International Journal of Nutrition, Pharmacology, Neurological Diseases

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 9  |  Issue : 3  |  Page : 87--96

Effect of Omega-3 Alone and in Combination With Dexamethasone or Methotrexate in Treatment of Experimentally Induced Arthritis in Rats


Mary Girgis Shahataa1, Ahmed A Elberry1, Ali A Abo-Saif2, Mai A.M Almoatasem3, Asmaa Mohammed M Hussein Elkelawy1,  
1 Department of Pharmacology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt
2 Department of Pharmacology, Faculty of Medicine, Alazhar University, Cairo, Egypt
3 Histology department, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt

Correspondence Address:
MD, PhD Ahmed A Elberry
Department of Pharmacology, Faculty of Medicine, Beni-Suef University, Beni-Suef, 62511
Egypt

Abstract

Aims: The aim of the present study is to evaluate the antiinflammatory and antioxidant effects of omega-3 fatty acids in the treatment of rheumatoid arthritis (RA) and its complementary effect in combination with the half-tested doses of the most common drugs used to treat RA as cortisone and methotrexate. Methods: Arthritis was induced by subcutaneous injection of three doses of 0.4 mL complete Freund’s adjuvant-antigen emulsion at three different limbs. Animals were divided into control group, arthritis group, omega-treated group (1 g\kg\d), dexamethasone-treated group (1 mg\kg\d), methotrexate-treated group (1 mg\kg\d), omega (1 g\kg\d) plus dexamethasone (0.5 mg\kg\d)-treated group, and omega (1 g\kg\d) plus methotrexate (0.5 mg\kg\d)-treated group. Serum cartilage oligomeric matrix protein, tumor necrosis factor alpha, interleukin-1B, myeloperoxidase, reduced glutathione, and malondialdehyde were assessed. A histopathological study of rat’s knee joints and an immunohistochemical study for expression of cyclooxygenase-2 in joint tissue were performed. Results: The results showed that omega treatment significantly corrected paw diameter and all biochemical markers, but the combination treatment of omega plus dexamethasone or methotrexate in lower doses showed more significant correction with the restoration of these parameters back to normal level except for tumor necrosis factor alpha. Also, there was an amelioration of pathological changes of knee joints with the restoration of the smooth articular surface besides the reduction in cyclooxygenase-2 expression that was showed by the immunohistochemical study. Conclusion: The current study provides evidence that omega-3 may have an ameliorative effect against experimentally induced arthritis and can improve the therapeutic effect of standard medications used in the treatment of RA.



How to cite this article:
Shahataa MG, Elberry AA, Abo-Saif AA, Almoatasem MA, Hussein Elkelawy AM. Effect of Omega-3 Alone and in Combination With Dexamethasone or Methotrexate in Treatment of Experimentally Induced Arthritis in Rats.Int J Nutr Pharmacol Neurol Dis 2019;9:87-96


How to cite this URL:
Shahataa MG, Elberry AA, Abo-Saif AA, Almoatasem MA, Hussein Elkelawy AM. Effect of Omega-3 Alone and in Combination With Dexamethasone or Methotrexate in Treatment of Experimentally Induced Arthritis in Rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2019 [cited 2019 Nov 18 ];9:87-96
Available from: http://www.ijnpnd.com/text.asp?2019/9/3/87/270037


Full Text



 Introduction



Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disorder affecting approximately 1% of people in the developed countries.[1] The pathogenesis of RA can be attributed to a complex interaction between genetic, hormonal, and environmental factors with continuous activation of immune system components. These factors lead to synovial hyperplasia, bone erosion, joint swelling, and deformity.[2]

Proinflammatory cytokines especially tumor necrosis factor alpha (TNF-α) and interleukins (ILs) as well as inflammatory enzymes including cyclooxygenase-2 (COX-2) produced by the T cells and macrophages are upregulated in synovium causing severe inflammation.[3] Continuous inflammation leads to cartilage destruction and release of cartilage oligomeric matrix protein (COMP).[4] On the other hand, there are releases of many oxidants from inflammatory cells as malondialdehyde (MDA) and myeloperoxidase (MPO) with depletion of important antioxidant as glutathione (GSH).[5]

Despite there is no cure for RA, a variety of medications are available aiming to halt the disease and decrease joint damage.[6] Although steroidal and nonsteroidal antiinflammatory drugs as well as disease-modifying antiarthritic drugs such as methotrexate (MTX) are used frequently in RA management, these agents are associated with many adverse drug reactions as cardiovascular and gastrointestinal side effects.[7] Cytokines inhibitors therapies are used in treatment of RA for inhibition of immune reaction, but the usage of these therapies have many limitations due to expensive cost and hypersensitivity, and only 40% of RA patients respond to such therapies.[8]

Omega-3 (OMEGA) fatty acids especially the long-chain docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) originating from fish oils are considered essential nutrients established to improve the quality of life and decrease the risk of early death. They function only via cell membranes in which they are incorporated by phospholipid molecules.[9] The dietary OMEGA polyunsaturated fatty acids are also considered to reduce pain and inflammation in tissues by its ability to compete with arachidonic acid for enzymatic metabolism inducing less production of inflammatory and chemotactic factors such as prostaglandins E2 (PGE2) and leukotriene B4 (LTB4) which in turn inhibit the activation of nuclear factor kappa light-chain-enhancer of activated B (NFκB), and thus the release of inflammatory cytokines such as IL-1β and TNF-α.[10]

Studies using fish oil in patients with inflammatory diseases reported decrease in IL-1 production by monocytes[11] and diminished circulating concentrations of IL-1β, TNF-α, and soluble receptor activator of NFκB ligand.[12] In addition, clinical trials with OMEGA supplementation have reported an improvement in tender joints on physical examination and morning stiffness.[13]

Accordingly, we aimed to evaluate the proposed antiinflammatory and antioxidant effects of OMEGA fatty acids in the treatment of RA and its complementary effect in combination with the half-tested doses of the most commonly drugs used to treat RA such as cortisone and MTX against adjuvant-induced arthritis in rats.

 Material and Methods



Drugs and chemicals

OMEGA (Super Omega; Regal Pharma, Cairo, Egypt) was provided in the form of soft gelatin capsules (1000 mg). The capsules were opened and the solution was collected (1 mL containing 1 g) and given orally by gavage according to body weight in a dose of (1 g\kg\d). Dexamethasone (Dexazone; Kahira Pharmaceuticals, Cairo, Egypt) and MTX (Methotrexate; Orion, Espoo, Finland) were provided in the form of tablets. The tablets were crushed and dissolved in 1 mL saline. It was given orally by gavage according to body weight in a dose of (1 mg\kg\d).

Complete Freund’s adjuvant (CFA)-antigen emulsion (Sigma-Aldrich, European Export; Chemical Company, Germany). Each milliliter contains 1 mg of Mycobacterium tuberculosis (H37Ra, ATCC 25177), heat killed and dried, 0.85 mL paraffin oil, and 0.15 mL mannide monooleate. Freund’s adjuvants may be used to produce water-in-oil emulsions of immunogens using gum acacia as an emulsifying agent. The mycobacteria in CFA attract macrophages and other cells to the injection site that enhances the immune response.

COMP, ELISA kits (Clinilab, Cairo, Egypt); TNF-α, ELISA kits (Glory Science, TX, USA); IL-1B, ELISA kits (Cusabio Biotech, TX, USA); MPO, colorimetric kit (Clinilab, Cairo, Egypt); Reduced GSH, colorimetric kit (Biodiagnostic, Giza, Egypt); and MDA, colorimetric kit (Clinilab, Cairo, Egypt).

Animals used

Seventy adult female albino rats weighing (170–200 g) were used. Animals were kept for 14 days for adaptation before being subjected to laboratory experiments. All experimental procedures of animal handling and drug administration were done according to guidelines of local ethics committee, Faculty of Medicine, Beni-Suef University, which comply with the guidelines of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 018-62, revised 2017). Animals were kept in separate cages, five animals per cage, at controlled temperature (26°C), and on a 12-h light–dark cycle. Water and food were given ad libitum.

Experimental design

Animals were randomly assigned into seven groups, 10 rats in each group. Group 1, control group received 0.5 mL saline only. Group 2, RA-untreated group, where RA was induced using modified method of arthritis described by Wahba et al.[5] by subcutaneous injection of three doses of CFA, each of 0.4 mL at three different limbs on days 1, 4, and 7 for 12 days. Group 3, RA group, treated with OMEGA (1 g\kg\d).[14] Group 4, RA group, treated with DEX (1 mg\kg\d).[15],[16] Group 5, RA group, treated with MTX (1 mg/kg/d).[17] Group 6, RA group, treated with OMEGA (1 g\kg\d) along with DEX (0.5 mg\kg\d). Group 7, RA group, treated with OMEGA (1 g\kg\d) along with MTX (0.5 mg/kg/d). All drugs were freshly prepared during the experiment in the concentration required and were given to the treated groups on day 13 of RA induction and continued to day 19 using an oral syringe with blunt curved edge to avoid injury of oral mucosa of rat and to allow proper swallowing of the drug.

Body weight measurement

Body weight was recorded on day 1 before RA induction, day 13 after induction of RA, and at the end of the experiment on day 20.

Paw thickness measurement

As in body weight assessment, paw thickness was measured on days 1, 13, and 20 of RA induction using Vernier scale.[18] The change in paw thickness is measured in millimeter using Vernier caliber and the effect of tested drugs is manipulated by calculation of the percentage of inhibition of paw edema as follows:

Percentage of inhibition of paw edema = (1−Vt\Vc) × 100

where Vc represents the increase in paw thickness of diseased group and Vt represents the change in paw thickness after drug treatment.[19]

Collection of serum blood samples

On the 20th day, rats were anesthetized with thiopentone (35 mg/kg; i.p.) and blood samples were collected by orbital puncture using heparinized microtubes. The rat was restrained, the neck gently scuffed, and the eyes made to bulge. Capillary tubes inserted medially, laterally, or dorsally to the eye. Blood was allowed to flow by capillary action into the capillary tube.[20] Up to 4 mL sample volume was obtained and the collected blood was then centrifugated at 3000×g for 20 minutes to separate sera that kept at −80°C for measurement of COMP, TNF-α, IL-1B, MPO, reduced GSH, and MDA.

Histopathological examination of rat knee joints

After collection of blood samples, animals were euthanized using carbon dioxide asphyxiation and knee joints of rats of different groups were extracted for histopathological study. Right/left knee joints from rats were collected into 10% neutral buffered formalin and then processed for complete decalcification in ethylene diaminetetraacetic acid (EDTA).[21] After decalcification, knee joints were embedded in paraffin, sectioned longitudinally at 5 µm, and stained with hematoxylin and eosin.[22]

Immunohistochemical study

COX-2 immunostaining using the avidin–biotin–peroxidase complex technique was performed. After blocking the endogenous activity of peroxidase using 10% hydrogen peroxide, the sections were incubated with 2 mL of ready-to-use rabbit monoclonal antibody against COX-2 for each section. Thereafter, the secondary antibody was applied (biotinylated goat antibody). The site of antibody binding was visualized after adding diaminobenzidine chromogen, which converts into a brown precipitate.[23] Evaluation of COX-2 expression was based on the extent of positivity. The presence of brown colored was indicative of positive reactivity. The COX-2 immunohistochemical staining score was assessed using the scoring systems by Jeong et al.[24]; in this scoring, COX-2 staining intensity in tissue was scored using a scale of 0 to 3 (0 = no staining or negative, 1 = weak staining, 2 = moderate staining, and 3 = strong staining).

Statistical methods

All statistical calculations were done using computer programs Microsoft Excel (Microsoft Corporation, NY, USA) and SPSS (Statistical Package for the Social Science) statistical programs (SPSS Inc., Chicago, IL, USA). Data were statistically described in terms of mean ± standard deviation (±SD). Descriptive statistics were done for quantitative data as minimum and maximum of the range as well as mean ± SD for quantitative parametric data.

Inferential analyses were done for quantitative variables using the analysis of variance test for comparison between two or more means of quantitative data. Values of P less than 0.05 were considered significant.

 Results



Body weight measurements

The present study revealed that CFA-induced RA significantly decreased the body weight in all groups on day 13 compared to the control group values. Although nonsignificant, OMEGA treatment showed some improvement in body weight on day 20 (206.2 ± 11.1) when compared to the control group values (219.0 ± 7.3). This improvement was significant when compared to the control group values. This improvement was significant and reached nearly to the control group values when OMEGA was combined with MTX (210.5 ± 5.9) [Table 1].{Table 1}

Paw diameter measurements

Induction of RA showed a significant increase in mean paw diameter in all groups that received CFA compared to control group values with percent ranged from 35% to 48% with no significant differences from each other. Treatment with OMEGA, for seven consecutive days resulted in significant alleviation in mean paw diameter by 14%, compared to that of RA group values. Similarly, the current results showed a significant alleviation in mean paw diameter by 26% and 28% in groups treated with combined regimen (OMEGA + DEX) and (OMEGA + MTX) compared to that of RA group with insignificant differences when compared to control group values [Table 2].{Table 2}

Biochemical study

COMP

Induction of RA in the RA group caused a significant increase in the serum level of COMP compared to the control group values. Administration of OMEGA significantly decreased the elevated serum COMP level compared to the RA group values. Combined treatment of OMEGA plus DEX and OMEGA plus MTX resulted in a more significant decrease in the elevated COMP level compared to the RA group values. Also, the results showed that the combination treatment could restore the COMP level back to normal levels [Figure 1A].{Figure 1}

Cytokines and inflammatory markers

Rats subjected to induction of RA by CFA showed significant increase in serum levels of TNF-α and IL-1B compared to the control group values. Administration of OMEGA, alone or in combination, resulted in significant decrease of these elevated serum levels of TNF-α and IL-1B compared to values of RA group, DEX group, and MTX group. In addition, the present study showed normalization of IL-1B values in group treated with OMEGA + MTX [Figure 1]B and [Figure 1]C.

With regard to MPO, there was significant increase in serum level of MPO in arthritic group compared to the control group values. Groups treated with OMEGA, alone or in combination, showed significant alleviation of this elevated values compared to diseased group. Besides, the level of MPO return nearly to normal levels only in groups that were treated with combination therapy [Figure 1D].

Oxidative markers

Induction of RA in the RA group caused a significant decrease in serum level of GSH and significant increase in level of MDA compared to the control group values. Treatment with OMEGA, alone or in combination, resulted in significant amelioration of both markers compared to the diseased group. Only the groups that were treated with OMEGA plus either DEX or MTX showed restoration of oxidative markers back nearly to the normal levels [Figure 1]E and [Figure 1]F.

Histopathological study

Results of hematoxylin and eosin staining

Histopathological investigations from arthritic group revealed distortion compared to the normal control group [Figure 2]A and evidence of arthritis that include distortion of the articular cartilage and loss of its regular rounded contour. In addition, some fields showed distorted cells in the superficial layer with the appearance of empty lacunae denoting dead cells and absence of cell nest [Figure 2]B. In contrast, it was found that treatment with OMEGA, DEX, and MTX showed amelioration of these pathological changes with restoration of the smooth articular surface. Moreover, treatment with combination therapy OMEGA plus DEX and OMEGA plus MTX resulted in restoration of the smooth rounded articular surface with subchondral plate of bone [Figure 2]F and hypercellularity of articular cartilage with colonization of chondrocytes [Figure 2]G.{Figure 2}

Immunohistochemistry

For expression of COX-2 in joint tissue, there was no positive reaction detected in chondrocytes as well as the synovial cells in the normal control group [Figure 3]A, whereas in arthritic group, there were many cells that exhibited positive reaction in the articular cartilage. The COX-2 staining was observed in chondrocytes with a cytoplasmic and prenuclear pattern of expression [Figure 3]B. Positive reaction was detected in some cells in the articular cartilage as well as in the synovial membrane in both the groups treated with either omega or MTX [Figure 3]C and [Figure 3]D). Few cells in the articular cartilage showed positive reaction in groups treated with DEX, omega plus DEX, and omega plus MTX [[Figure 3]E–G). In addition, the present results showed that there was a statistically significant difference between the seven studied groups regarding their immunohistochemical staining score as the control group had the highest frequency of score zero (90%) and the RA group had the highest frequency of score three (70%) [Figure 4].{Figure 3}{Figure 4}

 Discussion



CFA used in the current study is a method for RA induction as CFA administration can promote localized immunological and inflammatory reactions in the same pattern simulating clinical RA.[25] Induction of RA in the current study was evident by high immunohistochemical scoring and showed significant decrease in body weight and significant increase in paw diameter which is in agreement with the results of the previous studies.[7],[26],[27],[28] The decrease in body weight is attributed to inflammation, and inflammatory cachexia, increased immobility, skeletal muscle loss, pain, and febrile condition of rats.[26] The significant increase in the paw diameter of inoculated rats was observed due to the state of inflammation induced by CFA. On the other hand, the current study showed that the OMEGA treatment alone failed to alleviate significantly the body weight but this alleviation was significant when combined with MTX. This is in agreement with the previous studies recorded by de Castilho et al.[29] and Kaprinay et al.[30]

The current study showed that OMEGA significantly alleviated the increased paw diameter induced by RA which comes in agreement with the previous studies.[31],[32] Morris[33] explained this improvement in paw edema by the inhibitory actions of OMEGA on some mediators involved in the inflammatory response, such as histamine, bradykinin, lipid mediators derived from arachidonic acid, nitric oxide, and cytokines.

The present results also showed that treatment of RA-induced rats with DEX resulted in significant more reduction in body weight compared to that of RA group, but there was a significant improvement in paw edema compared to that of the RA group, whereas the combination of DEX in a half dose with OMEGA resulted in more reduction in paw edema compared to the RA group values with normalization of this parameter when comparing it to control group values. These results are in agreement with those mentioned by Mohamed and El-Hadidy,[34] Ma et al.,[35] and Dubey et al.[36] DEX improved paw edema by its antiinflammatory effects[36] and in support to our results, Dubey et al.[36] reported that DEX causes a reduction in body weight and food intake. The probable mechanism could be that DEX increases leptin mRNA expression in the adipose tissue and plasma leptin may play a role in DEX-induced anorexia.

On the other hand, the present study showed that treatment of RA-induced rats with MTX resulted in significant improvement in weight gain compared to RA group values; also there was significant reduction of paw edema compared to that of RA group with no significant differences compared to the control group values. These results come in agreement with the results obtained by previous investigators.[37],[38],[39],[40] The suggested mechanisms of MTX include inhibition of T-cell proliferation by affecting purine and pyrimidine metabolism and inhibition of transmethylation reaction necessary for T-cell cytotoxicity prevention, and alter the recruitment of monocytes to the inflamed joint by interfering with GSH metabolism.[41]

Indeed, many patients have a poor response to MTX and they suffer from distressing side effects from the application of long term and high dose. So, it is meaningful to reduce the dosages and unwanted effects of drugs while improving efficacy. Therefore, a rational combination of drugs should be used in the treatment of RA.[42] Based on that, in our study, we combined OMEGA with MTX in a lower dose and the results showed that this combination showed further decrease in paw edema and more improvement in weight gain that nearly equals to the normal levels.

Extracellular glycoprotein COMP is a member of the thrombospondin family of calcium-binding proteins. It is associated with cartilage breakdown and has been studied as a potential diagnostic and prognostic indicator as well as a marker of disease severity or the efficacy of treatment.[43] The present study showed that treatment with OMEGA could significantly improve the serum COMP level, and this improvement was evidenced by overall reduction in bone and cartilage destruction in the joints of animals treated observed in the current histopathological results.

Moreover, it seems from the present study that OMEGA administration to rats had antiinflammatory and immunomodulatory effects evidenced by significant corrections of disrupted serum levels of TNF-α, IL-1B, and MPO. These results come in agreement with the previous results.[44],[45],[46] The antiinflammatory properties of OMEGA arise from processes such as the partial replacement of arachidonic acid to act as a substrate for COX enzyme, resulting in inhibition of the production of proinflammatory eicosanoids such as prostaglandin E2; there is also a reduction in the chemotaxis of monocytes and neutrophils, modulating the inflammatory response in its earliest stages.[47]

The current results also show that treatment with OMEGA could improve the levels of oxidative stress markers in arthritic rats, evidenced by significant amelioration of the elevated MDA level, and the decrease of the antioxidant marker GSH. These results are in agreement with the results obtained by the previous researchers showing improvement in the oxidant status associated OMEGA treatment.[48],[49],[50] On the other hand, there have been conflicting reports on the effects of OMEGA supplementation on oxidant/antioxidant status in both animal and human studies.[51],[52],[53] In the light of our results, it seems possible that the decreased MDA levels, as a result of OMEGA supplementation, clearly show its protective effect against lipid peroxidation and oxidative stress. Interestingly, Barbosa et al.[54] suggested that OMEGA supplementation may have free radical scavenger activity. Administration of OMEGA may stimulate vitamin E incorporation into membranes to avoid lipid peroxidation resulting from increased membrane OMEGA content.[55]

As regard the COX-2 expression in joint tissues, the current study showed upregulation of COX-2 expression in articular cartilage of arthritic group including chondrocytes and synovial cells. These results are in agreement with that previously reported by Kang et al.[7],[56] Lee et al.[57] reported that the overexpression of COX-2 in articular tissue is a distinctive feature of inflammatory disease like RA. Inflammatory mediators including IL-1B and TNF-α appear to play a main role in modulation of COX-2 gene expression in joint tissue and subsequent release of prostaglandins resulting in cartilage destruction. Treatment of OMEGA and MTX resulted in moderate decrease in COX-2 expression, whereas treatment with DEX and combination therapy showed marked lowering in the expression of COX-2 in joint tissue comparing to arthritic group.

Simopoulos[58] concluded that the antiinflammatory properties of OMEGA fatty acids, especially EPA, are due to competition with arachidonic acid as a substrate for COXs and 5-lipoxygenase. In addition to their effects on prostaglandins, thromboxanes, and leukotrienes, OMEGA fatty acids suppress the production on IL-1B by suppressing the IL-1B mRNA, as well as the expression of COX-2 mRNA that is induced by IL-1B.

In conclusion, the current study shows that OMEGA ameliorates RA in rats’ joints and the possible mechanism of this antiinflammatory effect is mediated through COX-2 enzyme. In addition, OMEGA with half the dose of the standard treatment used in RA (MTX and DEX) exhibited the best results. Interestingly, it could be concluded that combination therapy potentiated the effect of single drug suggesting different mechanisms. Further studies are recommended to evaluate the clinical use of OMEGA in combination with other treatment modalities of RA to improve the therapeutic outcome of the standard medications and to decrease their side effects by decreasing their doses when combined with OMEGA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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