The Effect of Omega-3-enriched Fish oil on the Inflammation of Mice Colon Induced by AOM and DSS: Study on COX-2

 

Kusmardi Kusmardi1, Pater Dean Adare2*, Ria Kodariah1

1Department of Anatomic Pathology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya 6 Jakarta, Indonesia.

2Postgraduate Study Program of Biomedic, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya 6 Jakarta, Indonesia.

2Faculty of Medicine, University of Papua, Papua, Indonesia

*Corresponding Author E-mail: paterdean@gmail.com

 

ABSTRACT:

Introduction: Colorectal cancer is one of the leading causes of cancer deaths in some countries of the world. Inflammation has been recognized as one of the etiologies of colorectal cancer. Omega-3 is known to have anti-inflammatory properties. The aim of this tudy was to investigate the anti-inflammatory effect of omega-3-enriched-fish oil on inflammation of mice’s colon by induction of azoxymethane (AOM) and dextran sodium sulfate (DSS) by analyzing the expression of cyclooxygenase-2 (COX-2). Method: 30 Swiss Webster mice divided into 6 groups: (1) Negative control group (KN); (2) test group of fish oil with a dose of 1.5 mg / day (Dose-I); (3) test group of fish oil with a dose of 3 mg / day (Dose-II); (4) solvent-control group of corn oil (KP); (5) Positive control group of aspirin (ASP); (6) Normal group (KNO). Group 1-5 was induced with AOM 10 mg / kgBB intraperitoneally once, then 1 week later followed by administering 2% ad lib DSS in drinking water for 7 days. After the induction period,  continued treatment time based on group above for 6 weeks. Results: There were no significant diffenrence in the test group (Dosis-I dan Dosis-II) (p>0.05) related to COX-2 compared with all control groups. Significant differences in the expression of COX-2 were found by comparing all treatment groups with the normal group (p <0.05). Conclusion: omega-3-rich fish oils with dose of 3 mg and 1.5 mg were not able to inhibit inflammatory proces of AOM+DSS-induced colon mice.

 

KEYWORDS: AOM, COX-2, DSS, Fish oil, omega-3, inflammatory.

 

 

 

INTRODUCTION:

Colorectal cancer (CRC) is one of the main causes of death by cancer in several developed countries such as the United States, Australia, New Zealand, and Canada.1,2 In Indonesia CRC is the third most common cancer after breast cancer and lung cancer.3 The main trigger for causing CRC is still unknown. Previously proposed a theory of changes in the intestinal mucosa into adenomas that progress to adenocarcinoma.4 These changes are followed by genetic changes that lead to carcinogenesis.5

 

The role of inflammation in the CRC has led some researcher to find substances that have potential as chemopreventive agent on CRC. Some studies have shown that omega-3 which is one of the polyunsaturated fatty acids (PUFA) can reduce inflammation in humans.6 Omega-3 derived from fish oil are natural ingredients that can be obtained from fish rich in fat, such as salmon, tuna and mackerel.7 Omega-3 have inhibitory action against COX-2 which ultimately inhibits the accumulation of β-catenin which is considered to play an important role in the development and progression of CRC.8 Omega-3 can act as an alternative substrate of AA in COX-2, causing reduction of formation PGE2. In addition, omega-3 can bind to the COX-2 substrate channel and inhibit COX-2.9

 

This study aimed to evaluate the effect of omega-3 on inflammation of mice colon induced by AOM and DSS.

 

 

MATERIAL AND METHODS:

Animal Study:

30 Swiss Webster mice age 12-16 weeks and weighed 20-30grams were used in this study. Animals are obtained from Research and Development Agency, Ministry of Health, Republic of Indonesia (LITBANGKES). Test animals are placed in rooms with constant temperature and humidity, adequate lighting, and fed with pellets and drinking water ad lib. Mice were divided into 6 groups: Group I negative control, mice were given induction of AOM and DSS (KN); Group 2 mice were given AOM and DSS, and given fish oil with a dose of 1.5mg (Dose-I); Group 3 mice were given AOM and DSS, and were given fish oil dose of 3mg (Dose-II); Group 4 mice were given AOM and DSS, and were given corn oil as solvent control (KP); Group 5 mice were given AOM and DSS, and were given acetosal (ASP) as a positive control group; Group 6 is a normal group and is not given special treatment.

 

Tanaka’s animal model of collitis10 was used in this study. The induction was carried out at the beginning with 10mg/kgBB AOM injection once intraperitoneal, and continued a week later with 2% ad libitum DSS carried out for 1 week. After the induction period was completed. The treatment using test substance was carried out for 6 weeks longs. After the treatment period, the mice were terminated by decapitation. Mice colon tissue is taken, then the lumen is cleaned with water, and fixed by using Neutral Buffered Formalin (NBF) 10%.

 

Imunohistochemical Staining:

The analysis was carried out by immunohistochemical (IHC) staining, tissue samples were cut with a thickness of 3-5μm. After the deparafinization and rehydration, the sample was given citrate buffer 0.1M (pH 6.0) for 5 minutes in the microwave. Next, hydrogen peroxide is added 3% 5 minutes at room temperature. The sample was incubated with COX-2 antibody in PBS for 2 hours at room temperature in a humidity chamber, then incubated again for 1 hour. Samples were then added to secondary antibodies and incubated for one hour at room temperature, followed by addition of HRP-conjugated streptavidin and incubated for 30 minutes. For visualization, a sample of 3,3'-diaminobenzidine (DAB) was added then incubated for 10 minutes at room temperature. The sample was then counterstained with Harris hematoxylin, dehydrated, and glued.

 

Imunohistochemical Staining Analysis:

The slides that have been colored by IHC staining, are assessed for the intensity of COX-2 expressions with the help of a light microscope. In this study Leica DM750 microscope was used, and image documentation was done using a computer with Leica LAZ EZ software and a camera that had been integrated with Leica DM750 microscope. Five picture was taken per slide in 5 different field focused on the part of the colon epithelium in the crypta region.

 

Furthermore, the pictures taken were analyzed using ImageJ, a java-based public domain program developed by the National Institutes of Health (NIH).,The IHC profiler11 plugin in the ImageJ is used to analyze the results of the IHC output.

 

Statistical Analysis:

This study was analyzed statistically using SPSS ver. 16, and statistical tests were deemed significance at p<0.05. A one way ANOVA test was used to see the significance of each group compared to normal group.

 

RESULTS AND DISCUSSION:

Resut of HE Stained, we found an inflammatory cell in the crypt in almost all treatment groups. In the negative control group the inflammatory cells seemed to cover the crypt more and more, and the goblet cells had depleted and were hardly found. In the other treatment groups (Dose-I, Dose-II, KP, ASP) and normal groups, it appears that the goblet cell population is still dominant in filling colon crypt.

 

Figure 1: HE stained of mice colon for all treatment groups. 40x10 Magnification. [A] Negative Control Group, [B] Dose-I Group. [C] Dose-II Group, [D] Aspirin Positive Control Group, [E] Solvent Control Group, [F] Normal Group. Description: Red arrow show populations of goblet cells in crypts.

The COX-2 expression in each group was measured in optical density score as shown in Figure 1 below. Positive results were found in all groups. There appears to be an increase in the level of expression of the treatment group (ASP, Positive controle, Negative controle, Dose-I, and Dose-II) when compared to the normal group.

 

Figure 2. COX-2 Expression of each group

 

The finding above is in line with the one-way ANOVA test which found a significant increase between (p <0.05) COX-2 expression levels in each treatment group. This indicates that the mice model using AOM and DSS induction in this study succeeded in causing inflammation in the colon with a marked increase in COX-2 expression in all treatment groups. This is in accordance with the carcinogenesis model proposed by tanaka in which using mice with AOM injected and subsequently treated with DSS develop adequate tumors in as little as 7 weeks,12  which mean the inflammation can be triggered earlier than those time mentioned.

 

The results of the post hoc Tamhane test found no significant differences in the ASP, KP, D-1, D-II groups, when compared to the negative group (p> 0.05). Statistically, aspirin and the fish oil do not adequately reduce COX-2 expression. However, referring to figure 1 above, there is a tendency for suppression of COX-2 expression in the positive control group (ASP, KP) and the fish oil treatment group (Dose-I, Dose-II).This result may be due to the treatment time being too short (6 weeks), compared to study conducted by Kusmardi13 which stated in the second month, there is a decrease in the number of inflammation foci occurred between the control and low dose groups with medium and high dose groups (p<0,05). Statistically significant differences were only seen in the normal group compared to all other treatment groups (p <0.05). This means that a significant increase in expression occurred between the groups treated with AOM and DSS. A non-significant results were also obtained when comparing the treatment of the fish oil treatment group (Dose-I and Dose-II) to the control group (p> 0.05).

 

Figure 3. COX-2 IHC Stained of mice colon. 40x10 Magnification [A] Negative Control Group, [B] Dose-I Group. [C] Dose-II Group, [D] Aspirin Positive Control Group, [E] Solvent Control Group, [F] Normal Group.

 

In Figure 2, positive COX-2 stain were found in all treated groups with the presence of brown colored cells. In the negative control group (KN) it looked more brown when compared to the other groups, showing higher levels of COX-2 expression compared to the other groups. In all groups there are still quite a lot of blue epithelial cells with nucleus that is still clearly visible.

 

CONCLUSION:

Omega-3-rich fish oil at a dose of 1.5mg and 3 mg for 6 weeks in this study were unable to reduce the expression of COX-2 in the inflammatory model of colon-induced by AOM+DSS. However, there is a tendency it can decrease the expression of COX-2.

 

ACKNOWLEDGEMENT:

We would like to thank to The Directorate of Research and Public Services Universitas Indonesia Jakarta, and  The Ministry of Research,Technology, and Higher Education for the Research Grant.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 07.05.2019           Modified on 10.06.2019

Accepted on 01.07.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(11): 5265-5268.

DOI: 10.5958/0974-360X.2019.00911.9