الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Rheumatoid arthritis is a complex systemic and inflammatory autoimmune disease conferred by various genetic and environmental factors and associated with joint swelling, synovial hyperplasia, pannus formation, as well as, bone and cartilage destruction. It affects mostly individuals within the economically productive age range, ultimately imposing significant limitations on their functional ability that result in the loss of work capabilities.
Methotrexate, a corner stone component of RA therapy, as well as, a chemotherapeutic agent, for many types of cancer, has been found to ameliorate JAK/STAT pathway signaling and STAT phosphorylation in Hodgkin’s lymphoma human cell line. Tetrandrine, a drug used clinically, in China, for decades, in the treatment of hypertension, silicosis and autoimmune disorders, has been recently reported to modulate osteoclastogenesis via SYK inactivation.
The present work was designed to investigate the inhibitory effect of methotrexate on JAK/STAT activation, in adjuvant-induced arthritis, in rats, and to evaluate whether the combination of methotrexate and tetrandrine, as a SYK inhibitor, would improve the efficacy of methotrexate on JAK inhibition as well as on other inflammatory parameters.
In the present study, adult male Sprague-Dawley rats, weighing 140 to 150 g, were used. Rats were divided into five groups, of eight rats each. group 1 served as the negative control group. Adjuvant arthritis was induced, in the other 4 groups, by injection of 0.1 ml heat-killed mycobacterium butyricum suspension, of 12 mg/ml, in incomplete Freund’s adjuvant, intradermally, at the base of the tail. Adjuvant arthritis was allowed to progress for two weeks post-induction, and severity of arthritis was assessed by hind paw swelling measurement every other day. Rats were treated for 14 days as follows:
group 1: Eight normal rats serving as non-arthritic healthy controls, received the vehicle only (negative control group).
group 2: Untreated AA rats: Eight untreated AA rats received the vehicle only (positive control group).
group 3: AA rats treated with methotrexate, intraperitoneally, twice weekly, at a dose of 1 mg/kg/week.
group 4: AA rats treated with tetrandrine, orally, daily at a dose of 20 mg/kg/day.
group 5: AA rats treated with both methotrexate and tetrandrine at the aforementioned doses.
At the end of the study, rats were sacrificed by cervical dislocation and blood samples were collected from the posterior vena cava. Sera were separated, stored at -80 C and used for determination of:
1- Anticitrullinated protein autoantibody (ACPA)
Summary, C onclusion & Recommendations
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2- Cartilage oligomeric matrix protein (COMP).
3- Matrix metalloproteinase-9 (MMP-9).
4- Receptor activator of NF-κB ligand (RANKL).
5- Tumor necrosis factor-α (TNF-α).
6- Interleukin 6 (IL-6).
The subcutaneous tissue of the hind paw and surrounding the tibiotarsal joints were excised and stored at -80°C for determination of the following parameters by ELISA and western blot analysis:
1- p-JAK1, p-JAK2 and p-JAK3.
2- p-STAT3.
3- p-SYK.
Histopathological examination of hind paws was also performed to determine the effect of drug treatment on synovial inflammation and cartilage and bone erosion.
In the untreated AA group, a significant increase in rats’ hind paw swelling occurred progressively till the end of the study, accompanied by synovial hyperplasia, inflammation and erosion, on histopathological examination of the joint tissue.
Elevated serum levels of ACPA (a reliable biomarker for RA), cartilage oligomeric matrix protein (COMP) and matrix metalloproteinase-9 (MMP-9) (cartilage degeneration markers), receptor activator of NF-κB ligand (RANKL) (bone erosion marker), and the pro-inflammatory cytokines; tumor necrosis factor-α (TNF-α) and interleukin-6 IL-6) were also found in the untreated group. Both ELISA and western blot analysis revealed an increase in the activity of JAK/STAT and SYK tyrosine kinases pathways, as evident by the increase in the protein expression levels of p-JAK1, p-JAK2, p-JAK3, p-STAT3 and p-SYK.
In the present study, the inhibitory action of methotrexate on JAK/STAT signaling pathway was clearly demonstrated by the significant lowering of phosphorylated JAKs and STAT3 compared to the untreated group. Also, it exhibited a novel significant inhibitory effect, on SYK expression levels, which strongly support the role of these pathways in the mechanism of the anti-inflammatory action of methotrexate.
Tetrandrine proved to be potent and nearly as effective as methotrexate in amelioration of parameters of arthritis progression, inflammation and bone and cartilage erosion, as well as, in suppression of the increased SYK and JAK/STAT signaling compared to the untreated adjuvant arthritis group.
As both JAKs and SYK kinases are involved to a great extent in the pathophysiology of RA, the effect of targeting them, at the same time, by both methotrexate and tetrandrine, was consequently reflected in the significant improvement of all of the studied parameters, than either drug alone, indicating a broader anti-rheumatic action. However, further experimental and clinical trials are still required to support the obtained promising results.