The Therapeutic Effect of Shark Liver Oil in a Rat Model of Acetic Acid-Induced Ulcerative Colitis

Ulcerative colitis (UC) is one of the most well-known types of inflammatory bowel disease that manifests as recurrent inflammation of rectum and colon. The goal of this study is to evaluate the protective effects of shark liver oil (SLO) on acetic acid-induced ulcerative colitis in rats. Eighty induced UC rats were randomly divided into ten equal groups and received the following treatments for seven days: 1 ml of normal saline rectally, 1 ml of gel base (carboxymethyl cellulose) rectally, 10 mg/kg of Asacol rectally, 10 mg/kg of mesalazine orally, 5% gel form of SLO rectally, 10% gel form of SLO rectally, 200 mg of SLO orally, and 400 mg of SLO orally. We examined the oxidative stress indices, histopathological features, and body weight changes, as well as the function of the liver and kidneys at the end of treatment. Administration of 10% rectal and 400 mg oral SLO resulted in a significant weight gain. Also, glutathione peroxidase activity was significantly higher in 5% and 10% SLO-treated groups, and elevated superoxide dismutase activity in rats that received 5% SLO was observed compared to negative control and Asacol groups. While no significant changes were observed in most of the kidney and liver function markers, higher levels of aspartate aminotransferase were detected in the group that received 400 mg SLO orally compared to negative control and Asacol groups. Many histopathological signs of improvement were observed in mesalazine, Asacol, and SLO groups. There were no significant changes detected in the mean rank among different groups. Our data indicate that SLO supplementation could improve the amelioration of acetic acid-induced UC in rats due to its antioxidant effects.


Introduction
Ulcerative colitis (UC), as one of the main types of inflammatory bowel diseases (IBD), is a recurrent chronic disorder of rectal and colonic mucosa. e etiology of this disease is not entirely clear, but there are strong pieces of evidence that dysregulation of the immune response towards intraintestinal antigens could play a pivotal role in the initiation of UC and deterioration of patient's condition [1]. e incidence of UC has been the highest in Westernized nations since the past few decades, and it seems to be emerging in newly industrialized countries in Asia, South America, and the middle east [2]. is disease mostly initiates in the second and third decades of the patient's life and presents with diarrhea, abdominal cramps, rectal bleeding, tenesmus, and passage of mucus [3]. More importantly, UC increases the risk of colorectal cancer development in patients [4]. Although several environmental factors such as alteration of intestinal microbiota, exposure to antibiotics and air pollution, and smoking, as well as genetic factors, can predispose an individual to UC, there are firm shreds of evidence, emphasizing on the effect of the intestinal immune system dysregulation in the ulcerative colitis pathogenesis [5].
Current UC therapies include the application of 5-ASA agents, glucocorticoids, azathioprine, 6-mercaptopurine , and biological agents [6,7]. Many patients with UC have progressive courses that lead to hospitalization and surgical interventions [8]. Although new treatments, including biological agents, reduced the need to perform a surgery, the cost burden of such treatments is high for patients and healthcare systems [9,10]. erefore, physicians and scientists tend to use alternative medicine, including natural products, which have minimal side effects and on the other hand, reduce the cost burden of UC.
Shark liver oil (SLO) has been applied as a traditional marine natural product by Scandinavian people due to its possible therapeutic effects [11,12]. SLO is rich in alkylglycerols and squalene and also contains n-3 polyunsaturated fatty acids (N-3 PUFA) in lower amounts [13,14]. According to recent studies, SLO has multiple biological activities, including improving the immune system against infections by increasing neutrophil and macrophage activity [15,16], stimulating hematopoiesis, erythropoiesis [17], antitumor and antimetastasis activities [18,19], and alleviating the side effects of radiotherapy [20]. Due to lack of previous studies about therapeutic effects of SLO in UC and to find an alternative therapeutic agent for UC, we tried to evaluate the effects of SLO on acetic acid-induced UC in a rat model.

Ethical Statement.
is study was approved by the Ethics Committee of Fasa University of Medical Sciences (registration number: IR.FUMS.REC.1395.89), and all procedures were along the lines with the Helsinki Declaration of 2008 [21].

Preparation of SLO.
e liver of Centrophorus granulosus shark had been caught in the Persian Gulf during the autumn season and transferred on ice to the laboratory. e gallbladder was removed, and the rest of the liver was chopped and mixed. e mixed liver was weighed, and 500 gr of the mixture was placed in a cotton bag on an aluminum pan and cooked in a waterbath at 70-80°C for 30 minutes. en, the cotton bag was hand-pressed to release the oil-water extract, and then, centrifuge (3000 rpm, 15 min, and room temperature) to separate the oil from the water [22]. e density of SLO was 0.9 gr/ml. To prepare the gel form of shark liver oil, 5 ml and 10 ml of the prepared shark liver oil were added to 95 ml and 90 ml of the gel base (carboxymethyl cellulose), respectively.

Animals.
e Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, provided 80 male Sprague-Dawley rats (300 ± 20 gr weight). Rats were housed in separated cages in a limited-access room with equal light and dark cycle, the ambient temperature of 22 ± 2°C, and 50% relative humidity. All animals fasted 24 hours before induction of UC. erefore, their bowels were cleaned, and after the induction, standard chow diet and water were freely accessible for them.

Induction of UC.
UC was imitated by the administration of 2 ml of 3% acetic acid, transrectally, under light anesthesia of ether inhalation using a 2 mm diameter cannula that inserted 8 cm proximal to the anus. After that, rats were positioned in a supine Trendelenburg position for 1 minute to avoid acetic acid leakage [23].

Experimental Design.
After randomization, we allocated rats into eight groups (ten rats in each) as follows. All the animals received their treatments once a day for seven days.
(i) Group I (negative control 1) received 1 ml of normal saline rectally (ii) Group II (negative control 2) received 1 ml of gel base (carboxymethyl cellulose) rectally (iii) Group III (positive control 1) received 10 mg/kg of Asacol rectally (iv) Group IV (positive control 2) received 10 mg/kg of mesalazine orally (v) Group V received 1 ml of 5% gel form of shark liver oil rectally (vi) Group VI received 1 ml of 10% gel form of shark liver oil rectally (vii) Group VII received 200 mg (0.22 ml) of shark liver oil orally (viii) Group VIII received 400 mg (0.44 ml) of shark liver oil orally 2.6. Weighing and Sampling. We recorded animal weights before the experiment (day 0) and at days of 1, 3, 5, and 7 using a digital scale with 0.1 g precision. Weight changes percentages were calculated using following formula: (1)

Histopathological Evaluations.
All animals were sacrificed after seven days of treatment under deep ether anesthesia. Laparotomy was performed, and 8 cm of the distal of affected colon was excised and opened by longitudinal incision. After washing the tissue by normal saline, 6 cm of the specimen was fixed in 10% formaldehyde solution for histopathological evaluation. e remaining tissues were stored in liquid nitrogen till biochemical analysis. Formaldehydefixed tissues were embedded in paraffin. Blocks were divided into 5 μm thick sections and stained with hematoxylin and 2 Evidence-Based Complementary and Alternative Medicine eosin. We studied one slide for each animal.
e representative slides for each animal contained both UC-affected and normal regions, were studied under light microscopic, and reviewed by a pathologist in a blinded fashion. e inflammation severity, inflammation extent, crypt damage, percentage of involvement, and regeneration are evaluated as given in Table 1 [24].

Oxidative Stress Evaluation.
To biochemical analysis, 0.5 g of each frozen colonic tissue was mechanically homogenized in 5 ml of 0.05 M phosphate buffer saline pH 7.4 on ice to prevent heat shock. e homogenized samples were centrifuged (3500 rpm, 15 minutes, and 4°C), and their supernatants were collected and stored at −20°C for further evaluation of tissue total antioxidant capacity (TAC) by ELISA (ZB-TAC-96A, ZellBio GmbH, Germany) and activity of superoxide dismutase (SOD) by ELISA (ZB-SOD-96A, ZellBio GmbH, Germany) and glutathione peroxidase (GPx) by ELISA (BXC0551, Biorexfars, Iran).

Liver and Kidney Function Evaluation.
At the end of the experiment, 2 ml of peripheral blood was collected into a vacutainer tube from each rat. Samples were centrifuged at 3000 rpm for 15 min at 4°C, and then, the serum was aliquoted and stored at a −80°C freezer. e serum concentration of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, total protein, indirect and total bilirubin, creatinine, and urea were detected by the autoanalyzer BT-3000 (Biotecnica Instruments, Italy) using commercial kits and reagents (Pars Azmoon Co., Iran).

Statistical
Analysis. Data were expressed as mean and standard deviation (SD) or mean rank. SPSS version 21 for statistical analysis and GraphPad Prism 8.0 for drawing figure were used. e percentage of body weight change was analyzed by two-way ANOVA and Dunnett's multiple comparisons test. Antioxidant statuses and liver and kidney function tests were analyzed by one-way ANOVA and Tukey's post hoc test. Sum of histopathological scores in different groups were used as a cumulative scale which are presented in Table 2.
e histopathological scores were analyzed using the nonparametric test of the Kruskal-Wallis H test. P values that are lesser than 0.05 were considered as a significant difference.

Bodyweight.
We restricted rat's access to food for 24 h before the experiment begins. All groups showed bodyweight loss due to the NPO time. On the 7 th day, all groups showed increased weight compared to day 1 of experiment. However, groups that treated by 10% SLO and 400 mg SLO presented a significant percentage of weight gain compared to day 1 (P value <0.05) (Figure 1). Figure 2, GPx activity is significantly increased in 5% and 10% SLO groups compared to the normal saline group (P < 0.05 and P < 0.0001, respectively). In addition, 10% SLO-treated groups had a significantly higher GPx activity than gel base, Asacol, SLO 200 mg, and SLO 400 mg (P < 0.001, P < 0.05, P < 0.01, and P < 0.01, respectively).

Evaluation of Oxidative Stress Markers. As shown in
According to our results, SOD activity of the group treated by 5% SLO is significantly elevated compared to normal saline, gel base, Asacol, SLO 200 mg, and SLO 400 mg (P � 0.0001, P < 0.05, P < 0.05, P < 0.001, and P < 0.01). However, we observed no significant differences in TAC results among groups (P > 0.05). Figure 3, several histopathological abnormalities were observed due to acetic acid UC, induction including ulceration and loss of epithelial lining, goblet cell depletion, irregularity in crypt structure, and infiltration of inflammatory cells into colonic mucosa and submucosa. Certain histopathological features of colon tissues including inflammation severity, inflammation extent, crypt damage, percentage of involvement, and regeneration were evaluated.

Histopathological Examination and Microscopic Scoring for Ulcerative Colitis. As shown in
Although we observed many signs of improvement including regeneration in Asacol and SLO groups, the mean rank of pathological changes did not represent a significant difference among groups (Table 1).

Liver and Kidney Function Evaluation.
We examined creatinine, urea, AST, ALT, ALP, albumin, total protein, and indirect and total bilirubin levels to determine whether SLO treatments caused any kidney and liver dysfunctions or not. According to Table 3, a significant elevated AST level was detected in the group that received 400 mg of oral SLO compared to normal saline, gel base, and Asacol (P < 0.05). Our results showed no significant differences in other measured factors among groups.

Discussion
In this study, we focused on the antioxidant capacity of SLO and its effect on an acetic acid-induced ulcerative colitis in rat as a model. Our results showed that treating an animal model of UC by SLO can improve some aspects of oxidative stress and prevent the progress of the disease. Besides, SLO had significant positive effects on weight gain in 10% SLO and 400 mg SLO groups. However, no significant changes were observed in TAC activity or histopathological evaluations. Although it is possible that low dose and the short period of treatment were the reasons that we did not observe a Evidence-Based Complementary and Alternative Medicine significant improvement in colon tissue regeneration and other pathological aspects [25,26]. To our knowledge, this is the first time that SLO has been evaluated for its potential application as a UC therapeutic agent in the rat animal model. Previously, Hünka et al. indicated that cod liver oil prevents oxidative stress and enhances glucose and lipid metabolism in diabetic rats [27]. Moreover, according to many studies, SLO seems to have biological activities including immunological defense enhancement, antitumor and antimetastasis activities, and antibacterial effects [28][29][30].
Ulcerative colitis is known as a chronic immune-mediated complication with an increasing number of affected patients around the world. e main goal of UC therapeutic strategies is to prolong the remission or corticosteroid-free period and reduce the risk of hospitalization and colorectal neoplasia [6]. UC management is mainly based on antiinflammatory and immunosuppressant drugs, which have many adverse effects. Hence, there is an urgent need for better therapeutic approaches with high efficacy and lower side effects. Considering extensive research studies, oxidative stress has been proposed to play a crucial role in pathogenesis and exacerbation of this disease [31][32][33]. It has been documented that UC is associated with extensive amount of reactive oxygen species (ROS), which may be due to either overproduction or insufficient scavenging of ROS. e lack of balance between ROS production and antioxidant capacity may lead to oxidative stress in UC. Many researchers believe that the combination of antioxidant and anti-inflammatory natural agents could be a practical therapeutic approach for UC [34][35][36][37]. Shark liver oil has    Evidence-Based Complementary and Alternative Medicine been known for its antioxidant and anti-inflammatory effects [27,38]. SLO contains natural alkylglycerols and squalene, which are known for their immune modulatory and antioxidant activates, respectively [39].
Despite our results, this study has some limitations. First, since we did not assess the inflammatory markers, including interleukins and tissue necrosis factor, we cannot discuss the anti-inflammatory effect of SLO. Second, it is possible that  Figure 2: Effect of different treatment strategies on the biochemical assay of acetic acid-induced UC rats. Rats were sacrificed on day 7, and colon were dissected and homogenized and examined for GPx, SOD, and TAC. (a) Groups treated by 5% and 10% rectal SLO showed higher GPx activity. (b) SOD activity is significantly increased in the 5% SLO group. (c) No significant differences in TAC activity were detected among groups. e analysis was performed using one-way ANOVA and Tukey's post hoc test. Data are presented as mean ± SD. * P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001. the short period of treatment and low doses of SLO could be the reasons that we did not observe any significant improvement in the histopathological evaluations [25].

Data Availability
e data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.