الفهرس 
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Abstract
Rheumatoid arthritis (RA) is a systemic progressive autoimmune disease associated with a chronic inflammatory process, which not only affected joints but also several extra-articular organs, including the kidney, heart, lung, digestive system, skin, eye and nervous system.
The etiology of RA is still unclear despite the several mechanisms that have been suggested. Without suitable treatment, symptoms gradually worsen until the joint is irreversibly damaged causing permanent disability.
Therefore, the main target of RA therapy is to decrease inflammation, minimize joint damage and obtain remission with low incidence of side effects. Pharmacotherapy of RA incudes NSAIDs, glucocorticoids, and DMARDs, classified as conventional or biologic DMARDs
Leflunomide is preferred than other DMARDs, for the treatment of moderate to severe RA, because of its greater efficacy and low cost. Despite the protective effect of leflunomide, have been significant side effects, associated with its treatment such as hepatotoxicity, interstitial lung disease and commonly gastrointestinal dysfunction and chronic diarrhea raising the possibility of a specific leflunomide induced colitis.
Nowadays, studies are being conducted on complementary and natural medicine techniques as adjuvant therapy to raise the possibility of a full RA remission.
Ectoine is a natural compatible solute protectant, produced by halophylic bacteria. The molecular mechanisms of ectoine are not fully known, but it binds strongly to water molecules, forming a protective shield around proteins and other biochemical, so it has the unique ability to stabilize biological membranes, it has been successfully marketed as an ingredient in several over-the-counter medicines, mainly achieving epithelial protection. A significant anti-inflammatory effect was also documented for ectoine through its ability to significantly reduce the production of inflammatory cytokines, alleviating inflammatory responses in several diseases, such as, chronic obstructive pulmonary disease (COPD) inflammatory bowel disease (IBD).
The present work was designed to investigate the anti-inflammatory effect of ectoine, on adjuvant-induced arthritis, in rats, and to compare its efficacy with the commonly used DMARD; leflunomide. The combination of, the osmoprotectant and membrane stabilizer; ectoine and leflunomide was also evaluated a potential effect on the studied parameters of inflammation and on reported leflunomide-induced colonic changes.
In the current investigation, 40 adult male Sprague-Dawley rats weighing 170-200g were used. Rats were divided into five groups, of eight rats each. group I served as normal control. Adjuvant arthritis was induced in the other 4 groups, by injecting rats with 0.1 ml of complete freund’s adjuvant (CFA) containing 10 mg/ml of heat-killed Mycobacterium tuberculosis, subcutaneously at the base of the tail. Adjuvant arthritis was allowed to progress for two weeks’ post–induction, and arthritis severity was assessed by hind paw swelling measurement every other day, the body weights of all rats were measured at the beginning of the study and every other day till the day of sacrifice. Rats were treated for 14 days as follows:
group I: Eight normal control rats receiving saline (the vehicle) orally daily.
group II: Untreated arthritic rats (positive control) receiving saline orally daily.
group III: Adjuvant arthritis rats receiving leflunomide 10 mg/kg, orally, daily.
group IV: Adjuvant arthritis rats receiving ectoine 100mg/kg, orally, daily.
group V: Adjuvant arthritis rats receiving the combination of ectoine 100mg/kg, and leflunomide 10mg/kg, orally daily.
At the end of the study, rats were scarified by cervical dislocation. Blood samples were collected from the posterior vena cava through a laparotomy incision; sera were separated, stored at -80 and used for the determination of the following parameters:
A. Serum parameters:
• Serum anti-cyclic citrullinated peptide antibodies (Anti-CCP) by ELISA
• Serum interleukin 1B (IL-1B) by ELISA
• Serum interleukin 10 (IL-10) by ELISA
• Serum nitrate/nitrite (NOx)
B. Tissue parameters
1. The spleen and liver of all rats were cut off, dried on filter paper and weighed. The spleen and liver mass indices (ratio of spleen and liver weights in mg to animal weight in g) were taken as further markers of inflammation.
2. The subcutaneous tissue of the hind paws and surrounding the tibiotarsal joints were removed and homogenized in phosphate-buffered saline and stored at -80 °C for determination of the following parameters by enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturers’ instructions:
• Tumor necrosis factor-α (TNF-α)
• Nuclear factor kappa B (NF-κβ)
• Matrix metalloproteinases 9 (MMP-9) by PCR
3. The distal segment of the colon was removed, opened longitudinally and thoroughly washed, then homogenized in ice-cold distilled water for determination of the following parameters:
• Intracellular adhesion molecule (ICAM-1) by PCR
• TNF-αby ELISA
• Reduced glutathione (GSH)
• Malondialdehyde (MDA)
• Myloperoxidase activity
C. Histopathological Examination:
On day 29, hind paws and colon segments of rats were removed and processed for histopathological examination to determine the extent of joint and colon inflammation.
In the current study, the anti-inflammatory effect of the compatible solute ectoine was investigated for the first time in the adjuvant model of arthritis, in rats. Ectoine has proved to be effective in hindering arthritis progression, as evident by significant lowering of hind paws swelling, and in decreasing the accompanying extra-articular inflammatory markers; spleen and liver mass indices in a qualitative and quantitative pattern similar to the standard ant rheumatic drug; leflunomide.
In addition, the effects of ectoine treatment were evaluated in different tissues including the serum, joint and colon of adjuvant arthritis rats, where it exhibited a significant beneficial anti-arthritic, anti-inflammatory and anti-oxidant effects as compared to the positive control group. However, the anti-inflammatory effect of leflunomide remained superior to ectoine on most of the serum; Anti-CCP, IL-1B, IL-10, NOx and joint MMP9-NF-Kβ and TNF-α parameters.
In contrast, the effect of leflunomide was not as optimum in the colon, as it increased the inflammatory parameters ICAM-1, TNF-α and MPO and showing oxidant effect evident by the significant elevation of MDA and the reduction of GSH levels as compared to the untreated arthritic rats.
Such deleterious effects were significantly ameliorated upon its combination with ectoine with improvement of the inflammatory reactions caused by leflunomide upon histopathological examination of the colon. Moreover, the combination of both drugs significantly improved the serum and joint inflammatory markers better than either drug alone.
Taken together, the present results clearly indicate that leflunomide and ectoine combination could be a promising therapeutic option with a potential outcome in rheumatoid arthritis patients. However, more clinical studies are required to support these findings.