الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Rheumatoid Arthritis (RA) is a chronic autoimmune disease, which affects 0.51 % of the population worldwide. Primarily, RA affects females and its incidence increases alongside with age. It is characterized by a systemic inflammatory state, involving several organs, including blood vessels. Rheumatoid Arthritis has been linked to a substantially increased risk of cardiovascular disease (CVD), which accounts for 30-50 % of all deaths in patients with RA, mainly through accelerated atherosclerotic disease. Striking progress has been made in the understanding of the biology and epidemiology of CVD in the inflammatory rheumatic disorders. Although the prevalence of some traditional cardiovascular risk factors is increased, RA itself is presumed an independent risk factor for CVD. The high prevalence of atherosclerosis has been documented in even early stages of RA. It is believed, nowadays, that chronic inflammatory conditions promote atherosclerosis by means of modulation of traditional risk factors, as well as, direct biological impressions on the artery structure and functions. chronic inflammation contributes by increased synthesis of pro-inflammatory mediators (such as cytokines, chemokines, adhesion molecules) and autoantibodies, perturbations in T-cell subsets, hyper-homocysteinemia, oxidative stress, and abnormal vascular repair.
In view of such consideration, the present study was designed to assess the effect of methotrexate conjugated to gold nanoparticles on vascular dysfunction associated with rheumatoid arthritis in adjuvant-induced arthritis rat model. A pilot study was done to confirm the dose of Complete Freund’s Adjuvant (CFA) that can induce vascular dysfunction together with arthritis. The main study was conducted on 50 female Wistar albino rats of body weight ranging from 150–200 g. They were housed (10/cage) under standard laboratory conditions with 12-hr light/dark cycle and free access to standard chow and water throughout the study. Initially, animals were randomly divided into 2 main groups:
Normal control group (10 rats):
Animals received a single intradermal injection of 0.3 ml PBS at the base of the tail at the beginning then they received PBS subcutaneously in the dorsal flank (1ml/100 g body weight) and 1 ml Normal saline intraperitoneally as vehicles for 14 days, in the same schedule as the diseased rats.
Adjuvant-induced arthritis group (40 rats)
On day zero, rats in this group were subjected to induction of arthritis by CFA. The rats were restrained, and 0.3 ml was injected intradermally at the base of the tail. Ankle joint diameters (anteroposterior and mediolateral diameters) were measured every 3 days to check for the onset of the disease. By day14, arthritis became evident in the form of increased combined ankle joint diameters of about 6.4 mm2. At this point, rats were randomly divided into 4 subgroups of 10 rats each according to the received treatment:
- Subgroup a: Non-treated arthritis group
The rats received PBS (1ml/100 g body weight) subcutaneously and 1 ml Normal saline intraperitoneally, as vehicles for 14 days.
- Subgroup b: GNPs-treated rats
The rats received synthesized GNPs 500 µg per kg subcutaneously and 1 ml Normal saline intraperitoneally for 14 days.
- Subgroup c: Methotrexate-treated rats
The rats received Methotrexate 40 µg per kg intraperitoneally and PBS (1ml/100 g body weight) subcutaneously for 14 days.
- Subgroup d: Methotrexate/GNPs-treated rats
The rats received Methotrexate conjugated with GNPs 20 µg per kg subcutaneously and 1 ml Normal saline intraperitoneally for 14 days.
On day 29, rats were anesthetized with Sodium thiopental for blood collection before being sacrificed. The collected blood samples were used for assessment of serum levels of VCAM-1, CRP, total cholesterol (Total-C), high-density lipoproteins cholesterol (HDL-C), low-density lipoproteins cholesterol (LDL-C), and triglycerides (TG).
The thoracic aorta was dissected and divided into two parts. One part was fixed in 10% formalin together with the dissected femoral artery, and served for histological examination by hematoxylin and eosin (H&E), and orcein stains, and for immunohistochemical assessment of smooth muscle alpha actin (-SMA). The other part was carefully dissected, isolated, and rapidly put in a freshly prepared Krebs solution for in-vitro isometric smooth muscle tension studies.
Results of the present study revealed that treatment with methotrexate conjugated to gold nanoparticles (MTX/AuNPs) for 2 weeks significantly improved the induced vascular inflammatory and atherogenic process, and enhanced vascular structure and function. This was evident by the significant reduction in VCAM-1 and CRP levels versus the non-treated arthritis control rats and both AuNPs- and MTX-treated rats, and the significant improvement of the whole lipid profile versus the non-treated arthritis control rats and significant reduction in LDL-C level, and TC/HDL and LDL/HDL ratios compared to MTX-treated rats.
Biologically, MTX/AuNPs-treated rats’ isolated aortae expressed a significant improvement in vascular reactivity as evidenced by the significantly enhanced Ach-induced relaxant response versus all arthritis-treated and non-treated rats. Histological studies confirmed its beneficial effect as shown by the improvement of the structural architecture of both aorta and femoral artery; such as the reduction of inflammatory cells, the reconstruction of the elastic lamina, and the significant regain of -SMA immune-reactivity.
Overall, these results indicate that the conjugation of MTX to gold nanoparticles demonstrated a prominent synergistic action over each treatment alone. Findings of the present study could possibly highlight the promising potential of gold nanoparticles in enhancing methotrexate cellular delivery, maximizing its therapeutic effectiveness, and probably increasing its tolerability.