Rheumatoid Arthritis (RA) is a chronic autoimmune and inflammatory disorder which involves the multiple joints causing pain, immobility and stiffness [1]. About 24.5 million people were affected by RA in 2015 [2]. The existing management of RA includes NSAIDs, DMARDs (like methotrexate, cyclophosphamide, sulfasalazine, hydroxychloroquine), biological such as TNF inhibitors and interleukin-1 receptor antagonists [3].
These available drugs relieve the symptoms and also modify the progression of disease but are required for longer duration and may result into serious adverse drug events. Therefore, patients often seek some alternative modality of treatment which is effective as well as safe in comparison to available medicine. Therapeutic potential of Omega-3 (n-3) polyunsaturated fatty acids (n-3 PUFAs) in chronic inflammatory diseases have been well documented [4]. In Ayurveda, Withania somnifera (WS) has been used for arthritis, asthma, hypertension, inflammation and tumours for thousand years [5]. WS and omega-3 fatty acids have been shown effective for the treatment of RA but effect of combination of the two on cytokines level and joint inflammation has not been explored previously [4,6].
Therefore, the purpose of this study was to evaluate the efficacy of the combination of WS and omega-3 fatty acids on TNF alpha level and joint histopathology in the treatment of CFA induced RA in a rat model. CFA-induced arthritis in rats mimics human RA and characterised by chronic synovitis with inflammatory cell infiltration, pannus formation, bone erosion, and cartilage destruction [7].
Materials and Methods
This experimental (animal) study was conducted in King George’s Medical University, Lucknow, Uttar Pradesh, India, in 2014-15 with duration of one year. It was conducted on 36 healthy adult male Wistar albino rats, weighing between 150-250 gm after being approved by the Institutional Animal Ethics Committee (IAEC Ref. No. 67/IAH/Pharma-14). Animals were housed in Institutional Animal House Facility under standard laboratory conditions of temperature (25±2°C), humidity (55±5%) and 12 hours light-dark cycle controlled environment. Rats were provided pellet food and water ad libitum. Rats were divided into six groups containing six rats in each group.
Group I served as arthritis control, Group II and III received WS in dose of 500 mg/kg and 1000 mg/kg respectively [8]. WS were procured from Himalaya Healthcare Co, India. Group IV received Omega-3 polyunsaturated fatty acids in dose of 100 mg/kg [9]. Capsules of Omega-3 polyunsaturated fatty acid were purchased from Merck Germany. The combination of WS (1000 mg/kg) and Omega-3 polyunsaturated fatty acids (100 mg/kg) was given to rats of group V. Group VI served as standard treatment group and received Indomethacin 3 mg/kg. Indomethacin obtained from Jagsonpal Pharmaceuticals India.
Induction of rheumatoid arthritis: Baseline values were measured on day 0 and arthritis was induced in groups II to VI. CFA was injected (0.2 mL) intradermally in the footpad of their left hind paw. CFA was procured from Sigma Aldrich Chemical Co, USA. Development of arthritis is suggested by swelling appeared in animals’ limbs by day 10 [10]. Drug administration in rats was carried out from days 10 to 21. TNF-α was measured on day 0, 10 and 21. On day 21 all rats were sacrificed by using a high dose of Pentobarbitone (150 mg/kg i.p) [11]. The inflammed limbs were excised above the ankle joints and examined for a pathological finding of RA.
Measurement of TNF alpha: Blood samples (1 mL) were withdrawn on day 12 and 21 from the retro-orbital plexus and after serum being prepared by centrifugation for 15 minutes at 3000 rpm. Level of TNF-α was determined by Enzyme-Linked Immunosorbent Assay (ELISA) kits. An ELISA kit for TNF-α was purchased from Diaclone, France. Rat TNF-α ELISA kit was used as per manufacturer’s instructions.
Histopathological examination: The left hind paw joint from each rat was cut about 0.5 cm above and below the joint and decalcified by immersion in 3% nitric acid. Then joints were fixed in 10% neutral buffered formalin for 2 days. The decalcified specimens were dehydrated in alcohol series, cleared in xylene and embedded in paraffin. Sections were serially cut at 5-μm thickness and stained with Haematoxylin and Eosin (H&E) and evaluated microscopically.
Destruction of bone and cartilage was evaluated by using the following scoring system [12].
Synovial proliferation:
Grade 0- no proliferation
Grade 1- mild proliferation (2-4 layers of reactive synoviocytes)
Grade 2- moderate proliferation (4 plus layers with increased mitotic activity)
Grade 3- severe proliferation (with invasion of joint space)
Cellular infiltration:
Grade 0- no changes
Grade 1- few focal infiltrates
Grade 2- extensive focal infiltrates
Grade 3- extensive infiltrates invading capsule with aggregate formation
Cartilage erosion:
Grade 0- no changes
Grade 1- superficial and localised cartilage degradation in more than one region
Grade 2- deep and localised cartilage degradation
Grade 3- extensive deep cartilage degradation at several locations
Pannus formation:
Grade 0- no changes
Grade 1- pannus formation (up to 2 sites)
Grade 2- pannus formation (up to 4 sites with infiltration)
Grade 3- pannus formation (> 4 sites)
Statistical Analysis
The data was analysed by one-way analysis of variance (ANOVA) followed by Dunnett’s test and Tukey test. The Dunnett’s test was used to compare each mean with control mean. Statistical significance was based on p-value less than 0.05. Data analysis was performed using SPSS version 16.
The percentage change from baseline to follow-ups was also calculated for each group. All values were expressed as mean±SEM.
Results
Effect of CFA induced arthritis on serum inflammatory factor TNF-α: Induction of arthritis significantly increased the level of TNF-α in all the groups as compared with baseline (day 0) value (p<.001) [Table/Fig-1]. In the absence of any treatment, values of TNF-α kept on rising in control group till the end i.e., 21 day. The mean % change in TNF-α levels from day 10 to day 21 was 30.01 which was significant (p<0.001).
TNF-α (pg/mL) values as mean ± SEM on day 0, 10 and 21.
| Groups | Days of study | Day 0-10 | Day 10-21 |
|---|
| Day 0 | Day 10 | Day 21 | Mean % change | p-value | Mean % change | p-value |
|---|
| TNF-α | I | 10.46+0.01 | 61.68±0.10 | 80.12±0.02 | 489.32 | <0.001 | 30.01 | <0.001 |
| II | 11.23±0.01 | 60.72±0.03 | 38.03±0.01 | 440.50 | <0.001 | -37.35 | <0.001 |
| III | 11.19±0.05 | 59.51±0.07 | 32.36±0.02 | 431.79 | <0.001 | -45.63 | <0.001 |
| IV | 10.29±0.02 | 60.43±0.01 | 33.23±0.01 | 487.20 | <0.001 | -45.02 | <0.001 |
| V | 10.10±0.02 | 60.46±0.25 | 26.25±0.02 | 498.67 | <0.001 | -56.57 | <0.001 |
| VI | 10.86±0.01 | 59.25±0.02 | 20.35±0.03 | 445.24 | <0.001 | -65.65 | <0.001 |
Effect of Withania somnifera (Ashwagandha) on serum inflammatory factor TNF-α: After treatment with WS in doses of 500 mg/kg and 1000 mg/kg, TNF-α levels decreased significantly (p<0.001) [Table/Fig-1]. The mean % change were -37.35 and -45.63 respectively. WS at both doses decreased the level of TNF-α as compared with arthritic control group (p<0.001) but better effect was seen with higher doses [Table/Fig-2].
Comparison of TNF-α (pg/mL) values of arthritic control group with treatment groups on day 21.
| Groups | Day 21 |
|---|
| Mean difference | p-value |
|---|
| TNF-α | I Vs | II | 42.16 | <0.001 |
| III | 47.84 | <0.001 |
| IV | 46.97 | <0.001 |
| V | 53.94 | <0.001 |
| VI | 59.85 | <0.001 |
Effect of Omega-3 fatty acids on serum inflammatory factor TNF-α: TNF-α levels decreased significantly (p<0.001) after treatment with Omega 3 fatty acids at the dose 100 mg/kg. The mean % change was -45.02 [Table/Fig-1]. Omega 3 fatty acids decreased the level of TNF-α as compared with arthritic control group (p<0.001) [Table/Fig-2].
Effect of combination of Withania somnifera (Ashwagandha) and Omega-3 fatty acids on serum inflammatory factor TNF-α: TNF-α levels decreased significantly (p<0.001) after treatment with combination of Withania somnifera (1000 mg/kg) and Omega-3 fatty acids (100 mg/kg). The mean % change was -56.57 [Table/Fig-1]. Combination of WS and Omega-3 fatty acids decreased the level of TNF-α as compared with arthritic control group (p<0.001) [Table/Fig-2].
Effect of indomethacin on serum inflammatory factor TNF-α: TNF-α levels decreased significantly (p<0.001) after treatment with indomethacin (3 mg/kg). The mean % change was -65.65 [Table/Fig-1].
Histopathological Analysis
Histology of joints of arthritic control rats showed greater score for synovial proliferation, cellular infiltration in the sub synovial region, cartilage erosion and pannus formation compared to treatment groups. Treatment with the combination group showed minimal synovial cell infiltration and less erosion of cartilage and bone. WS (1000 mg/kg) and omega-3 fatty acids treated rats showed moderate synovial cell infiltration and significantly reduced bone and cartilage destruction. WS (500 mg/kg) reduced the histological scores to a lesser extent when compared to higher dose of WS [Table/Fig-3].
Histopathological grading in control and experimental groups.
| Histopathological changes | Arthritic control | WS (500 mg/kg) | WS (1000 mg/kg) | Omega 3 Fatty-Acids (100 mg/kg) | WS 1000 + Omega 100 mg/kg | Indomethacin (3 mg/kg) |
|---|
| Synovial Proliferation |
| Grade 0 | - | - | - | - | 1 | 2 |
| Grade 1 | - | - | 3 | 2 | 3 | 3 |
| Grade 2 | - | 4 | 3 | 4 | 2 | 1 |
| Grade 3 | 6 | 2 | - | - | - | - |
| Total | 18 | 14 | 9 | 10 | 7 | 5 |
| Mean | 3 | 2.33 | 1.5 | 1.66 | 1.16 | 0.83 |
| Cellular Infiltration |
| Grade 0 | - | - | - | - | 2 | 4 |
| Grade 1 | - | 2 | 5 | 3 | 3 | 2 |
| Grade 2 | 1 | 3 | 1 | 3 | 1 | - |
| Grade 3 | 5 | 1 | - | - | - | - |
| Total | 17 | 11 | 7 | 9 | 5 | 2 |
| Mean | 2.83 | 1.83 | 1.16 | 1.5 | 0.83 | 0.33 |
| Cartilage Erosion |
| Grade 0 | - | - | - | - | - | 2 |
| Grade 1 | - | 1 | 2 | 1 | 3 | 4 |
| Grade 2 | 1 | 3 | 3 | 5 | 3 | - |
| Grade 3 | 5 | 2 | 1 | - | - | - |
| Total | 17 | 13 | 11 | 11 | 9 | 4 |
| Mean | 2.83 | 2.16 | 1.83 | 1.83 | 1.5 | 0.66 |
| Pannus Formation |
| Grade 0 | - | - | 1 | 1 | 1 | 2 |
| Grade 1 | - | - | 2 | 1 | 3 | 3 |
| Grade 2 | - | 4 | 3 | 4 | 2 | 1 |
| Grade 3 | 6 | 2 | - | - | - | - |
| Total | 18 | 14 | 8 | 9 | 7 | 5 |
| Mean | 3 | 2.33 | 1.33 | 1.5 | 1.16 | 0.83 |
Synovial proliferation score: Maximum synovial cell proliferation was observed in arthritic control rats [Table/Fig-4]. Arthritic rats treated with WS (500 mg/kg and 1000 mg/kg) showed decline in synovial cell proliferation score (14 and 9 respectively) in comparison to arthritic control group. Omega-3 fatty acids treated group also showed greatly reduced score (10) as compared to arthritic control group. Combination of WS and omega 3 fatty acid showed minimal synovial proliferation score (7) in comparison to other test groups [Table/Fig-5].
Control group showed ulceration and damage of cartilage and pronounced synovitis (X20).
Withania somnifera and Omega-3 fatty acid treated group showed intact cartilage and synovium (X20).
Cellular infiltration score: Cellular infiltration score was maximum (17) in arthritic control rats in comparison to treatment groups. Score of 11 was observed with the lesser dose of WS (500 mg/kg). WS (1000 mg/kg) treated rats showed a score of 7 [Table/Fig-6]. Omega treated rats showed a score of 9. Arthritic control group showed extensive infiltrates invading the capsule with aggregate formation while few focal infiltrates were found in the combination group (score of 5) [Table/Fig-7].
Withania somnifera treated group showed moderate infiltration of macrophages and plasma cells (X20).
Withania somnifera and Omega 3 fatty acid treated group showed mild infiltration of plasma cells and macrophages (X20).
Cartilage erosion score: The cartilage destruction score was found maximum (17) in arthritic control rats as compared to that of the treatment groups [Table/Fig-4]. The combination group (score of 9) showed superficial localised cartilage degradation while deep cartilage degradation was present in arthritic control group [Table/Fig-4,5].
Pannus formation score: Mean Pannus formation score was maximum (18) in arthritic control group. Extensive pannus formation was seen in more than 4 sites in arthritic control group. Pannus formation up to 2 sites was present in the combination group.
Discussion
RA is a chronic inflammatory and autoimmune disorder characterised by progressive joint destruction leading to severe disability. It mainly affects the distal joints symmetrically which become tender, swollen and stiff. Stiffness usually worsen in the mornings. Extra-articular manifestations also present in RA affecting eyes, skin, lungs, heart and blood vessels [13].
Anti-inflammatory and antioxidant activities of WS have been demonstrated in earlier studies [14,15]. In our study, Serum TNF-α was significantly increased in all groups after 10 days of injecting CFA to induce arthritis and inflammatory changes in rats. TNF-α has a significant role in mediating inflammation and bone degradation and it is found elevated in synovial fluid in RA [16,17].
Role of TNF-α in pathogenicity of RA is endorsed by the fact that neutralisation of monoclonal anti-TNF antibodies diminish collagen-induced arthritis in mice [18]. TNF-α antagonists, like infliximab, etanercept, adalimumab and Certolizumab Pegol (CZP) have been used for the treatment of RA. In our study, WS in both doses (500 mg/kg and 1000 mg/kg) showed significant reduction in TNF-α (p<0.001). A previous study showed that administration of WS extract for 10 days significantly decreases TNF-α concentration [19]. Malekshahi MA et al., showed that short-term omega-3 fatty acid administration decreases serum levels of TNF-α which is in agreement to our study which also showed decrease in TNF-α level after omega-3 fatty acid administration in dose of 100 mg/kg [20].
Combination of WS (1000 mg/kg) and omega-3 fatty acid (100 mg/kg) significantly decreased the TNF-α expression indicating synergistic anti-inflammatory effect on pathology of the disease. Combination of WS and omega-3 fatty acids was more effective than individual drugs alone. Though the mean % change in TNF-α levels was lower than that of indomethacin, both group showed statistically significant decrease in its levels.
Inter group comparison analysis showed a dose dependent response with maximum mean percent reduction in group VI (-65.65%) and minimum in group II (-37.35%). Group V showed a higher percent reduction in TNF-α as compared with groups II, III and IV.
TNF increases the expression of chemokines and adhesion molecules and there by attracts recruitment of neutrophils [21]. Local inflammation is also induced by TNF-α leading to pannus formation and bone destruction [22].
Histopathological evaluation revealed similar findings as observed by Gupta A et al., [6]. Histology of the joints of arthritic control rats showed synovial cell proliferation, pannus formation and infiltration of mononuclear and neutrophil infiltration in sub synovial region. Pannus formation caused destruction of bone and cartilage. Elevation of synovial cell infiltration was observed in arthritic control group. Arthritic rats treated with combination of WS and omega-3 fatty acids showed minimal cell infiltration, pannus formation and no sign of bone and cartilage destruction. Reduction in mean histological score in the combination group may be attributed to its anti-inflammatory and antioxidant properties [23].
Limitation
Animal model of disease does not exactly represent the real disease in human beings so further clinical studies should be conducted. Though ideally both the doses of WS should be chosen in combination with omega-3 fatty acid, only higher dose of WS was chosen (to reduce the number of animals). As this study was of short duration and evaluated only treatment of disease, other studies of longer duration need to be planned to explore the preventive role of WS and omega-3 fatty acids in RA.
Conclusion
The results of the present study are encouraging and may reveal the importance of the WS and omega-3 fatty acids which suppress the changes produced during adjuvant induced arthritis. Combination of WS and omega 3 fatty acids was more effective than individual drugs alone.