INTRODUCTION:

The community often uses Tuna because of its high nutritional content, relatively low price, and ease of obtaining it. Tuna contains essential fatty acids such as omega-3 fatty acid and several other fatty acids that play an important role in the body's metabolism. Tuna also contains several vitamins, namely vitamins A, B1, and B6, which have the potential as antioxidants that can play a role in reducing oxidants and free radicals in the body. And the selenium content in Tuna can increase enzymatic antioxidant activity in the body.^1-3Rheumatoid arthritis is a chronic disease caused by an autoimmune process that often occurs in middle-aged women and results in joint damage and damage to several important organs in the body.

This situation decreases the sufferer's quality of life, so it needs effective, efficient, and relatively affordable treatment.

People with Arthritis experience a decrease in endogenous antioxidant levels, and an inflammatory process occurs, which will result in the secretion of cytokines. The inflammatory process that occurs results in the formation of free radicals so that the body absorbs endogenous antioxidants, which will decrease the activity and amount of endogenous antioxidants in the body.^4,5

C-reactive Protein (CRP) is one of the biomarkers used to diagnose and evaluate rheumatoid arthritis (RA) disease activity. CRP may be a part of the pentraxin protein family, which consists of five 23-kDa subunits and can increase up to 1,000-fold or more with inflammation, infection, and tissue injury. In arthritis rheumatoid patients, CRP is not only a by-product of the inflammatory response but also plays a proinflammatory and prothrombotic role. CRP levels correlate with morning stiffness, fatigue, pain grip strength, disability, andarticular index.^6,7

This study used an animal model of adjuvant arthritis, namely experimental animals that were induced with CFA so that signs of arthritis occurred. Experimental animals were divided into three groups: the untreated group, the CFA-induced group, and the treatment group, namely the CFA-induced after there were signs of arthritis given Tuna fish extract orally for seven days and 14 days. Furthermore, the experimental animals were sacrificed and examined for CRP, and edema thickness.

Oral administration of Tuna fish extract is expected to be able to repair the damage caused by arthritis caused by CFA induction. The vitamin content in Tuna is expected to have the potential as an antioxidant that can reduce free radicals and formed oxidants. The selenium content in Tuna will increase the ability of endogenous antioxidants to reduce oxidants and free radicals to prevent cell damage. The content of omega-3 fatty acid and omega-6 fatty acid are expected to act as an anti-inflammatory in animal models of adjuvant arthritis so that the inflammatory process can decrease and joint edema will decrease. Reducing inflammation will also reduce proinflammatory cytokines that trigger the formation of oxidants and free radicals. It is hoped that the administration of Tuna fish extract can reduce one of the markers of inflammation, namely CRP levels, and reduce joint edema.

This study aimed to determine the effect of Tuna (Thunnus obesus) fish extract on inflammation in the legs of animal models of adjuvant arthritis.

MATERIAL AND METHODS:

This study utilized the post-test-only control gather plan strategyconducted at the research facility of Biochemistry, Medical faculty of Hang Tuah University, Surabaya, Indonesia. We utilized 24 male Rattus norvegicus which were partitioned into three groups (each group containing 8 rats): P1groups of no treatment rats, P2 groups of adjuvant arthritis rats, the rats of this group were induced with CFA, P3 groups, which are rats with adjuvant arthritis who were given Tuna fish ethanol exticate orally for seven consecutive days from the primary day of the emergence of symptoms of adjuvant arthritis, and given with the dosage of 12grams/kg of body weight, beginning on the 22nd day. At the last day of the study, the CRP and swelling feet joints of all groups of rats were measured (on day 29).

Adjuvant Arthritis rats induced by Complete Freund's Adjuvant (CFA):

Complete Freund's Adjuvant (CFA) was injected into the rats intra-dermally with 0.1ml at the base of the tail. After 14 days, the rats were given 0.1ml CFA booster at the proper and cleared out feet. After seven days, the symptoms of adjuvant arthritis happened within the shapeofredness, pain, and swelling in the feet joints. This arthritis animal model is called Adjuvant Arthritis (AA) and has been commonly utilized as an animal model of rheumatoid arthritis (RA).^8-10

Thunnus obesus ethanol Extract administration procedure:

Ethanol extract from Tuna fish (Thunnus obesus) produced procedure are:

Tuna (Thunnus obesus) were washed, chopped, and dried until the water substance was 20-30%. Every 116 grams of Tuna was extricated using the maceration strategy with 1.5 liters of 85% ethanol. The extricate was concentrated for roughly 2 hours using a filtered rotary vacuum evaporator at 40^oC. The extricate was washed with 100ml chloroform three times, and the upper layer (non-lipid fraction) was then removed from the ethanol division and dried utilizing N2, and a Tuna extricate was shaped.

The Administrative procedure for giving Tuna ethanol extract:

The rats were weighed and after that given Tuna ethanol extract at a dose of 12g/kg BW; the nasogastric tube for pediatric was embedded through the mouth until it come to the rat's stomach.

Procedure for taking blood and measuring CRP:

The rats were sacrificed by being anesthetized with ketamine when the time came, after being properly anesthetized, i.e. not moving anymore, placed on the surgical table in a supine position and the four limbs were fixed. Surgery is performed on the thorax region so the thoracic region is opened and the heart and ascending aortic blood vessels are visible. A syringe of 3 ccs is used for blood collection from the ascending heart/aorta, with the maximum amount possible, and is mixed with EDTA.^11,12

The procedure for measuring the thickness of edema with a plethysmometer:

Experimental animals are placed and held in such a way, the legs measure the volume of edema using a plethysmometer coupled with a digital balance which will measure the increase in edema volume.¹³

RESULT:

The mean and standard deviation of the ankle joint edema volume of rats in P3 group were smaller than those in P2 group, but still larger than P1group show in table 1.

Table 1: Mean volume of rat ankle joint edema (cm3)

Groups	Mean±standard deviation (cm3)
P1	0.684 ± 0.053
P2	1.32 ± 0.166
P3	0.78 ± 0.188

Note:

P1 : untreated rats

P2: arthritis adjuvant rats

P3 : arthritis adjuvant and treated with Tuna extract rats

Data on the volume of edema of the joints of the feet by the ANOVA test showed a significant difference between group P1 without treatment and group P2 with adjuvant arthritis (p=0.002), but there was no significant difference with P3 group (p = 0.245). The ANOVA test results between group P2 and group P3 show a statistically significant difference (p = 0.034). Figure 1 showing the difference rat ankle joint edema between group.

A B

Figure 1: Differences in the volume of rat paw edema. A Normal rat leg, B Rat leg with arthritis adjuvant, C Rat leg with arthritis adjuvant that received Tuna extract

The average and standard deviation of CRP levels in group P3 AA and treated withTuna extract were smaller than those in group P2 with adjuvant arthritis, but still higher than group P1 without treatmentshow in table 2.

Table 2: Average CRP levels

Group	Average ± standart deviation (mg/L)
P1	0.039 ± 0.0064
P2	0.179 ± 0.0196
P3	0.116 ± 0.0151

Note:

P1 : untreated rats

P2 : arthritis adjuvant rats

P3 : arthritis adjuvant and treated with Tuna extract rats

The CRP data were analyzed non-parametrically using Kruskal Walis, and if there was a difference, the Man-Whitney test was continued. The results of the Kruskal Walis test showed a significant difference between groups.

The comparison test results showed that CRP levels between group P1 without treatment and group P2 with adjuvant arthritis (p = 0.001) and group P3 arthritis treated with Tuna extract (p = 0.022) showed significant results. The comparative test of CRP levels in group P2 of adjuvant arthritis with group P3 of arthritis treated with Tuna extract showed significant results (p=0.022).

DISCUSSION:

Tuna is a fish that people in Indonesia widely consume. Tuna contains amino acids, fatty acids, mineral, and antioxidants that are beneficial for the health of the human body. In this study, animal models used Tuna fish extract to treat inflammation due to arthritis.

Effect of Tuna fish extract on leg edema of adjuvant arthritis experimental animals:

In animal models of adjuvant arthritis, edema and other markers of inflammation occur in the feet due to CFA induction.^14,15

Fatty acids are crucial for maintaining good health, and some of them are utilized to make eicosanoids as bioregulators in several processes in cells. Omega-3 fatty acid intake will modulate proinflammatory cytokines, reduce the secretion of interleukin-6 (IL-6), Interleukin-1β (IL-1β), Tumor necrotizing factor-α (TNF-α) by endotoxin-stimulated mono-cytes cells. Inhibition of proinflammatory cytokine secretion improves the symptoms and signs of inflammation in the form of leg edema in rheumatoid arthritis patients.Tuna is known to contain polyunsaturated fatty acids including omega-3 that play an important role in the synthesis of prostaglandins and trigger wound healing. Tuna contains omega-3 fatty acid (DHA and EPA), polyunsaturated fatty acids, and arachidonic acid, which acts as a precursor to prostaglandins and thromboxane.

Rheumatic arthritis is a chronic disease due to autoimmune. It is believed that the pathogenesis is due to an increase in free radicals resulting in oxidative stress and damage. In several studies, it was found that rheumatic arthritis patients experienced a decrease in endogenous antioxidant activity in the body. Tuna contains vitamin C vitamins B1, B6, and B12 which function as antioxidants, as well as selenium content in Tuna which helps increase the activity of endogenous enzymatic antioxidants in the body. The antioxidant content in Tuna plays a role in preventing oxidative stress, oxidative damage, and reducing inflammation processes in adjuvant arthritis.^5,16,19

In this study, the content of unsaturated fatty acids in Tuna was proven to reduce the inflammatory process through inhibition of cox-2, resulting in a decrease in the volume of leg joint edema in animals tested for adjuvant arthritis. This study is in line with research that the consumption of marine fish at least 2x a week can reduce disease activity in patients with rheumatoid arthritis.^2,20,21

Effect of Tuna fish extract on CRP levels in adjuvant arthritis experimental animals:

CRP is an immune regulator, not only as a biomarker widely used to determine the occurrence of an acute inflammatory process. CRP measurements were used for diagnostic criteria according to the 2010 ACR/EULAR and evaluation of therapy in patients with AR. Serum CRP is an acute phase reaction, which is mainly produces by hepatocytes under the control of proinflammatory cytokines, particularly interleukin-6. Increased levels of CRP are associated with the clinical picture of rheumatoid arthritis patients in the form of pain, stiffness in the morning, fatigue, and disability. CRP levels will increase thousands of times in conditions of infection, inflammatory processes, and tissue damage.^11,22-26

In some rheumatoid arthritis patients, CRP levels will increase earlier before the onset of clinical symptoms. This indicates that the immune response in patients with rheumatoid arthritis is the beginning of the course of the disease. CRP levels are frequently determinedly hoisted in patients with RA. Different components impact serum CRP levels in patients with RA. Body fat, dietary quality, stress, and female hormone levels too been showed up to affect CRP levels in patients with RA.^2,11,12,27

CRP is one of the biomarkers used to evaluate therapy in AR patients. A decrease in CRP levels in the serum of RA patients indicates a reduction in disease activity. Omega-3 fatty acid intake inhibits T cell proliferation, reduces antigen presentation through MHC-II and decreases proinflammatory cytokine production so that it can modulate the autoimmune inflammatory response in rheumatoid arthritis patients. Tuna extract contains DHA, and EPA polyunsaturated fatty acids, which act as an anti-inflammatory and anti-oxidant that prevents oxidative stress. This study found that administering Tuna fish extract reduced CRP levels of adjuvant arthritis experimental animals.^1-3-28

CONCLUSION:

This study concluded that administering Tuna fish extract at a dosage of 12 g/kg BW could reduce the leg edema and serum CRP levels of adjuvant arthritis experimental animals.

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Received on 07.12.2022 Modified on 24.05.2023

Research J. Pharm. and Tech 2023; 16(12):5867-5871.

DOI: 10.52711/0974-360X.2023.00950