Sepia officinalis ink mitigates gastric ulcer via modulation of antioxidant/anti-inflammatory pathways

Abstract

Background

Sepia officinalis ink is a bioactive secondary metabolite rich in melanin granules, which has a wide range of nutritional and therapeutic values and also has been used to prevent various gastrointestinal disorders. Gastric ulcer, the most common gastrointestinal disease, is characterized by severe gastric mucosa damage, and its prevention is currently one of the main goals of clinical and experimental studies. Thus, the present study was focused on evaluating the potential gastroprotective efficacy of Sepia officinalis ink extract (SOIE) against ethanol-induced gastric ulcer in rats.

Results

The current results revealed that SOIE administration at the two selected doses improved significantly gastric mucosa integrity as indicated by the significant (P < 0.05) amelioration in gastric secretion indices (pH and volume) and the marked decrease in the ulcer index. Moreover, SOIE could counteract the gastric oxidative stress induced by ethanol via a marked decline in malondialdehyde content as well as a significant (P < 0.05) increment in glutathione content and antioxidant enzymes activities (catalase and glutathione-s-transferase). Additionally, SOIE treatment caused a significant (P < 0.05) reduction in gastric nitric oxide content. Respecting morphological and histopathological studies, SOIE treatment at 200 mg/kg body weight caused marked healing of gastric lesions as indicated by no hemorrhagic bands or injuries observed as well as significantly reduced severity score of ulcer.

Conclusions

SOIE could be used as a promising alternative antiulcerogenic compound to treat severe gastric lesions.

1 Background

The gastric ulcer is one of the most prevalent chronic gastrointestinal diseases with high morbidity of about 5–10% of the world´s population, being a major substantial threat to human health care organizations in the current century [1, 2]. The gastric ulcer is characterized by chronically inflamed lesions of the mucosal epithelium as a result of excess secretion of gastric acid as well as aggressive pepsin activity and ultimately, leading to severe gastric mucosal damage [3]. The damage of gastric mucosa is mainly caused by infection with Helicobacter pylori as well as the long period to the steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) administration and alcohol abuse [4]. These aggressive factors can disrupt gastric defensive and repair barriers, especially mucus-bicarbonate (HCO₃⁻) barrier, prostaglandins level, and epithelial renewal, eventually leading to mucosal irritation and ulceration [5].

The most commonly used ulcer model is abusive ethanol ingestion that mimics various features of acute human gastric ulcer condition [6]. Ethanol consumption is a highly damaging factor aggravating gastric mucosal damage via digestion of the mucosal layer and eventually, exposing the mucosa to the proteolytic as well as hydrolytic actions of HCL and pepsin, which in turn, leads to ulcer formation [7]. Furthermore, Aziz et al. [8] demonstrated that severe gastric lesions after ethanol consumption may be due to overproduction of oxygen free radicals (ROS), especially superoxide anion, hydroxyl radicals, and lipid peroxides which ultimately resulting in the decrease in cellular antioxidant levels. Besides overproduction of ROS, ethanol consumption-induced microvascular injuries by reducing blood flow and stimulating the production of pro-inflammatory cytokines [6]. Thus, the ethanol-induced acute gastric ulcer serves as a significant model for the assessment of prospective gastro-defensive potency of therapeutic natural products [9].

In the clinical studies, the conventional approach for the management of gastric ulcer focuses mainly on reducing the secretion of gastric acid as well as re-enforcing gastric mucosal protection via utilizing proton pump inhibitors, H₂ receptor antagonists, and prostaglandin analog as the mainstay remedy [10]. But, the long utilization of these drugs is facing major drawbacks due to their various undesirable adverse reactions which reduce their treatment efficacy [7]. Thus, identifying more effective as well as safe antiulcer agents is urgently desired for the prevention and treatment of gastric ulcer via controlling the formation of ROS as well as secretion of gastric acid. Recently, natural pigments from marine sources have become attractive as pharmaceutical agents due to their antioxidant potency in the treatment of gastric ulcer [11]. Apart from these effective marine pigments, Sepia officinalis ink is a bioactive secondary metabolite rich in melanin granules that are released as a self-defense for escaping from predators. It contains plenty of bioactive molecules such as protein, peptidoglycans, amino acids, lipid, and tetrodotoxin [12]. Additionally, sepia ink has a higher amount of trace elements which are essential nutrients for tissue repair and improving immune and digestive function [13]. Furthermore, sepia ink is considered a multifunctional marine bioactive material due to its wide range of biological activities such as antioxidant, anti-radiation, and antibacterial as well as antitumor [14, 15]. Respecting the nutritional and therapeutic values of Sepia officinalis ink, the current investigation is proposed to demonstrate the potential antiulcerogenic efficacy of Sepia officinalis ink against an ethanol-induced gastric ulcer in rats.

2 Materials

2.1 Sepia officinalis collection

The fresh cuttlefishes, Sepia officinalis, were collected from a fishmonger at Abu–Qir Bay on the Mediterranean coast, Alexandria, Egypt. Then, the samples were maintained in an icebox containing ice cubes until transported to the laboratory. At the laboratory, the samples were instantly washed under running tap water and then sterile water. Thereafter, the animals were posteroventrally dissected to acquire the ink gland, and then, the ink was gently squeezed into sterile brown containers.

2.2 Preparation of Sepia officinalis ink extract (SOIE)

The SOIE was prepared according to the method adopted by Fahmy et al. [16]. The collected ink was diluted immediately with an equal volume of distilled water and then mixed sufficiently. After dilution, the admixture was concentrated and lyophilized to a black residue using a lyophilizer (LABCONCO, shell freeze system, the USA).

2.3 Experimental animals

Adult male Wistar albino rats, Rattus norvegicus, weighing 150—170 g which aged forty-two days were used in the current study. Animals were purchased from the National Research Centre (NRC), Egypt. They were grouped and housed in spacious polypropylene cages (three animals/cage) bedded with sawdust and nesting (Kleenex tissues) material. The animal house was well-ventilated and maintained at a temperature of 23 ± 2 °C within a natural day/night cycle via the experimental period. Animals were nourished by commercial pellet diet (Agricultural- Industrial Integration Company, Giza, Egypt) ad libitum and drinking tap water (2 bottles fitted in each cage) ad libitum. Rats were acclimatized to animal house conditions for one week before the initiation of the study.

2.4 Ethical consideration

In the present study, experimental protocols and procedures accomplished were approved by the Cairo University, Institutional Animal Care and Use Committee (IACUC) (Egypt) (CU/I/F/47/21). All the experimental procedures were carried out in agreement with international guidelines for the care and use of laboratory animals.

2.5 Induction of gastric ulcer

The acute gastric ulcer induction was performed as Alam et al. [17] previously described, by using a single intragastric dose of absolute ethanol administration. Firstly, the animals were fasted for 24 h before gastric ulcer induction to prevent coprophagia, but they had free access to water up to 2 h before the experiment. The ethanol ulcerated rats were orally administrated absolute ethanol (100%, 5 ml/kg body weight).

2.6 Experimental design

Twenty-four adult male albino rats, Rattus norvegicus, were randomly divided into 4 groups (six rats/ each group) and treated as shown in Fig. 1.

Fig. 1

Schematic presentation of experimental design

Group 1: served as a control group; rats administrated orally distilled water.

Group 2: served as an ulcerated group; rats administrated orally a single dose of absolute ethanol (5 ml/kg body weight). After one hour, rats were administrated orally distilled water.

Group 3: Rats administrated orally a single dose of absolute ethanol (5ml/kg body weight). After one hour, rats were administrated orally SOIE at a dose of 100 mg/kg body weight.

Group 4: Rats administrated orally a single dose of absolute ethanol (5ml/kg body weight). After one hour, rats were administrated orally SOIE at a dose of 200 mg/kg body weight [18].

2.7 Animal handling

One hour after administration of all treatments, the rats were euthanized using an overdose of sodium pentobarbital, and their stomachs were immediately removed from six rats/group. The stomach samples were dissected and carefully opened along the greater curvature, where their contents were collected for volume and gastric pH determination as gastric ulceration indices. Then, the gastric tissue was rinsed gently with phosphate buffer saline (PBS) to remove any blood clots. Firstly, all gastric tissue samples were examined macroscopically to calculate gastric ulcer index, as Saheed et al. [19] described. Secondly, each stomach sample was divided into two parts; one-part was homogenized for the oxidative stress analysis, while the other part was immersed in 10% formalin for histopathological examination.

2.8 Evaluation of gastric mucosal injury

2.8.1 Determination of gastric acidity (pH measurement)

The gastric content was collected in centrifuge tubes and then centrifuged at 1000 r/min for 10 min at 4 °C. The resultant supernatant was used for pH measurement as an indicator of gastric acidity utilizing a digital pH meter (PB-11; Sartorius, Sartorius AG, Göttingen, Germany) as demonstrated by Bigoniya and Singh [20].

2.8.2 Morphological examination of gastric lesions

After collecting the gastric contents, the stomach was opened and distended on a clean white background and photographed to view the morphological alterations, induced by different treatments. Photographs were taken using a digital camera (10 megapixels 5× zoom). Furthermore, the degree of ulceration was graded by examination of the gastric cavity as adopted by Haule et al. [21] as follows:

Gastric mucosa lesions	Degree of ulcer severity
No lesions	Zero
Hyperemia	0.5
Hemorrhage spots	1
1–5 small ulcers (1–2 mm)	2
More than 5 small ulcers or one intermediate lesion (3–4 mm)	3
Two to more intermediate lesions or one gross lesion (> 4 mm)	4
Perforated ulcers	5

2.8.3 Gross gastric lesions assessment

The gastric mucosa ulcer seemed as elongated bands of hemorrhagic lesions parallel to the long curve of the stomach. The length of the ulcer (mm) was measured by a planimeter under dissecting microscope (magnification: 1.8×). Then, the ulcer index was determined based on ulcer length as follows: if the ulcer length is: ˂ 1.0 mm = 1, between 1 and 2 mm = 2, ≥ 3 mm = 3 [22].

Then, the ulcer index for each animal was calculated by the following equation [23, 24]:

$${\text{Ulcer}}\;{\text{index}} = {\text{ mean}}\;{\text{ulcer}}\;{\text{score}} \times {\text{ percentage}}\;{\text{of}}\;{\text{ulcerated}}\;{\text{animal}}$$

Furthermore, the ulcer preventive index was calculated by the following formula based on the recommendation of Mahmood et al. [25]:

$${\text{Preventive}}\;{\text{index}} = \frac{{{\text{Ulcer}}\;{\text{ index }}\;{\text{of}}\;{\text{ ulcerated}}\;{\text{ group}} - {\text{ Ulcer}}\;{\text{ index}}\;{\text{ of}}\;{\text{ treated }}\;{\text{group}}}}{{{\text{Ulcer}}\;{\text{ index }}\;{\text{of}}\;{\text{ ulcerated }}\;{\text{group}}}} \times 100$$

2.9 Evaluation of causative factors of SOIE gastroprotection

2.9.1 Assessment of gastric mucosal free radicals and antioxidant markers

Gastric mucosa was homogenized (10% w/v) in an ice-cold 0.1 M Tris HCl buffer (pH 7.4) and centrifuged at 3000 RPM/min for 15 min at 4 °C. The resultant supernatant was used for estimation of malondialdehyde (MDA), glutathione reduced (GSH), catalase (CAT), glutathione-s-transferase (GST) using the commercially available kits.

2.9.2 Assessment of Nitric oxide as an inflammatory marker

The NO level in the gastric tissue homogenate was measured using colorimetric kits obtained from the spectrum. Measurement of NO absorbance was performed at a wavelength of 450 nm.

2.10 Histopathological evaluation

Gastric tissue samples were washed in phosphate buffer saline and then fixed in 10% neutral buffered formalin (pH 7.0) for 24 h. Following fixation, gastric tissue samples were processed conferring to the established methods assumed by Nanji et al. [26] to obtain paraffin sections. The embedded paraffin sections were cut at 5 μm using an optical rotary microtome and then stained with hematoxylin–eosin (H&E). The sections were examined by a light microscope for assessment of severe morphological changes such as congestion, hemorrhage, edema, and erosions. Additionally, gastric microscopic damage was scored in a 0–14 range according to the criteria as previously described [27]. Concisely, the gastric tissue was assessed for epithelial cell loss (score: 0–3), edema in the upper mucosa (score: 0–4), hemorrhagic damage (score: 0–4), and the presence of inflammatory cells (score: 0–3). A summation of the four histopathological scores provided the total microscopic score.

2.11 Statistical analysis

All results were expressed as means ± standard error (SEM) of six animals. For variances evaluation between the groups, one-way analysis of variance (ANOVA) followed by the Duncan post hoc test was used to compare between two group means using IBM SPSS statistic 22 software (SPSS Inc., Chicago, IL, the USA) software. Values of P < 0.05 were regarded as statistically significant.

3 Results

3.1 The volume and pH of gastric content as gastric secretion indices

Table 1 demonstrates that a single dose of ethanol (5 ml/ bodyweight) induced severe gastric ulcer in rats. This is indicated by a significant (P < 0.05) increase in the volume of gastric content when compared with the control group. Conversely, a significant (P < 0.05) decline in pH was noticed after ethanol administration as compared to the control group, indicating the increased secretion of gastric acid. Interestingly, ulcerated rats treated with SOIE at selected doses (100 and 200 mg/kg body weight) showed a significant (P < 0.05) amelioration in the gastric secretion indices, revealing its healing potency (Table 1).

Table 1 Gastric juice volume and pH in all the experimental groups

3.2 Gross morphological change of gastric mucosa

Regarding morphological examination of gastric mucosa, Fig. 2a demonstrates that control rats showed normal gastric mucosa architecture which exhibited no signs of hemorrhagic lesions or ulceration. In contrast, the rats treated with a single dose of ethanol (5 ml/kg body weight) demonstrated severe damage and extensive visible hemorrhagic necrosis of the gastric mucosa (Fig. 2b). Furthermore, the severity score of the ulcerated group was significantly increased to 4.67 ± 0.21 in comparison with the control group (Fig. 2e). Meanwhile, SOIE treatment at a low dose (100 mg/kg body weight) and high dose (200 mg/kg body weight) caused a marked recovery from gastric ulcer as indicated by no hemorrhagic bands or injuries observed as well as significantly reduced severity score of ulcer to 1.92 ± 0.42 and 1.08 ± 0.20, respectively, as compared to untreated ulcerated rats (Fig. 2c–e).

Fig. 2

Gross morphological change of gastric mucosa. a The normal control group exhibited a normal gastric mucosa architecture which exhibited no signs of hemorrhagic lesions or ulceration. b Ulcerated rats showed extensive and severe hemorrhagic gastric mucosal lesions. c Ulcerated rats treated with SOIE (100 mg/kg body weight) exhibited less hemorrhagic lesions of the gastric mucosa compared with the control group. d Ulcerated rats treated with SOIE (200 mg/kg body weight) showed a marked recovery as indicated by no hemorrhagic bands or injuries. e Gastric severity score of different ulcerated groups. Values are expressed as mean ± SEM (n = 6). Values with different superscript letters are significantly different (P < 0.05). Blue arrow = hemorrhagic band. SOIE₁₀₀: Sepia officinalis ink extract (100 mg/kg body weight); SOIE₂₀₀: Sepia officinalis ink extract (200 mg/kg body weight)

3.3 Ulcer index and preventive index

Rats administrated orally single dose of absolute ethanol (5 ml/kg body weight) showed severe gastric mucosal lesions as indicated by a marked ulcer index which was 266.4 ± 12.23 (Table 2). Conversely, SOIE treatment at a low dose (100 mg/kg body weight) markedly reduced the ulcer index to 73.77 ± 21.03 resulting in a 68% reduction in gastric lesions induced by ethanol, as compared to the ulcerated group (Table 2). Interestingly, SOIE treatment at a high dose (200 mg/kg body weight) has a high antiulcer potency comparable to that of SOIE treatment at a low dose, and this is indicated by a significant reduction in ulcer index (16.67 ± 8.60) resulting in 94% inhibition in gastric lesions induced by a single dose of ethanol (Table 2).

Table 2 Percentage of ulceration, ulcer index, and preventive index in all the experimental groups

3.4 Oxidative/antioxidant pathway of SOIE

3.4.1 Gastric mucosal free radical (MDA)

Table 3 reveals that ethanol administration induced oxidative stress status in gastric tissue as confirmed by a significant (P < 0.05) increment in the gastric MDA content as compared to the control group. Meanwhile, ulcerated rats treated with SOIE at the two selected doses showed a significant (P < 0.05) decline in their gastric MDA when compared with untreated ulcerated rats (Table 3).

Table 3 Gastric oxidative/antioxidative stress markers and nitric oxide in all experimental groups

3.4.2 Gastric antioxidant markers

The gastric GSH content and antioxidant enzyme activities (CAT and GST) were significantly decreased in rats administrated a single dose of absolute ethanol in comparison with the control group (Table 3). Meanwhile, SOIE treatment at two doses (100 and 200 mg/kg body weight) caused a marked amelioration in antioxidant status as indicated by a significant (P < 0.05) increase in the gastric GSH content as well as antioxidant enzyme activities when compared with an ulcerated group (Table 3).

3.5 Gastric nitric oxide as an inflammatory marker

Table 3 reveals that ethanol administration induced inflammation in gastric tissue as confirmed by a significant (P < 0.05) increment in the gastric NO content as compared to the control group. Meanwhile, the NO content of ulcerated rats treated with SOIE at the two selected doses significantly decreased when compared with untreated ulcerated rats (Table 3).

3.6 Histopathological studies

Regarding gastric architecture, Fig. 3a, b reveals normal histology of gastric tissue which exhibited normal histological structure of gastric mucosa and submucosa in the control group. Relative to control, the gastric architecture of untreated ulcerated rats showed serious histopathological alterations, and this was evidenced by multifocal ulcerative areas in the glandular mucosa that is characterized by desquamation of the epithelial lining accompanied by hemorrhages and accumulation of necrotic tissue and hemosiderin pigment (Fig.3c, d). Besides the multifocal ulcerative area, dense inflammatory cells infiltration in the mucosa and submucosal layers was observed in the untreated ulcerated rats (Fig.3e, f). Additionally, the submucosa layer of ulcerated rats revealed a marked expansion with abundant edema, congested blood vessels, excessive hemorrhages, and inflammatory cell infiltration (Fig. 3g–j). Conversely, the ulcerated rats treated with SOIE at 100 mg/kg body weight demonstrated a slight improvement in gastric structure confirmed by mild epithelial sloughing with fewer inflammatory cells infiltration in the mucosa and submucosal layer (Fig.3k, l). Meanwhile, ulcerated rats treated with SOIE at 200 mg/kg body weight showed a pronounced recovery in the gastric mucosa and this is evidenced by the normal histological structure of glandular mucosa and submucosa (Fig. 3m, n).

Fig. 3

Photomicrographs of the rat's gastric mucosa architecture of different experimental groups stained with hematoxylin and eosin. a, b Control group showing the well-preserved normal histological structure of stomach mucosa at low and high magnification. c, d Ulcerated group showing severe alterations in gastric mucosa with higher power of ulcerative mucosal surface associated with deposition of hemosiderin pigment (black arrow). e, f Gastric mucosa of ulcerated rats showing necrosis of glandular mucosa with dense inflammatory cell infiltration. g, h Untreated ulcerated rats showed congested blood vessels in the mucosa and submucosal layers with inflammatory cells infiltration (black arrow). i, j Gastric submucosa of the ulcerated group showing the expansion of the submucosal layer with edema, congested blood vessels and hemorrhages (black arrow). k, l Ulcerated rats treated with SOIE (100 mg/kg body weight) showed a slight improvement in mucosa structure confirmed by slight normalization of mucosa with fewer inflammatory cells infiltration in the glandular mucosal with moderate submucosal edema (black arrow). m, n Ulcerated rats treated with SOIE (200 mg/kg body weight) showed a pronounced recovery in the gastric mucosa, and this was evidenced by the normal histological structure of glandular mucosa and submucosa. M: Mucosa, SM: Submucosa

3.7 Histological Score

Regarding histological alterations score, ulcerated rats showed a significant (P < 0.05) increment in their total histopathological alterations as compared to control or treated groups (Fig. 4). Meanwhile, SOIE treatment at the selected doses caused a significant (P < 0.05) decline in total lesions score comparable to that of the untreated ulcerated group. Interestingly, SOIE treatment at a high dose (200 mg/kg body weight) is more effective than SOIE treatment (100 mg/kg body weight) as it can restore the abnormal histological alteration in gastric tissue confirmed by a significant (P < 0.05) reduced in total lesions score as compared to SOIE₁₀₀ group (Fig. 4).

Fig. 4

Total lesion score in different ulcerated groups. Values are expressed as mean ± SEM (n = 6). Values with different superscript letters are significantly different (P < 0.05). SOIE₁₀₀: Sepia officinalis ink extract (100 mg/kg body weight); SOIE₂₀₀: Sepia officinalis ink extract (200 mg/kg body weight)

4 Discussion

Gastric ulcer is a progressive gastrointestinal disease that is characterized by severe gastric mucosa damage constituting one of the main causes of morbidity and mortality worldwide [28]. Despite the remarkable progress in its treatment strategies, gastric ulcer is still a major ongoing global healthcare problem [29]. So, discovering novel therapeutically effective as well as safe natural antiulcer remedies is urgently desired for the prevention and treatment of gastric ulcer. Alcohol consumption is considered the major leading cause of gastric mucosa corrosion in humans. Therefore, absolute ethanol abuse is a convenient experimental model for animal gastric ulcer models to stimulate gastric mucosal damage. Because, it can solubilize the gastric wall mucus and penetrate the gastric mucosa rapidly, producing severe gastric lesions, which are reliable typical characteristics of human gastric lesions [30]. So, the ethanol model can provide unique insights into gastritis pathology. Additionally, the ethanol abuse model is widely used to investigate the antiulcer efficacy of novel therapeutics intended to be utilized for gastric ulcer healing [31]. Therefore, the present study is designed to evaluate the antiulcer potency of Sepia officinalis ink extract (SOIE) against ethanol-induced severe gastric lesions in rats.

Concerning gastric ulcerogenesis, volume and pH of gastric secretion are essential aggressive factors facilitating gastric mucosal injury where an excess of gastric acid secretion is contributing to the development of gastric ulcers [32, 33]. The current study demonstrated that administration of ethanol caused a significant (P < 0.05) increase in gastric acid secretion as indicated by a decrease in pH as well as an increase in gastric juice volume. These findings confirmed the digestion of gastric mucosa via stimulation of pepsin secretion as Liu et al. [7] reported. The current study is attributed the marked change in the volume and pH of gastric secretion to a hemorrhage accompanied by gastric mucosal damage induced by ethanol which in turn reduced the blood flow to the stomach mucosa and subsequently, increase the flow of Na⁺ and K⁺ into the lumen leading to a significant (P < 0.05) increase in gastric acid secretion[34]. Also, Anandan et al. [35] suggested that the increased acid secretion may be a consequence of increased permeability of the gastric mucosa.

de Martel et al.[36] suggested that the reduction in gastric juice secretion is considered a useful strategy in the prevention and treatment of gastric ulcer. In this study, SOIE treatment at 100 and 200 mg/kg body weight significantly attenuated gastric acid secretion as indicated by significant (P < 0.05) reduction in the gastric volume and pH, as compared to ulcerated rats. These results suggested a gastric mucosal protective effect of SOIE via inhibition of gastric acid secretion and, subsequently, reduced the activity of pepsin which is a risk factor for gastric ulcer as Rozza et al. [37] stated. The present findings are similar to those reported for melanin extract of marine animals [38, 39]. The efficiency of SOIE in increasing gastric pH level may be credited to the presence of polysaccharides in SOIE as Cao et al. [40] and Zuo et al. [41] demonstrated. They revealed that squid ink polysaccharides can increase the mucus contents and thus prevent sodium and potassium ions flow into the lumen and pepsin secretion. Furthermore, the increase in mucus contents may prevent ulcer formation by back diffusing of hydrogen ions and subsequently enhanced the buffering of gastric acid and descending stomach wall friction via peristalsis [42]. Additionally, the current study suggested that ulcer healing activity of SOIE may be related to the acid-neutralizing capability of SOIE by the gradual release of its glucosamine residues into the gastric mucosa as confirmed by elevation in gastric pH [43]. This postulation is supported by Santhosh et al. [43] who demonstrated that the neutralization of acid secretion in the stomach could be accelerated ulcer healing.

In the present study, a high severity degree of ulceration was observed in rats treated with absolute ethanol. This was confirmed by macroscopical and histopathological findings which revealed severe damage and extensive visible hemorrhagic necrosis of the gastric mucosa appeared as severe congestion in the submucosa as well as extravasation of RBCs among gastric mucosal villi, resulting in a high ulcer index in the ulcerated group. The current findings may be due to acute ethanol administration which induced gastric micro-vessel disturbance and blood flow stasis leading to the hemorrhage shock and ultimately induced necrotic gastric injury as Lustenberger et al. [44] suggested. These results are consistent with the previous findings which clarified that oral administration of absolute ethanol-induced mucosa damage is characterized mainly by elongated macroscopic lesions with intense hemorrhage and hyperemia which finally leads to necrotic gastric damage [45, 46].

Interestingly, treatment of ulcerated rats with SOIE at 100 and 200 mg/kg body weight prominently attenuated gastric mucosa damage and promoted healing of gastric mucosa lesions induced by ethanol as indicated by a significant (P < 0.05) reduction in severity degree of ulceration and a marked increase in the preventive index as compared to ulcerated rats, suggesting its potent antiulcerogenic ability. Furthermore, in macroscopic and histological evaluation, SOIE treatment at the selected doses ameliorated severe mucosa damage induced by ethanol and this was evidenced by a marked reduction in gastric lesions and enhanced the healing of gastric mucosa. These results are in agreement with the earlier results reported by Mimura et al. [38] and Mimura et al. [39]. The antiulcerogenic potency of SOIE may be due to its low molecular weight melanoprotein that can enhance glycoprotein synthesis, major constituents of the mucous barrier, in the gastric mucosa which in turn protects gastric mucosa against gastric secretion as Derby [47] suggested. Thus, the present study suggested that a possible mechanism for gastric mucosal protection by SOIE is reinforcing the resistance of the mucosal barrier which creates a protective coating that prevents direct contact between ethanol and the epithelium.

Ethanol-induced gastric lesions are significantly associated with increased oxidative stress via overproduction of reactive oxygen species (ROS). Thus, the generation of ROS and subsequent oxidative stress is a major mechanism in the pathogenesis of gastric tissue damage and ulcerogenesis induced by ethanol [48, 49]. Extreme generation of ROS causes increased lipid peroxidation (LPO), leading to suppression of antioxidant enzyme activities and GSH consumption. Lipid peroxidation is a consequence of ROS reaction against mucosal cell membrane and produces significant levels of MDA that leads to severe oxidative damage of gastric mucosal layers resulting in hemorrhagic lesions [50, 51]. In the present work, absolute ethanol-induced oxidative stress was confirmed by a significant (P < 0.05) elevation in lipid peroxidation in gastric mucosa which was paralleled by the reduction in GSH and antioxidant enzymes (catalase and GST) in ulcerated rats, which is consistent with previous studies [7, 48, 52]. According to Yadav et al. [53], ethanol-induced oxidative stress may be due to its ability to exaggerate neutrophils activation, which in turn generates a surge of ROS, especially O₂^·−, H₂O₂, HO^·, and ONOO⁻ leads to increased lipid peroxidation of gastric mucosa membrane and subsequent depletion in GSH content as well as antioxidant enzymes activities. Subsequently, the GSH depletion has resulted in increased susceptibility of the gastric mucosal cell to oxygen metabolites and acid-mediated mucosal cell damage [35].

Attractively, the current study revealed that SOIE treatment ameliorated ethanol-induced gastric ulcer formation in a dose-dependent manner as indicated by its pronounced efficacy in preventing free radical-mediated oxidative damage by enhancing the activity of antioxidant enzymes (CAT and GST) and restoring the depleted GSH levels simultaneously with reducing MDA levels. Therefore, this finding indicated that the gastroprotective efficacy of SOIE may be related to its antioxidant activity against free radical species. The antioxidant effect of the SOIE could be attributed to its strong free radical scavenging activity due to the presence of polysaccharides and melanin that act as free radical scavengers [54, 55]. Additionally, melanin of SOIE acts as a powerful SOD due to the presence of dihydroxyindole (DHI) which catalyze the disproportionation of O₂^•− to H₂O₂ and O and thus prevent the free radical chain reaction triggered by O⁻₂ [56]. Besides melanin and polysaccharides, SOIE contains a considerable amount of taurine which exhibits antioxidant properties [57]. The current findings were by earlier reports that SOIE exhibited antioxidant activity [54, 56].

Besides oxidative stress, an ethanol-induced gastric ulcer caused an inflammatory response associated with disturbing in nitric oxide (NO) pathway [58]. NO is regarded as a double-edged mediator of mucosal defense that exerts either protective or destructive effects depending on the extent of NO synthesis as Kamar et al. [59] stated. Previous reports have revealed that NO derived from constitutive nitric oxide synthase plays a significant role in the maintenance of normal gastric mucosal integrity by reducing neutrophils infiltration [60], whereas NO derived from inducible nitric oxide synthase (iNOS) contributes to ulcer formation via the production of oxygen-derived radicals [61]. Therefore, overproduction of NO from iNOS indicates a strong detrimental action in gastric ulcer formation. The current investigation demonstrated a marked elevation in gastric NO production in ulcerated rats when compared with control rats. This finding may be attributed to stimulation of iNOS by ethanol consumption, which reacts with superoxide to form peroxynitrite, a potent cytotoxic oxidant causing lipid peroxidation of gastric tissue which in turn accelerates gastric mucosa damage as Shin et al. [62] reported that ethanol consumption induces the gastric expression of iNOS. An intense detrimental action of ethanol on the production of NO is in accordance with previous reports [63, 64]. On the other hand, ulcerated rats treated with SOIE at the two selected doses displayed a significant (P < 0.05) reduction in gastric NO level suggesting the anti-inflammatory action of SOIE. The potent gastroprotective efficacy of SOIE may be attributed to its ability to downregulate the iNOS mRNA expression leading to decreased production of gastric mucosal NO as Guo et al. (2004) suggested. This result is consistent with previous studies of Abdelfattah et al. [11] and Kamar et al. [59].

While the obtained results are valuable findings, the present study has some limitations within findings need to be interpreted. First, as in most gastric ulcer studies, the current research was limited by evaluating the exact mechanism of SOIE in the healing process of gastric mucosa.

5 Conclusions

Based on the aforementioned parameters, SOIE at both doses attenuated the detrimental effect of ethanol on gastric mucosa that may be due to its antioxidant pathway via the enhancement antioxidant defense. Additionally, ulcer healing potency of SOIE may be also via the anti-inflammatory pathway by the downregulation of NO production and iNOS expression. The gastroprotective efficacy of SOIE may be attributed to the presence of bioactive constituents as polysaccharides and melanin. Therefore, these findings provide convincing evidence to support using SOIE as a promising alternative antiulcerogenic compound to treat severe gastric lesions. However, further studies are urgently needed to explore the exact mechanisms of its action on maintaining normal gastric mucosal barrier integrity.