الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
RA is traditionally considered as a chronic, inflammatory autoimmune disorder that causes the immune system to attack the joints. It affects 1% population all over the world and associat with severe morbidity, functional impairment, permanent disability and increased mortality.
CIA model in rats is similar to RA in many aspects, which is perhaps the most commonly used model for RA today. Intradermal injection of collagen emulsified in IFA leads to a severe, erosive poly-arthritis developing within 2–3 weeks after immunization followed by a subsequent chronic relapsing phase. With these considerations in mind, we address a question of whether DDB can reveal anti-rheumatic activity and whether this drug could be recommended for treatment of RA. Thus, this study aimed to explore the effects of DDB alone and in combination with MTX on some inflammatory and angiogenic mediators in CIA model of RA.
Angiogenesis is involved in the pathogenesis of inflammatory diseases. RA has been associated with the perpetuation of new vessel formation that enables leukocyte transendothelial migration into the synovial tissue. There are numerous angiogenic mediators and inhibitors in the RA synovium. There is imbalance in RA yielding to increased turnover of capillary formation. One can block angiogenesis by either stimulating the synovial production of endogenous angiostatic mediators, or by externally administering synthetic inhibitors of neovascularization. There is evidence that the blockade of angiogenesis may lead to the suppression of synovial inflammation and pannus formation.
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Over recent decades, MTX was established as DMARD and immunomodulator with the best combination of long-term effectiveness and safety profile in the treatment of RA. Because of the limited success of MTX in the prevention of RA development and the potential risk for infection, malignancies and autoantibody production, the exploration of new anti-rheumatic drugs with high efficacy and less toxicity are eagerly needed. Now it has become the mainstream for RA therapy combining drug administration with adjuvant therapies. Traditional Chinese medicine which mainly includes Chinese herbs, acupuncture and massage is a new approach for the treatment of RA.
DDB is a synthetic analogue of Schizandrin C, one of compounds isolated from Fructus schizandrae. DDB has anticancer activity and inhibits malignancy differentiation effects on cancer cells. In an in vitro study, DDB was confirmed to have multidrug resistance chemo-sensitizing effect on a panel of cancer cell lines. VEGF, a growth factor that binds to heparin in the synovial endothelial cell, plays a central role in the regulation of neovascularization. DDB decreased the mRNA levels of VEGF demonstrated that DDB, at non-cytotoxic concentrations, has a significant anti-invasion effect in VEGF inhibition.
CIA was induced in Wistar rats using bovine collagen-II dissolved in 0.05M acetic acid. It was emulsified in an equal volume of IFA. Rats were immunized intradermally at the base of the tail with 200μl of emulsion, thus day 0 being the day of the first immunization. On day 10, rats were given a booster injection intradermally on the other side of the tail with 100μl emulsion of collagen-II and IFA.
The rats were randomly divided into 5 groups (10 animals each). Group (1) was normal age-matched controls (neither immunized nor treated rats). Group (2)
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was CIA rats. In all treated groups (3-5), treatment received from the day after onset of arthritis (day 12). Group (3) was DDB-treated (CIA rats were orally administered with 200mg DDB in 1 ml Tween-20/kg body weight daily for 10 successive days as previously described. Group (4) was MTX- treated (CIA rats were treated with MTX dissolved in phosphate buffered saline at a dose of 1 mg/kg/week, administered intraperitoneally in a single installment as described by. Group (5) received a duel therapy (combination of MTX and DDB) where CIA rats administered orally with DDB (200 mg/kg) and intraperitoneally with MTX (1 mg/kg).
Ankle joints of rats were imaged with digital X-ray machine to show the destruction of joints. Fore and hind paw and knee joints were removed above the ankle joint then processed for H&E staining. Plasma levels of VEGF, PDGF, IL-8, IL-4, TNF-α, and COX-2 were quantified by ELISA. NO levels were detected by Griess reagent).
The results of x-ray and histopathology showed that, in CIA group, severe destructive abnormalities with all the metatarsal bones showing definite bone erosion as well as swelling of soft tissues and marked bone marrow edema, osteoporosis could be seen. Joints from rats treated with MTX exhibited moderate reduction of damage. Joints from rats treated with DDB alone and combination therapy exhibited significant inhibition of damage, which closely resembled the joints from the normal rats.
A remarkable increase in VEGF, PDGF and IL-8 secretion levels in RA group in relation to normal controls. A statistically significant reduction in VEGF level was observed in all treated rats for DDB, MTX and DDB+MTX groups in relation to CIA rats. The maximum inhibition in VEGF production was recorded in MTX followed by DDB+MTX group.
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A slight reduction in PDGF secretion level was recorded in all treated groups, however, it is statistically insignificant. Concerning IL-8 production, although its level in CIA group was significantly ameliorated in DDB and DDB+MTX treated groups, it was significantly increased after MTX treatment as compared to normal controls.
Significant increase in TNF-α, IL-4 and COX-2 were produced by CIA compared with normal rats. TNF-α that highly increased in CIA rats was significantly reduced by treatment with DDB alone or combined with MTX. Pronounced increase in IL-4 secretion level in CIA group of rats was totally diminished by treatment. Significant reduction of IL-4 in all treated groups was demonstrated in relation to CIA group. Elevated level of COX-2 in CIA group was abrogated after treatment with DDB, MTX or combined therapy. A statistically significant increase in NO production in CIA group before and after treatment was observed in relation to normal controls.
Comparable levels of NO were found in all treated group as compared to CIA rats. Regarding angiogenesis, our results showed a remarkable reduction in various angiogenic factors (especially VEGF and IL-8) after treatment with DDB either alone or combined with MTX, compared to MTX treatment. Although MTX treatment showed diminution in VEGF and PDGF; IL-8 was increased. This elevation in IL-8 coincides with MTX treatment was totally abrogated by DDB treatment.
These preliminary results provide a close insight about the role of DDB in controlling the RA complications. In the present experiment, both DDB and MTX efficiently suppressed the CIA model after the onset of arthritis with a clear role of DDB as an anti-angiogenic/anti-inflammatory drug. Furthermore, DDB significantly prevented bone/cartilage destruction and inflammation in CIA
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indicating to its anti-arthritic/anti-inflammatory efficiency. Combination with MTX could enhance the therapeutic effect of DDB. However, much work has to be done to identify the whole vision about the role of DDB in RA therapy.