease. There are numerous methods to ameliorate the signs and symptoms of IBD, but there is no method to permanently cure this disease [2]. This leads to a search for new therapeutic strategies.
Obestatin is reported in many studies to possess protective and therapeutic effects on gastrointestinal tract inflammatory diseases [10, 11, 14]. Chemically, obestatin is a ghrelin-associated peptide formed of a 23-amino acid peptide derived from the post-translational processing of the preproghrelin gene [61]. Biologically, obestatin was identified in several organs such as the stomach, endocrine pancreas, adipose tissue, the lung, liver, skeletal muscle, mammary glands, and the male reproductive system [23, 26, 40]. Nevertheless, the stomach comprises the major source of circulating obestatin [13, 23].
In experimental models, administration of obestatin exhibited numerous therapeutic effects mediated through activation of receptors like the G-protein-coupled receptor [60] and the glucagon-like peptide 1 receptor in adipocytes and pancreatic beta cells [24, 25]. The main reported effects were suppression of the motility of the gastrointestinal tract, inhibition of cellular oxidative stress induced by molecular H2O2 [57], and regulation of the secretion of insulin [24, 25]. Accordingly, obestatin was considered a promising therapeutic agent in managing many clinical syndromes [27, 35, 62], particularly those related to the digestive system [47, 57]. Moreover, the role of obestatin in augmenting the healing of gastric ulcers via increasing both blood flow in gastric mucosa and cell proliferation has been reported [17]. Inflammatory bowel disease is another example with the advantage of using the ratio of obestatin to ghrelin in the serum as a corresponding marker for monitoring the severity of the accompanying inflammatory process [2, 30].
Experimentally development of colitis and related gastric complications induced by acetic acid [39] or dextran sodium sulfate [43] were significantly suppressed following administration of obestatin. Even though these studies proved the therapeutic effect of obestatin, the exact protective mechanisms, and the associated histopathological changes, are still in need of deeper exploration.
From the aforementioned studies relating to the pathogenesis of UC and the proposed therapeutic effect of obestatin, this study aims to explore the biological roles of obestatin as an antifibrotic, anti-inflammatory, and antioxidant agent on a murine model of acetic acid (AA)-induced colitis, and to investigate whether this action is associated with changes in the expressions of proliferating cell nuclear antigen (PCNA), nuclear factor kappa B (NFkB), and caspase-3, and further to explore the concomitant changes in the levels of oxidative stress parameters, antioxidant enzymes, tissue myeloperoxidase (MPO) activities. The expressions of ghrelin and fibrogenic markers like collagen and fibronectins in mucosal tissues of control and in AA-induced colitis rats are also pursued.
MATERIALS AND METHODS
A total of 40 healthy male albino Wistar rats weighing 200–250 g were obtained from the Animal breeding unit at (Taibah University). All the study steps were strictly designed and followed by the Ethical Committee of Taibah University Research Board and were in line with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH Publication 85-23 Rev. 1985).
All animals were kept in cages at room temperature under a 12 h light-dark cycle. Prior to induction of colitis, the animals were fasted for 16 h with free access to water through ad libitum. Then, the animals were provided with normal basal diets containing 21.1% of protein, 5.1% of fat, 60.0% of carbohydrates, 3.9% of fibre, 7.9% of minerals, and 2.0% of vitamins.
The rats were randomly categorized into four groups (10 rats each). Group I (control) was treated with saline, group II (treated with obestatin only), group III (AA-induced colitis), and group IV (AA-induced colitis + obestatin). Starting 24 h before the rectal enema, the rats were treated with intraperitoneally (i.p.) saline (groups I, II and III) or obestatin (group IV) twice per day for 4 consecutive days. Rat obestatin (Yanaihara Institute, Shizuoka, Japan) was given at dose of 16 nmol/kg [14, 17, 39].
After pentobarbital (30 mg/kg i.p.) anaesthesia, 1 mL of 3.5% (v/v) AA diluted in saline was used to induce colitis in groups III and IV by a rectal enema using a polyethylene catheter containing AA solution inserted into the rectum at 4.5 cm from the anus. Rats were kept in a vertical position during installation and for 1 min after installation to avoid solution leakage. Animals of groups I and II received 0.5 mL of saline 0.9% by the same procedure.
Fourteen days after the induction of colitis, rats were again anesthetized with pentobarbital (30 mg/kg i.p.) and sacrificed to collect the serum and colon tissue samples from all groups for biochemical and histopathological investigations. After excision of the colon, it was examined thoroughly for microscopic, immunohistochemical, and biochemical examinations.
The colon samples were fixed in 10% buffered formaldehyde and embedded in paraffin. Paraffin sections were processed and stained with haematoxylin and eosin, Alcain blue, and Masson trichrome stains according to the standard methodology. The stained sections were examined under a light microscope (Nikon Eclipse E600W, Japan), and the represented areas were photographed.
The haematoxylin and eosin-stained sections were further examined by the pathologist, uninformed about the treatment given. The grading of colonic damage (ulceration, inflammation, depth of the lesion, and fibrosis) and the number of goblet cells were determined using a scale of Vilaseca as described in detail previously [37, 54]. The standard techniques of staining of caspase-3, PCNA, and NFkB were followed as provided by the instructors [1, 18, 59].
The homogenates of colonic tissue were subjected to the estimation of oxidative stress antioxidant system markers, including malondialdehyde (MDA), total oxidant status (TOS), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (TAC) levels as previously analysed [19, 50]. Tissue MPO activity was identified in colonic tissue samples by colorimetric assays as previously reported in the literature [61]. The amount of MPO present in tissue samples that caused a change in absorbance measured at 460 nm for 3 min referred to one unit of MPO enzyme activity [28]. Ghrelin concentrations were estimated in the homogenates of the colonic tissue samples using commercially available EIA assay kits (Phoenix Pharmaceuticals, Burlingame, CA) as fully characterised in previous work by Ghomraoui et al. [22]. Hydroxyproline was estimated in acidified colonic tissues by adding 5 mL of 6N HCl for 3 h at 130°C, as reported previously in 1963 by Bergman and Loxley [6]. For assessment of fibronectin, a glass homogenizer was used to homogenize colonic tissues with 5–10 mL of ice-cold phosphate buffered saline (0.01 mol/L, pH 7.0–7.2). An ultrasonic cell disrupter was applied to the resulting suspension to destroy hard cell wall compartments for future extraction. Then, centrifugation of the homogenates for 5 min at 5000 g and the residue was used using an immunoassay ELISA kit (ABIN1874233, Atlanta, GA30338, USA) at a wavelength of 450 nm on a spectrophotometer. The fibronectin concentration was then determined by comparing the optical density of the samples to the standard curve as described by Attallah et al. [4].
Statistical analysis
All data were expressed as mean ± standard deviations (SD). The results obtained were statistically analysed by GraphPad Prism (version 7). In addition, a one-way ANOVA test followed by Tukey’s post hoc analysis was applied to compare and identify the significance between groups. The statistical significance was assigned at a p-value < 0.05.
RESULTS
In groups, I and II, tissue sections of rats exhibited normal colonic structure with intact colonic mucosa and numerous goblet cells on the surface epithelium and crypts (Fig. 1A, B). Notable pathological changes present in group III involved all colonic laminae (epithelial, propria, muscularis mucosae, and the submucosa). These laminae could not be distinguished from each other due to extensive inflammatory cell infiltration and ulceration (Fig. 1C). In addition to severe goblet cell depletion, an absence of crypts was evident. Treatment with obestatin in group IV did not completely alleviate the lesions but just restricted them (Fig. 1D). The mucosa retained its unharmed appearance with active goblet cells detected on the surface and the crypt’s epithelium.
The mucous secretions of the goblet cells in groups I and II stained Alcian blue stain demonstrated numerous deeply stained goblet cells lining the crypts and an intact film of mucus on the surface epithelium (Fig. 2A, B). On the other hand, group III shows nearly absent goblet cells and mucous, as evidenced by an extreme reduction in the intensity of Alcian blue stain. Group IV revealed few goblet cells and restored the intensity of the stain, particularly on the surface (Fig. 2C, D).
Masson trichrome stain of groups I and II detect minimal bluish stained collagen fibres in the lamina propria, muscularis mucosae, and the submucosa (Fig. 3A, B). Group III, on the other hand, showed increased expression of fibrosis in all colonic laminae (Fig. 3C). Group IV still shows extensive collagen fibres expression (Fig. 3D).
The immunohistochemical examination of PCNA of the mucosa of the colon of group I exhibited weak brown cytoplasmic positive immunoreaction in the epithelium and few cells in the lamina propria (Fig. 4A). the picture was more or less the same in group II (Fig. 4B). Group III exhibited marked increased immunoreactivity in both epithelial cells and cells of lamina propria (Fig. 4C). On the other hand, sections in the colonic mucosa of group IV demonstrated a decrease in positive immunoreaction compared to groups III (Fig. 4D).
NFkB immune staining of the group I demonstrated scanty brownish cytoplasmic positive immunoreaction in both the surface epithelium and the cells of lamina propria (Fig. 5A). Group II showed a similar presentation (Fig. 5B). Whereas sections of group III disclosed widespread marked immunoreactivity in all layers of colonic mucosa (Fig. 5C), while colonic mucosa of group IV displayed a limited presentation of the immunoreactivity to the mucosa and nearly absent reaction in the lamina propria (Fig. 5D).
Caspase 3 expression in the colon mucosal area showed a nearly absent reaction in both groups I and II (Fig. 6A, B). Group III showed a strong and diffuse reaction in all layers of the colonic mucosa (Fig. 6C). Group IV shows very weak reactivity in comparison (Fig. 6D).
The grading of colonic injury and a number of goblet cells was scored in Table 1. The results showed that normal rats treated with saline or obestatin showed no mucosal damage. However, rats with colitis induced by AA (group III) showed significant mucosal damage and remarkably decreased goblet cells number compared to control or obestatin-treated rats (group I and II). Group IV, on the other hand, showed a significant improvement in the colon mucosal damage score compared to group III rats. Additionally, the number of goblet cells was significantly increased in group IV compared with group III.
|
Groups |
I |
II |
III |
IV |
|
Microscopic grade |
0.89 ± 0.2 |
0.92 ± 0.1 |
13.6 ± 1.8a |
5.3 ± 2.6b |
|
Goblet cell |
51.3 ± 12.5 |
50.9 ± 11.9 |
18.7 ± 12.3a |
42.8 ± 15.2b |
The expression levels of MDA, TOS, and the activity of SOD, CAT, and TAC in tissue samples of group I and rats of the experimental groups are tabulated (Table 2). In groups I and II, the results showed no significant effect. However, group III showed a significant reduction in the concentration of MDA, TOS, and an increase in the activity of SOD, CAT, and TAC, respectively. In group III, AA induced a significant increase in the levels of MDA, TOS and a reduction in the activity of SOD, CAT, and TAC compared to groups I and II. In rats with colitis treated with obestatin (group IV), there was a significant (p < 0.003) reduction in the levels of MDA, TOS with more increased in the activity of antioxidant enzymes, SOD, CAT, and total cellular TAC.
|
Groups |
I |
II |
III |
IV |
|
MDA [nmol/g] |
4.1 ± 0.46 |
3.52 ± 0.38a |
7.5 ± 2.8b |
3.6 ± 1.3c |
|
TOS [µmol/g] |
1.86 ± 1.2 |
1.76 ± 1.5a |
4.8 ± 12.3b |
2.8 ± 1.4c |
|
SOD [U/mg protein] |
2.4 ± 0.98 |
2.8 ± 0.91a |
0.51 ± 04b |
3.6 ± 1.9c |
|
CAT [k/g protein] |
1.8 ± 0.65 |
1.9 ± 0.84a |
0.36 ± 0.45b |
1.6 ± 1.1c |
|
TAC [mmol/L] |
2.1 ± 0.48 |
2.8 ± 0.25a |
0.45 ± 0.25b |
2.3 ± 1.4c |
The expression levels of tissue ghrelin and MPO is shown in Figure 7. A significant increase in the tissue activity of MPO and a decrease in ghrelin levels in group III rats compared to control rats treated with saline only. In AA-induced colitis rats treated with obestatin (group IV), a significant reduction in the activity of MPO with an elevation in the levels of tissue ghrelin. A significant increase in the expression levels of fibronectin and hydroxyproline as markers of fibrosis (Fig. 8) was noted in group III compared to groups I and II. However, when treated with obestatin compared to group III, rats of group IV exhibited a significantly reduced expression of both fibronectin and hydroxyproline.
DISCUSSION
The current study results established the curative and protective effect of i.p. obestatin on the histopathological structure of the colon and the mucosal inflammatory and cellular antioxidant levels.
The model of colitis induced by AA investigates common colon diseases such as IBD as it induces a similar picture of inflammation together with ulceration [55]. The models of AA-induced colitis inject the acid with the tip of the catheter positioned at variable distances (from 1.2 [36] to 8 cm [44]) proximal to the anus verge. In this study, an intermediate depth of catheter insertion at 4.5 cm from the anus verge was chosen.
The curative outcome of obestatin was confirmed on the histopathological level as obestatin significantly amended the lesions grades, ulcerations, inflammatory cell infiltrations, and the loss of crypts induced by AA. The number of the goblet cells was significantly increased, and goblet cells were redetected with increased mucous secretions on the surface and crypt epithelium as evidenced by Alcian blue stain when compared with the AA-induce UC as likewise reported [31]. Obestatin significantly reduced colitis by increasing blood leukocytes and mucosal concentration of proinflammatory markers and leading to reduced histological signs of colonic damage [53]. These results also showed that obestatin improved the mucosal blood flow in the colon and decreased the local and systemic inflammatory processes.
As a marker of cell proliferation, PCNA immune stain was markedly increased in AA-induced colitis and decreased with obestatin. Similar results were reported [53]. In NFkB immune stain as a marker of coordination of chronic inflammatory condition of the colon also reported a marked increase in the AA-induced colitis and an apparent decrease in NFkB immunoreactivity with the use of obestatin indicating tissue induced anti-inflammatory effect [5]. At the same time, the indicator of apoptosis, caspase-3, was also markedly decreased in the obestatin treated group after induction of UC compared to the AA-induced colitis group. This indicator was helpful in a study on the heart proving the protection of the cardiac muscles with prior use of obestatin [3].
The administration of obestatin improved antioxidant status, reduced inflammation, increased the expression levels of tissue ghrelin, and reduced the activity of tissue MPO. At the same time, this curative effect was proved in AA-induced colitis, whereas fibrosis markers, inflammatory MPO markers, and oxidant markers such as MDA, TOS were significantly reduced, and the antioxidant enzymes SOD, CAT, and total TAC were significantly increased. The objective antifibrotic impact of obestatin was proven in our study by the marked inhibited expression of Masson trichrome stain in the treated group compared to the AA-UC group. These results are in line with others who reported that obestatin displayed preventive and therapeutic effects on acute pancreatitis [9–11], chronic gastric ulcers [17], and some models of colitis [39].
Oxidative stress is an important component of mucosal injury pathogenesis [32] which activates cellular apoptosis [21, 51], as evidenced in our study by the increased expression of caspase-3 as a marker of apoptosis. In contrast, deregulation or excessive apoptosis disrupts equilibrium and manifests variable clinical presentations [41, 48]. That is why most studies have focused on proposed treatment modalities with antioxidant and anti-inflammatory properties.
Colonic injury in this study was detected in the AA-colitis model with a significant increase in oxidative stress markers and histopathological changes. Oxidative stress is the key factor of AA-associated UC, where the overload of oxygen-free radicals induces disruption and apoptosis [15]. In addition, AA significantly augmented the reduction in the level of the antioxidant enzyme. Similarly, a decrease in the activity of tissue SOD, CAT, and the total cellular TAC was reported in rats with AA-colitis [20]. The inhibition of lipid peroxidation and improvement of cellular antioxidant status is one of the anti-inflammatory actions of obestatin and ghrelin as previously described [29, 34].
Ghrelin, an alternative product of preproghrelin, also had a therapeutic effect on the gastrointestinal tract [56]. It has a protective effect on gastrointestinal mucosa against damage caused by harmful factors [8, 29, 33] and potentially inhibits colitis development. Ghrelin has a protective effect on gastrointestinal mucosa against damage caused by harmful factors [7, 56] and potentially inhibits the development of colitis. Tissue ghrelin levels were identified to confirm the protective effect of obestatin against AA-UC and confirmed a significantly increased tissue ghrelin level in concomitant to the previously reported protective effect of ghrelin on gastrointestinal mucosa against damage with harmful factors [7, 29, 33].
In our study, the expression of tissue MPO was reported to be directly proportional to the number of inflammatory cell infiltration [12, 43]. It was suggested that obestatin inhibits polymorphonuclear cell recruitment during the progression of chronic colitis. Obestatin decreased tissue MPO levels significantly in rats with AA-colitis in the current study. At the same time, the histopathological examination reported that the inflammatory cell infiltration was markedly reduced. Accordingly, our results with others provided further evidence that obestatin exhibits anti-inflammatory actions and confirmed its curative, protection, and healing properties in this model.
Limitations of the study
We used Wistar rats as they are good models when studying colitis. However, some of the causes behind controversy may be the animal or species-related difference and this is one of the study limitations. Moreover, the effect related to multiple doses and long term use of acetic acid and obestatin may cause differences in results. Furthermore, the sex difference in the inflammatory response may be cause controversial results. We used male animals because although females have higher innate and adaptive immune responses than males, this contributes to increased susceptibility to inflammatory and autoimmune diseases in females compared with males. So it is recommended to repeat the experiment with multiple doses of drugs and with different animals and species with different sex.
Clinical applications
We recommend the use of obestatin in management of colitis through its anti-inflammatory, antioxidant, antiapoptotic and antifibrotic effect with regulation of regeneration of colic mucosa.
CONCLUSIONS
The current study concludes that pretreatment with obestatin at a dose of 16 nmol/kg inhibits the development of acetic acid-induced colitis. This effect significantly proceeded via a reduction in cellular inflammatory and overproduction of both cellular fibrosis and oxidative stress markers with a potential improvement of antioxidant status of the colonic cells. These data recommended that obestatin was independent anti-inflammatory, antioxidant, and antifibrotic against AA-induced colitis.
