Anti-inflammatory activity of omega-3-polyunsaturated fatty acid oil extract on acute inflammation in animal models.

doi:10.21203/rs.3.rs-1761276/v1

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Anti-inflammatory activity of omega-3-polyunsaturated fatty acid oil extract on acute inflammation in animal models.

https://doi.org/10.21203/rs.3.rs-1761276/v1

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Safety concerns for persistent long-term use of anti- inflammatory drugs must be considered when prescribing pain relief medications for acute, chronic and degenerative pain conditions due to the side effects. Therefore supplementation using natural forms of anti-inflammatories such as omega − 3- fatty acids (n-3 fatty acids) is more about therapeutic safety than it is of efficacy. In this study, standard methods (Carageenan, Arachidonic acid and PGE2) were used to determine the nutritional intervention of n-3 fish oil extract (due to its eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA contents) in experimental animal models for acute inflammatory models. Obtained results showed marked inhibition of inflammation by the fish oil brought about by EPA and DHA content of the oil. Resolution of carragenan-induced paw edema inflammation after 24hrs was 78.57%, 57.14%, 50.00%, 100.00% and 92.85% percentage inhibition for groups 1, 2, 3, 4, and 5 respectively. Arachidonic acid-induced ear edema was 30, 24, 27, 37 and 40% inhibition while 44.16, 52.48, 61.34,74 and 80.05% were for PGE-2 assay. The results show that the active components in omega-3 polyunsaturated fatty acids, which are eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), are capable of interrupting the prostaglandin metabolic pathway by competing with arachidonic acids for the COX active site thereby inhibiting their synthesis.

Applied Biochemistry

Docosahexanoic acid

Eicosapentanoic acid

Inflammation

Inhibition

Fatty acids are important components of the human body, having biological, structural and functional roles. Besides their role as source of energy, they act as main constituents of cellular membranes¹¹. They assure the fluidity, flexibility, permeability of the membrane as part of membrane phospholipids and also assure passive transport through the membrane and are interconnected with other membrane proteins. Among fatty acids, omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) seem to be the most important, due to their multiple biological roles, such as influencing the inflammatory cascade, reducing oxidative stress, and in neuro and cardiovascular protection¹¹. They also serve as precursors of bioactive mediators such as eicosanoids (prostaglandins, leukotrienes, and thromboxanes) and steroid hormones (cholesterol)¹⁹. Inflammation is the immune system’s response to harmful stimuli, such as pathogens, damaged cells, toxic compounds or irradiation and it acts by removing injurious stimuli and initiating healing process. It is therefore a defense mechanism that is vital to health⁴.

Omega-3 fatty acids (also known as n-3 fatty acids) are a group of naturally occurring lipids, which are present in high concentrations in certain fish and plants such as flax seed oil, perilla oil and others⁵. The term omega-3 ("n-3","ω-3") signifies that the first double bond exists at the third carbon-carbon bond from the terminal methyl end (ω) of the carbon chain. There are three most common omega 3 fatty acids viz. Alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). ALA is present in certain vegetable oils (flaxseed and canola), whereas EPA and DHA are present in fish and in more concentrated amounts in fish oil¹³. They are found in varying ratios in fish, particularly in cold water and pelagic species such as menhaden, mackerel, oil sardine and salmon². Omega-3 fatty acids have been found to possess anti- inflammatory activity in human and animal models, though mechanism of action is still widely being researched, a number of research and in-depth studies have proven this fatty acid to be one of the natural supplements with effective anti-inflammatory activity providing excellent therapeutic safety.

Chemicals and drugs

The chemicals and reagents used for this study were of analytical grade and were obtained from Merck (Germany), (Sigma, St. Louis, Missouri, USA), BDH Chemicals Limited (Poole, England), May and Baker Ltd, (England), Riedel-de-Haen, (Hannow, Germany), Hopkins and Williams (Essex, England) except otherwise stated. Omega -3 fatty acid capsules were gotten from Ibadan, Nigeria.

Animals

Healthy male and female albino rats (120 – 200 g) of Wistar strain and mice (40 – 65 g) were obtained from the Animal Unit of the Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State of Nigeria. The Animals were maintained at standard lighting and temperature conditions in the Animal House for one week to acclimatize in solid-bottom cages before commencement of the experiment. Food pellets and water were provided ad libitum to eliminate the effect of stress. Animals were handled according to prescribed ethical guidelines for Care and Use of Laboratory Animals, Kogi State University, Anyigba- Nigeria.

Methods

Drug treatment

Content of one 200 mg capsule was dissolved in 10 ml phosphate buffer saline (PBS) solution to obtain a 20 mg/ml concentration⁸. The required amount of drug (1 ml) was then given to the rats orally.

Experimental design

Animals were distributed in six groups of five each (n = 5) as follows;

Group 1 received a dose of 180 mg EPA + 120 mg DHA, Group2 received 360 mg EPA + 240 mg DHA Group 3, 540 mg EPA + 360 mg DHA, Group 4 was administered a combined dose of 180 mg EPA+ 120 mg DHA + 20 mg Celecoxib, Group 5 was given 20 mg celecoxib as the standard group while Group 6 took 2 ml PBS as the vehicle control group. All doses were administered orally and per kilogram body weights of the rats and mice.

Acute Inflammation studies

Carrageenan-induced paw edema

This method was carried out as described by Winter²⁰ with slight modifications from more recent sources³. The animals were divided into six groups (n = 5), deprived of food overnight but given water ad libitum prior to the experiment. One hour after drug dosing and 6 weeks of omega-3 fatty acids supplementation, test rats were then challenged by a subcutaneous injection of 0.1ml of 1% solution of carrageenan in sodium chloride into the sub-plantar side of the left hind paw following inhalation of 5% isoflurane (v/v) for 15 seconds while the vehicle control group was injected with the carrageenan solution. The basal paw thickness was then measured using a vernier caliper⁹. The paw thickness was measured again at 1, 2, 3, 4, and 5 hours after the challenge. The increase in paw thickness was then calculated as a percentage compared with the basal thickness (the zero hour). The difference of average values among treated and untreated groups was calculated for each time interval and evaluated statistically. The percent inhibition was calculated using the formula as follows: % edema inhibition = [1−(Tt/Tc)]×100

(Tt and Tc are edema thickness in the test and control groups, respectively).

Arachidonic acid – induced ear edema

This model is based on the principle of metabolism of arachidonic acid by cyclooxygenase (COX), as described by Romay¹⁷, leading to the generation of prostaglandins and thromboxanes that mediate pain and edema associated with inflammation. Inhibition of these mediators by the extract and test drug is then evaluated. In this method, Inflammation was induced by topical application of arachidonic acid (2 mg in 20 μml of acetone) to both surfaces of the right ear of each mouse. The left ear was considered as control. One hour later, the thickness of the ear was determined using a Digital Caliper. The percentage of the ear edema was calculated based on the left ear which is the control¹⁸.

PGE₂- Induced Hind Paw Edema

This model was used for the determination of anti-inflammatory activity following the modified method of Kasahara¹⁰. Animals were grouped in six groups of five each and were given either oil extract, standard drug or just physiological salt solution in the case of the control group. One hour after oral administration of the extract, drug or vehicle, each rat received 5µl of freshly prepared suspension of 1mg/ml PGE-2 in Tyrode’s solution by injection into the sub-plantar tissue of the right hind paw. For the vehicle control group, 5µl of Tyrode’s solution was injected into the left hind paw. Thereafter, edema was measured at 15minutes interval for 75minutes. Difference in the thickness of the footpad was measured relative to time zero¹.

Statistical analysis

All analyses were performed using graph pad Instat software using a 2- way ANOVA and subjected to Bonferroni test for the separation of means. Results were expressed as Mean ± SEM. Differences between means were considered statistically significant at p < 0.05.

Inflammation is a beneficial host response to external changes or cellular injury that leads to the activation of a complex array of inflammatory mediators, finalizing the restoration of tissue structure and function¹⁵. Anti-inflammatory potential of omega-3-fatty acids was tested and evaluated by supplementation using animal models. The anti- inflammatory effect was tested in different doses of omega- 3 fatty acids to obtain varied doses of EPA and DHA from 1000mg fish oils and these doses was administered according to the body weights of the rats in kg. Anti-inflammatory models used to evaluate acute inflammation include carrageenan-induced paw edema, arachidonic acid- induced ear edema and prostaglandin E2- induced paw edema.

The process of change observed in paw edema induced by carrageenan is an effective method used to evaluate acute inflammation in rats (Fig 1). The reaction is usually biphasic²², with the first phase occurring from 0- 2 hours, characterized with the release of histamine and bradykinin from mast cells, alerting the immune system that an injury or challenge has occurred. This leads to increase in vascular permeability resulting in the exudation of fluids from the blood into the interstitial space²². This is then accompanied by a second phase which starts from the third hour and is often characterized by the production of the inflammatory mediator, prostaglandins¹³. Inhibition of prostaglandin synthesis is the point of action of the oil extract as well as standard used in this study.

The results of the mean paw thickness (MPT) of the carragenan-induced paw edema in the rat model (Fig 1) for groups 1, 2, 3, 4, 5 and 6 for zero hour (pre-induced state) are 3.33 ± 0.14; 3.38 ± 0.06; 3.42 ± 0.21; 3.24 ± 0.07; 3.15 ± 0.03 and 3.19 ± 0.27 mm respectively. After one hour of induction, inflammation set in with MPT as 4.2 6± 0.28; 4.60 ± 0.07; 4.80 ± 0.31; 4.23 ± 0.09; 4.35 ± 0.04 and 4.92 ± 0.35 mm. Resolution of inflammation continue until 24hrs later when it was almost completely resolved with MPT as 3.36 ± 0.21; 3.44 ± 0.21; 3.49 ± 0.13; 3.24 ± 0.27; 3.16 ± 0.23 and 3.33 ± 0.28 mm representing 78.57%, 57.14%, 50.00%, 100.00% and 92.85% percentage inhibition for groups 1, 2, 3, 4, and 5 respectively. This result (Fig 3) showed the superiority of supplementation as shown in group 4 having resolved the inflammation 100%. Inhibition of prostaglandin synthesis is the point of action of the oil extract as well as the standard used in this study. The oil extract used was found to effectively inhibit and resolve edema at the end of the observation period. The oil extract used was found to effectively inhibit and resolve edema at the end of the observation period and pattern of resolution is in agreement with that reported by Girish⁷.

It was noticed from Fig 2 that the paw thickness did not show significant changes even after 2 hours of administration of the extract. Resolution however, became noticeable after 3 hours. The reduction in paw thickness was very remarkable after 4 and 5 hours of administration of the extract. After 24hours, it was completely resolved and the rate of reduction of paw thickness in groups 4 and 5 showed a simultaneous resolution showing the importance of supplementation as compared to the standard.

In a long list of mediators, including histamine, serotonin, the kinins, complement, etc, the metabolites of arachidonic acid (AA) have become the recent focus of attention. Alone or in appropriate combination, AA products of the cyclooxygenase and lipooxygenase pathways are capable of producing the characteristic signs of inflammation: vasodilatation, hyperemia, pain, edema, and cellular infiltration²¹. Table 1 shows the effect of fish oil extract on the metabolism of AA, which leads to the synthesis of prostaglandins and thromboxanes that can start the inflammation cascade leading to pain and exudation of fluid that causes edema. Topical application of AA on the ears of mice increased ear thickness, redness and edema as a result of the release of inflammatory mediators which are recruited to the inflamed site, causing the characteristic swelling within the dermal region and increased vascular permeability often associated with inflammation⁶. Reports^{6, 16} have shown that direct application of AA to the ear of mice causes an immediate vasodilatation and erythema, leading to development of intense edema.

The synthesis of leukotriene and prostaglandins, serotonin and histamine are responsible for the formation of the ear edema observed¹⁶. Arachidonic acid-induced ear edema in the rat models is given by ear weight and percentage inhibition. These were gotten to be, 0.118 ± 1.00, 0.129 ± 2.03, 0.123 ± 1.50, 0.107 ± 2.10, 0.101 ± 1.40 and 0.169 ± 3.60 mg corresponding to 30.18, 23.67, 27.22, 36.69 and 40.24% percentage inhibitions for groups 1, 2, 3, 4 and 5 respectively (Table 1). The weight of group 4, which is the supplemented group and that of 5, the positive control group as well as percentage inhibitions, were not statistically different (p<0.05). Result obtained shows marked inhibition of edema in the test groups when compared to the control group (p<0.05).

Evaluation of anti- inflammatory activity is often a common requirement in the development of novel compounds for the prevention of inflammation. In this study, omega-3 fish oil extract was tested using prostaglandin E-2 (PGE2) as the phlogistic agent to measure edema in rat paw. Subcutaneous injection of freshly prepared PGE-2 in tyrode solution into rat paw leads to the massive mobilization of AA from the membrane and its subsequent metabolism through the COX metabolic pathway to produce PGs that mediate pain and edema. Most prostaglandin reactions effectively kick off after a period of slow reaction, which is the first phase. This is the phase where sensory nerves are alerted of any threat. This phase is quickly followed by a phase of intense pain, swelling (edema) and redness characterized by increase in vascular permeability, exudation of fluid and leukocytes infiltration to the challenged tissue.

Omega-3 fish oil extract inhibited PGE2-induced paw edema significantly (p<0.05) and the percentage inhibition were monitored after every 15 minutes for 75 minutes (Table 2 and Fig 4). Groups 4 and 5 showed no difference all through (group 4:5; 77.93:74.50; 68.57: 77.40; 65.07:75.19; 74.65:80.62 and 82.90:92.38 %) when compared but significantly different when compared to the control at p<0.05 although this was not the case for the vehicle control group, which had the worst hit from the induction. The standard group however, showed maximum inhibition of edema (Table 2) although the other test groups also showed remarkable synergy in activity compared to the standard.

The results of the PGE2 – induced edemain the rat models was a dose – dependent fashion with the vehicle control exhibiting the highest effect of edema. Groups 4 and 5 led the way as the paw thickness goes back to the pre – inducement state as shown in Fig 4. This explains the effectiveness of the oil extract in combination with the standard drug as a potent anti – inflammatory compound, capable of inhibiting the lipoxygenase and cyclo-oxygenase pathways that bring about the synthesis of prostaglandins and other mediators of inflammation¹².

This study demonstrate that dietary omega-3 polyunsaturated fatty acid intake inhibits the production of prostaglandins both in-vitro and in-vivo, especially when supplemented with a standard drug. We therefore conclude that the age-long usage of omega-3 as a dietary supplement in the management of inflammatory conditions is safe and has therapeutic advantage.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgement

We acknowledge Kogi State University and Tertiary Education Trust Fund (TETFUND) for providing funding for this research. We also acknowledge the technical assistance of the staff of Biochemistry laboratories of the Kogi State University, Anyigba Nigeria.

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Table 1: Anti-inflammatory effect of Omega-3-polyunsaturated fatty acid oil extract on Arachidonic Acid-induced ear edema in albino rats.

Treatment Groups	Ear weight (mg)	Percentage Inhibition (%)
1	0.118±1.00^b	30.18
2	0.129±2.03^c	23.67
3	0.123±1.50^b	27.22
4	0.107±2.10^a	36.69
5	0.101±1.40^a	40.24
6	0.169±3.60^d	-

Values are reported as Mean ± SEM of five determinations. Columns with the same superscripts are not statistically significant (p < 0.05). Percentage inhibition was calculated with respect to the control. For this analysis, the concentration of standard Celecoxib used was 10 mg/kg body weight of the mice.

Table 2: Percentage Inhibition of Inflammation as a result of induction with PGE-2

Treatment groups	Percentage Inhibition (%)
Treatment groups	15 min	30 min	45 min	60 min	75 min
Group 1	47.00	59.10	41.76	46.78	44.19
Group 2	66.18	50.67	52.31	54.42	51.10
Group 3	71.63	65.67	62.09	70.79	57.75
Group 4	77.93	68.57	65.07	74.65	82.90
Group 5	74.50	77.40	75.19	80.62	92.38
Group 6	-	-	-	-	-

Percentage inhibition of inflammation by samples at 15 minutes interval and relative to the control. After 60 minutes, all groups showed an increase in inhibition, this is the point where significant decrease in paw thickness was observed. This increase continued for rats in groups 4 and 5 until the end of the observation period. Generally, after the investigation period, all groups inhibited the edema in a dose – dependent manner.

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Anti-inflammatory activity of omega-3-polyunsaturated fatty acid oil extract on acute inflammation in animal models.

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