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Anti inflammatory effect of thymoquinone in comparison with methotrexate on pristane induced arthritis in rats.

Byline: Rizwan Faisal, Sadia Chiragh, Abdul Jalil Popalzai and Khalil Ur Rehman

Abstract

Objective: To determine the anti-inflammatory effects of thymoquinone on body weight, clinical score of inflammation, total leukocyte count and differential leukocyte count in arthritic rats and compare it with that of methotrexate.

Methods: The study was conducted at the Post-Graduate Medical Institute, Lahore, from March to September 2013, and comprised female Sprague-Dawley rats randomised into four equal groups; group A (healthy control), group B (positive control), group C (thymoquinone treated) and group D (methotrexate treated). Arthritis developed in Group B, C and D within two weeks after a single intra-dermal injection of pristane. Body-weight measured on electronic balance in grams and clinical score of inflammation scored on macroscopic scoring system were monitored on every alternate day while total leukocyte count and differential leukocyte count were taken at day 0, 16 and 30.

After day 15, groups A and B were given 0.5ml of distilled water by intra-peritoneal injection daily for 15 consecutive days; group C was given thymoquinone 2mg/kg by intra-peritoneal injection daily for 15 consecutive days, and group D received methotrexate 0.5mg/kg by intra-peritoneal injection, daily for 15 consecutive days. SPSS 20 was used fo r statistical analysis.

Results: The 32 rats in the study were randomised into four groups of 8(25%) each. In group A the body-weight continued to increase and reached a mean of 144.1310.8% of the baseline at day 30. In group B the weight reduced to 93.134.19% at day 16 and to 88.36.97% at day 30. In groups C and D the weight reduced to 87.257.69% and 88.57.07% respectively at day 16; then the animals in the two groups regained their weight which increased to 108.6310.89% and 103.386.25% respectively at day 30. The score was zero in group A throughout the study period. The score of group B, which was zero at day 0, reached a mean of 160 at day 16. In groups C and D, the mean score increased till day 16 and reached 161 and 160 respectively, and then reduced to 52 and 41 at day 30 respectively.

Conclusion: Evaluation of data supported the anti-inflammatory activities of thymoquinone, so it may be investigated as an effective anti-inflammatory drug in rheumatoid arthritis.

Keywords: Rheumatoid arthritis, Pristane, Thymoquinone, Methotrexate. (JPMA 65: 519; 2015)

Introduction

Rheumatoid arthritis (RA) is a chronic, progressive, systemic inflammatory disorder affecting the synovial joints and typically producing symmetrical arthritis that leads to joint destruction, which is responsible for deformity and disability. The consequent morbidity and mortality has a substantial socioeconomic impact. The prevalence of RA is consistent worldwide, affecting about 0.5-1% of the population. Women are affected more than men at ratio of 3:1. RA progresses in three stages. The first stage is the swelling of the synovial lining, causing pain, warmth, stiffness, redness and swelling around the joints. Second is the rapid division and growth of cell, or pannus, which causes the synovium to thicken. In the third stage, the inflamed cell releases enzymes that may digest the bone and cartilage, often causing the joints to lose shape and movement.

The most commonly prescribed medication for RA treatment is non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs, corticosteriods and immunosuppressant drugs. The goal of these drugs has been to relieve pain, to decrease joint inflammation and to prevent joint destruction. Among disease-modifying anti-rheumatic drugs, methotrexate (MTX) remained the cornerstone for RA. These drugs are known to produce various side effects, including gastrointestinal (GI) disorders, immunodeficiency, hormonal disturbances and increased tendency to cause infections. Accordingly, reduced side effects should be considered while designing improved therapeutics for RA.

Nigella Sativa has been used traditionally in herbal medicine for the treatment of various diseases. Thymoquinone (TQ) is major active agent of Nigella Sativa.It has antioxidant effects and has been shown to protect against heart, liver and kidney damage,2 has anti- cancer,3 anti-bacterial,4 anti-convulsant,5 anti- inflammatory,6 anti-tussive,7 anti-asthmatic8 and anti- diabetic effects.9

Considering the anti-inflammatory property, the current study was conducted to evaluate the anti-inflammatory activity of TQ in arthritic rats and to compare it with that of MTX.

Subjects and Methods

The comparative study was conducted at the Post- Graduate Medical Institute PGMI), Lahore, from March to September 2013. Adult healthy female Sprague Dawley rats weighing 120-220 grams were kept in the PGMI Animal House in iron cages under hygienic conditions. Room temperature was maintained at 252C and they were fed rat chow and water ad libitum. They received care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals.10 They were kept for acclimatisation for one week.

The sample size was estimated by using power and precision 3.0 software and the rats were randomly divided into four equal groups; A, B, C and D. The rats were numbered 1, 2, 3 and 4; all rats having number 1 were included in group A, while the numbers 2, 3 and 4 rats formed groups B, C and D respectively.

All the four groups received rat chow and drinking water for 30 days. Group A was given 0.5ml of distilled water at the base of tail on day 0. After day 15, group A was given 0.5ml of distilled water by intra-peritoneal injection daily for 15 consecutive days. Groups B, C and D were given a single intra-dermal injection of 0.5ml pristane on day 0 at the base of tail.11 After day 15, group B was given 0.5ml of distilled water by intra-peritoneal injection daily for 15 consecutive days; group C was given TQ 2mg/kg dissolved in distilled water by intra-peritoneal injection daily for 15 consecutive days12 and group D received MTX 0.5mg/kg intra-peritoneal injection daily for 15 consecutive days.13

Parameters measured were body-weight, clinical score of inflammation, total leukocyte count (TLC) and differential leukocyte count (DLC). The weight of the animals was not similar at day 0 after random allocation so it was converted into percentage and was considered as 100%. Then the trend of change in weight was studied in percentages. Arthritis development was monitored by a macroscopic scoring system for the four limbs ranging from 0-4, where 0 = no swelling, no redness, no joint involvement; 1 = swelling and redness of one joint; 2 = two joints involved; 3 = more than two joints involved; and 4 = severe arthritis in the entire paw.14 (Minimum score = 0 and maximum = 4 for one limb; the score of 4 limbs was collectively taken so it became minimum score = 0 and maximum = 16.)

Clinical scoring of arthritis and body-weight for all the rats was performed at day 0 and then on every alternate day. Scores at day 0, 16, 24 and 30 were analysed statistically.

At day 0, 15 and 30, 1ml blood was collected by cardiac puncture under light anaesthesia (inhalation of chloroform). Blood was put in the test tubes containing Ethylenediaminetetraacetic acid (EDTA) and checked for TLC and DLC.

Data was entered and analysed using SPSS 20. Mean and standard deviations were used as descriptive measures in quantitative variables like body-weight, inflammatory score, TLC, and DFC for the four groups at different reading times. Data was tested for normality by using Shapiro's Wilkes test, and homogeneity of variance was tested by using Leven's statistics. Data following normal distribution and having homogeneity of variances were compared among groups by using one-way analysis of variance (ANOVA), and Tukey's test was used for post hoc analysis. Data deviating from normality and homogeneity was compared among groups by using Kruskal Wallis ANOVA, and Mann Whitney U test was used for post hoc analysis. Normally-distributed data was compared between times by using paired t-test. P less than 0.05 was considered statistically significant.

Results

The 32 rats in the study were randomised into four groups of 8(25%) each. In group A the body-weight continued to increase and reached a mean of 144.1310.8% of the baseline at day 30. In group B the weight reduced to 93.134.19% at day 16 and to 88.36.97% at day 30. In groups C and D the weight reduced to 87.257.69% and 88.57.07% respectively at day 16; then the animals in the two groups regained their weight which increased to 108.6310.89% and 103.386.25% respectively at day 30 (Figure-1).

The clinical score of inflammation was zero in group A throughout the study period. The score of group B, which was zero at day 0, reached a mean of 131 at day 8, and increased to 160 at day 16. Then it remained constant on 161 and 151 at day 24 and day 30 respectively. In groups C and D, the mean score increased till day 16 and reached 161 and 160 respectively. At day 24 the mean score reduced to 81 and 92 with further reduction to 52 and 41 at day 30 in the two groups respectively (Figure-2). The score was normal in group A, marked in group B, moderate in group C, and mild in group D (Figure-3).

Mean TLC at day 0 was 5025509/cmm in group A, 5263941/cmm in group B, 4738590/cmm in group C and 4913671/cmm in group D. In group A, TLC remained unchanged over time. In group B at day 15 and day 30 TLC was 12563795/cmm and 117381108/cmm (pless than 0.001). In groups C and D, TLC at day 15 was 13150924/cmm and 13450875/cmm (pless than 0.001). At day 30, TLC in groups C and D was 6938886/cmm and 5938760/cmm (pless than 0.001) (Table-1 and 2).

When pair-wise comparison was made between the groups at day 15, the mean TLC was significantly higher in groups B, C and D compared to group A (pless than 0.001 each). There was no significant difference between groups B, C and D at day 15. At day 30, group B had significantly higher mean TLC compared to group A, C and D (pless than 0.001 each). There was no significant difference between groups C and D (p=0.119) and A and D (p=0.298), but difference between groups A and C was significant (p=0.002) (Table- 3). Neutrophil percentage at day 0 was the highest 32.32.2 in group C and lowest 30.12.8 in group A. In group A, it remained unchanged over time. In group B, the neutrophil percentage at day 15 and day 30 was 35.12.5 and 34.42.7 respectively which was significantly higher than day 0 (pless than 0.001 and p=0.008). The difference between day 15 and day 30 was insignificant (p=0.080).

In groups C and D the neutrophil percentage at day 15 was 36.42.4 and 34.42.1 which was significantly higher than day 0 (pless than 0.001 each). At day 30,in groups C and D it was 33.82.2 and 30.62.3 which was significantly lower than day 15 (pless than 0.001 each).

Comparison made at day 15 between the groups and the percentage of neutrophil were significantly higher in groups B, C and D compared to group A (pless than 0.001 each). The difference among groups B, C and D was insignificant (p=0.663; p=0.900; and p=0.277). At day 30, groups B and C had significantly high neutrophil compared to group A (p=0.001;p=0.003) while difference between groups D and A was insignificant (pgreater than 0.05). In group A, lymphocyte count (LC)

Table-1: Effect of Thymoquinone on total and differential leukocyte count (TLC and DLC) in blood of prisaine-induced arthritic rats (n=8) (meanstandard deviation).

Day###Day 0###Day 15###Day 30

Group###A###B###C###D###A###B###C###D###A###B###C###D

###33.82.2# 30.62.3

TLC mm3###5025509###5263941 4738590 4913671###5175542###12563795####13150924####13450875# 5163632 117381108# 6938886# 5938760

Neutrophil %age###30.12.8###32.02.3 32.32.2 30.31.9###29.01.7###35.12.5####36.42.4####34.42. 1# 29.41.6 34.42.7#

Lymphocyte %age###67.93.3###66.43.2 66.52.8 69.52.0###69.11.6###61.52.7####61.13.9####63.03.5# 69.02.0 62.42.6####65.03.0# 68.12.6

Neutrophil: Lymphocyte###0.450.06###0.480.06 0.490.05 0.440.04###0.420.03###0.570.07####0.600.07####0.550.06# 0.430.03 0.550.06# 0.520.06# 0.450.05

Table-2: Comparison of weight, clinical score of inflammation, Total leukocyte count (TLC) and neutrophils among groups at various reading times (ANOVA).

Parameter###Sum of Squares###Df###Mean Square###F###Sig.

Weight###Day 16###Between Groups###7191.625###3###2397.208###62.193###less than 0.001

###Within Groups###1079.250###28###38.545

###Total###8270.875###31

###Day 30###Between Groups###13383.000###3###4461.000###55.159###less than 0.001

###Within Groups###2264.500###28###80.875

###Total###15647.500###31

Clinical score of inflammation###Day 16###Between Groups###1.3###2###0.7###2.3###0.122

###Within Groups###6.0###21###0.3

###Total###7.3###23

###Day 30###Between Groups###614.3###2###307.2###141.8###0.000

###Within Groups###45.5###21###2.2

###Total###659.8###23

Total leukocyte count###Day 16###Between Groups###375748437.5###3###125249479.2###196.870###less than 0.001

###Within Groups###17813750.0###28###636205.4

###Total###393562187.5###31

###Day 30###Between Groups###209323750.0###3###69774583.3###93.322###less than 0.001

###Within Groups###20935000.0###28###747678.6

###Total###230258750.0###31

Neutrophils###Day 16###Between Groups###253.8###3###84.6###17.86###less than 0.001

###Within Groups###132.6###28###4.7

###Total###386.5###31

###Day 30###Between Groups###139.8###3###46.6###9.52###less than 0.001

###Within Groups###137.1###28###4.9

###Total###277.0###31

Remained unchanged over time. The lymphocyte percentage was at day 0 the highest 69.52.0 in group D and the lowest 66.43.2 in group B. In group A the lymphocyte percentage remained unchanged over time. In group B LC at day 15 and day 30 was 61.52.7% and 62.42.6% which was significantly lower than day 0 (pless than 0.001 each). In groups C and D the lymphocyte percentage at day 15 was 61.13.9 and 63.03.5 which was significantly lower than day 0 (pless than 0.001). At day 30 the lymphocyte percentage in groups C and D was 65.03.0 and 68.12.6 which was significantly higher than day 15 (pless than 0.001 each).

Pair-wise comparison made between the groups at day 15 showed mean LC was significantly lower in groups B, C and D compared to A (pless than 0.001; pless than 0.001; p=0.002 respectively). There was no significant difference among groups B, C and D at day 15 (pgreater than 0.05 each). At day 30, groups B and C had significantly lower LC compared to group A (pless than 0.001 and p=0.022 respectively). Group D had non-significant difference with group A (pgreater than 0.05).

In group A, the neutrophil-to-lymphocyte ratio remained unchanged over time. At day 0 it was the highest 0.490.05 in group C and the lowest 0.440.04 in group D. In group A, the ratio remained unchanged over time. In group B at day 15 and day 30 it was 0.570.07 and 0.550.06 which was significantly higher than day 0 (pless than 0.001 and p=0.003 respectively). The difference

Table-3: Pair-wise comparison of weight (percent of day 0) among groups at various reading times (Tukey's test).

Parameter###Dependent Variable###(I) group###(J) group###Mean Difference (I-J)###Std. Error###P-value

Weight###Day 16###Group A###Group B###30.75()###3.10###less than 0.001

###Group C###36.63()###3.10###less than 0.001

###Group D###35.38()###3.10###less than 0.001

###Group B###Group C###5.88###3.10###0.254

###Group D###4.63###3.10###0.457

###Group C###Group D###-1.25###3.10###0.977

###Group C###Group D###3.75###3.70###0.743

###Day 30###Group A###Group B###55.75()###4.50###less than 0.001

###Group C###35.50()###4.50###less than 0.001

###Group D###40.75()###4.50###less than 0.001

###Group B###Group C###-20.25()###4.50###0.001

###Group D###-15.00()###4.50###0.012

###Group C###Group D###5.25###4.50###0.652

Clinical score of inflammation###Day 24###Group B###Group C###7.50()###0.74###less than 0.001

###Group D###7.00()###0.74###less than 0.001

###Group C###Group D###-0.50###0.74###0.782

###Day 30###Group B###Group C###9.75()###0.74###less than 0.001

###Group D###11.50()###0.74###less than 0.001

###Group C###Group D###1.75###0.74###0.067

Total leukocyte count###Day 16###Group A###Group B###-7387.5()###398.812###less than 0.001

###Group C###-7975.0()###398.812###less than 0.001

###Group D###-8275.0()###398.812###less than 0.001

###Group B###Group C###-587.5###398.812###0.467

###Group D###-887.5###398.812###0.141

###Group C###Group D###-300.0###398.812###0.875

###Day 30###Group A###Group B###-6575.0()###432.342###less than 0.001

###Group C###-1775.0()###432.342###0.002

###Group D###-775.0###432.342###0.298

###Group B###Group C###4800.0()###432.342###less than 0.001

###Group D###5800.0()###432.342###less than 0.001

###Group C###Group D###1000.0###432.342###0.119

Neutrophil###Day 16###Group A###Group B###-6.1###1.1###less than 0.001

###Group C###-7.4###1.1###less than 0.001

###Group D###-5.4###1.1###less than 0.001

###Group B###Group C###-1.3###1.1###0.663

###Group D###0.8###1.1###0.900

###Group C###Group D###2.0###1.1###0.277

###Day 30###Group A###Group B###-5.0###1.1###0.001

###Group C###-4.4###1.1###0.003

###Group D###-1.3###1.1###0.675

###Group B###Group C###0.6###1.1###0.942

###Group D###3.8###1.1###0.011

###Group C###Group D###3.1###1.1###0.041

Between day 15 and day 30 was insignificant (p=0.059).

In groups C and D, the neutrophil-to-lymphocyte ratio at day 15 was 0.600.07 and 0.550.06 which were significantly higher than day 0 (pless than 0.001 each). At day 30, the ratio in group C and D was 0.520.06 and 0.45.10.5 which were significantly lower than day 15 (pless than 0.001 each).

Pair-wise comparison made between the groups at day 15 showed that the neutrophil-to-lymphocyte ratios were significantly higher in groups B, C and D compared to A (pless than 0.001; pless than 0.001; and p=0.001 respectively). There was no significant difference among groups B, C and D at day 15 (pgreater than 0.05). At day 30, groups B and C had significantly higher ratio compared to group A (pless than 0.001 and p=0.006 respectively). Group D had no difference with group A.

Discussion

RA is the most common form of polyarticular inflammatory arthritis characterised by persistent synovial inflammation, bony erosions and progressive articular destruction leading to varying degrees of physical disability.15

Pristane (synthetic mineral oil) is often used to induce arthritis in rats with interesting immunological and genetic features. Pristane-induced arthritis (PIA) is of great importance for the understanding of the pathogenesis and genetics of human autoimmune diseases, such as RA.13

In the present study, arthritis was induced by pristane and monitored by body-weight, clinical score of inflammation, TLC andDLC.PIA in rats is characterised by an early acute phase of severe inflammation.16 In the present study, induction of arthritis was associated with decrease in body-weight and rise in clinical score of inflammation and TLC. DLC demonstrated increase in neutrophil percentage and decrease in lymphocyte percentage, indicating acute phase of disease. Both the standard drug MTX and test compound TQ significantly suppressed inflammation as seen from the significant increase in body-weight, decrease in clinical score of inflammation and TLC, and normalisation of DLC.

When comparison was made between TQ and MTX, it was observed that increase in body-weight and decrease in clinical score of inflammation were statistically same in both groups, but they did not return to normal at the end of the study. TLC and DLC improved with both drugs, but the effect of MTX was more marked and cell counts returned to normal values in this group.

Many studies have been carried out on TQ in order to explore its anti-inflammatory effects. One study investigated the preventive effects of TQ on collagen- induced arthritis in rats. A significant decrease in the incidence and severity of arthritis by clinical and radiographic assessments was found in recipients of the therapy compared with the controls. In our study also, clinical score of inflammation improved.11

Another study evaluated the effects of TQ on polyarthritis index and pain response score in Freund's adjuvant arthritis in rats. Biochemical parameters like Leukotriene B4 (LT-B4), interleukin 6(IL-6) were also considered. TQ- treated rats showed significant reduction in polyarthritis index, pain response, IL-6 and LT-B4 compared to the non- treated rats.17

In another study, the anti-inflammatory effect of TQ on adjuvant-induced arthritis in rat model was studied. Signs of inflammation on the claw and radiological signs were searched for and tumour necrosis factor (TNF)-alpha and IL-1beta were measured. The results of the control and other groups were compared and TQ significantly suppressed arthritis both clinically and radiologically.18

Improvement in clinical score of inflammation is in accordance with our study.

Studies suggest multiple possible anti-inflammatory mechanisms of TQ. It is found to inhibit the production of nitric oxide, a pro-inflammatory mediator, and thus anti- inflammatory action might be mediated partly through this mechanism.19 TQ also inhibits the synthesis of prostaglandins (PGEs) and LTs by blocking the cycloxygenase (COX) and lipoxygenase (LO) pathways of arachidonate metabolism, and is thus responsible for anti- inflammatory activity.20 TQ highly reduces the levels of IL- 1AY, IL-6, TNF-a and interferon gamma (IFN-) which are considered to be important pro-inflammatory mediators.21

TQ administration, at a dose of 2mg/kg/day for 15 days improved clinical features of PIA in rats. This effect was comparable to that of MTX.

Conclusion

TQ had anti-inflammatory effects in pristane-induced arthritis and can be investigated as potential anti-arthritic agent.

Acknowledgements

We are grateful to Prof. Dr Z. K. Shinwari, Chairman, Biotechnology Department, QAU, for guidance and assistance.

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