الفهرس 
IntroductionOnly 14 pages are availabe for public view
Abstract
Rheumatoid arthritis is considered one of the most common autoimmune diseases. The disabilities caused by rheumatoid arthritis have extensive impacts on the quality of life, with loss of productivity due to damaged and deformed joints inhibiting fine movements of the hand. The overall data on the therapeutic role of manganese in rheumatoid arthritis is inadequate. Additionally, therapeutic properties of nanoparticles are driving the revolution in the development of novel drugs; it is encouraging for further research. from the other side, despite the known efficacy of low dose radiotherapy in rheumatoid arthritis treament, irradiation regimens are still not well established. Also, the interrelationship between ionizing radiation and the immune system is complex and need further research efforts using a new irradiation protocol to be elucidated.
Accordingly, the present study was aimed to evaluate the possible anti-inflammatory and anti-arthritic effects of manganese bionanoparticles (MnBNPs) either alone or in combination with low dose γ-irradiation on an adjuvant induced arthritis model in female rats. In order to achieve this purpose, manganese nanoparticles were biologically prepared using ginger aqueous extract at the national center for radiation research and technology (NCRRT) laboratories.
Characterization of MnBNPs was performed using visual observation, ultraviolet-visible spectral analysis (UV-Vis), transmission electron microscopy (TEM), dynamic light scattering (DLS) and Fourier transform infrared (FTIR) analysis. These
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analytical procedures were carried out to confirm good production of nanoparticles in solution as well as to determine the physicochemical properities of the prepared nanoparticles such as size dimensions, distribution, shape and the specific site of interaction of Mn ions on bio-molecules. Reaction mixture used for biosynthesis of manganese nanoparticles was observed for visual color change. The color changing from pale green to dark green was due to metal ion reduction. The intensity of color increased with time due to capping of stabilizing agent. The bioreduction of aqueous manganese ions was then easily followed by UV-visible spectrophotometer. Ultraviolet-Visible spectrum of MnBNPs showed a surface plasmon absorption band with a maximum absorbance at critical wavelength 283.5 nm. The appearance of absorption edge at 283.5 nm was a clear indication of the formation of manganese nanoparticles.
Analysis of TEM photos showed that MnBNPs were of different sizes in nano scale (50-100 nm) and different shapes mostly spherical shape. Dynamic light scattering analysis confirmed that MnBNPs were distributed in nano scale and their size ranged from 58.77 to 105.70 nm. Besides, it revealed that about 40% of the biologically synthesized manganese nanoparticles were of size 68.06 nm (the main particle diameter). By comparing Fourier transform infrared spectrum of ginger aqueous extract used in the green biosynthesis and the synthesized MnBNPs, shifting in signal wavenumber per centimeter of the major peaks was observed indicating interaction between bio-molecules of ginger and aqueous manganese ions which resulted in reduction of manganese ions and formation of Mn nanoparticles (MnBNPs).
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The determination of MnBNPs the median lethal dose (LD50) in vivo was then performed using the ”up-and-down” procedure. Acute toxicity study was carried out on 32 healthy female Wistar albino rats. Rats were orally administered different doses of MnBNPs ascendingly and observed for toxicity signs and mortality over 24 hr. Results showed that oral administration of MnBNPs caused some toxicity symptoms and only 25% mortality at a dose of 90 mg/kg body weight. The percentage mortality of experimental rats was 50% at dose 110 mg/Kg body weight after 24 hrs of the treatment with MnBNPs. Based on these data, the median lethal dose of biologically synthesized manganese nanoparticles in normal female rats was 110 mg/kg. Consequently, MnBNPs has proved to possess moderate toxicity according to toxicity scale. Therefore, the safe dose that used in the current study was 1/10 LD50 (0.37 mg/kg body weight for 30 consecutive days).
The anti-inflammatory effect of MnBNPs and/or low dose radiotherapy was evaluated by using adjuvant induced arthritis model in female rats. In the present study, a total number of seventy female Wistar albino rats (180±20g) were equally divided into seven main groups of 10 rats each as follows:
Adjuvant free groups:
group I
Normal Control group (NC): Rats of this group served as controls and were neither treated nor irradiated. Healthy rats were daily received 1ml of distilled water via oral tube till the end of experiment.
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group II
Manganese Bionanoparticles group (MnBNPs): Rats of this group were received 1ml (0.37mg/Kg body weight) of MnBNPs via oral administration for 30 consecutive days starting from the 15th day of the experiment.
group III
Irradiated group (IRR): Rats of this group were whole body exposed to a fractionated dose of γ-radiation at a dose level of 0.25Gy, four times at the 15th, 23th, 30th and 37th days of the experiment up to total dose of 1Gy and were received 1ml distilled water via oral gavage for 30 consecutive days starting from the 15th day of experiment.
Adjuvant induced groups:
group IV
Arthritic group (AR): Each rat of this group was inoculated subcutaneously with a single dose of 0.1ml of complete Freund‟s adjuvant (CFA) containing 100 μg dry Mycobacterium tuberculosis into the dorsal root of the tail. The day of adjuvant injection is referred to as day zero. Rats were orally received 1ml distilled water for 30 consecutive days starting from the 15th day of the experiment.
group V
Arthritic irradiated group (AR+IRR): Rats were inoculated by adjuvant inducer (CFA) as described in group IV. Starting from the 15th day, each arthritic rat was orally received 1ml distilled water for 30 consecutive days and exposed to whole body gamma radiation as in group III on the 15th, 23th, 30th and 37th days following CFA inoculation. group VI
Arthritic Manganese Bionanoparticles treated group (AR+MnBNPs): Rats of this group were inoculated by adjuvant inducer (CFA) at day zero as described in group IV and were orally treated with 1ml (0.37mg/Kg body weight) of MnBNPs for 30 consecutive days starting from the 15th day following CFA inoculation.
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group VII
Arthritic, Manganese Bionanoparticles treated, irradiated group (AR+MnBNPs+IRR): Rats were inoculated by adjuvant inducer CFA as described in group IV. At the 15th day following CFA inoculation, rats were first orally received 1ml (0.37mg/Kg body weight) of MnBNPs for 30 consecutive days, 30 min latter rats were exposed to whole body γ-irradiation as group III at the 15th, 23th, 30th and 37th days of the experiment.
Clinical assessment of arthritic parameters includes arthritic index (arthritis symptoms) and change in body weight were observed and recorded for all experimental rats every 5 days over the entire period of experiment for all animal groups.
At the end of the experiment after 24 hours from the last dose of MnBNPs (seven days from the last dose of γ-irradiation) and after an overnight fast, all experimental rats were anesthetized with diethyl ether and immediately whole blood samples were withdrawn from each rat by heart puncture. Each blood sample was divided into two parts; the first part was allowed to clot at 37oC for 15 min in a plain tube. The clotted blood was then centrifuged at 4,000 rpm for 10 min. The separated serum was stored at -20oC until used for the assessment of alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities, creatinine, total antioxidant capacity (TAOC), rheumatoid factor (RF), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels. While the other part of blood sample was taken on a tube contains di-sodium ethylenediaminetetraacetic acid (EDTA) for total leukocytic count (TLC).
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Immediately after the animals were sacrificed, heart tissue of each animal was quickly excised, washed with ice cold isotonic saline and blotted dry with a filter paper. Heart samples were then weighed and homogenized in 10 volumes of ice-cold phosphate buffered saline pH 7.5, containing 1 mM/L Na2EDTA using an electric tissue homogenizer to yield ultimately (10% W/V) whole tissue homogenates. The homogenates were centrifuged at 10,000 rpm for 10 min at 4˚C to remove cell debris and the supernatant was aliquoted and stored at -20oC until used for the assay of antioxidant markers including superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels.
The results obtained were statistically analyzed and can be summarized as follows:
All arthritic rats showed a significant arthritis symptoms when they were injected with complete Freund‟s adjuvant (CFA), including paw swelling, erythema, higher arthritis indices and obvious weight loss as compared to normal control. These symptoms were significantly improved after treatment with either MnBNPs or low dose γ-irradiation. The combination between MnBNPs and γ-radiation produced the most pronounced improvement compared to γ-irradiation or MnBNPs alone.
The results also revealed that subcutaneous injection of CFA into rats induced a marked rise in liver enzymes activities (ALT & AST) in comparison with the normal control group. On the other hand, treatment of these arthritic rats with either γ-irradiation or MnBNPs reduced this elevated activities of ALT and AST and
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resulting in a significant improvement in comparison with arthritic untreated group. The combination between MnBNPs and γ-radiation for the treatment of arthritic rats produced the most pronounced improvement compared to γ-irradiation or MnBNPs alone. Neither oral administration of manganese nanoparticles by dose 0.37 mg/kg body weight for 30 successive days nor whole body γ-irradiation 0.25Gy/Week for four weeks significantly altered liver enzymes activities compared to the normal control group values indicating that they had no deleterious effect on the normal liver and didn‟t cause any liver impairment.
Regarding creatinine level in serum, results showed that subcutaneous injection of CFA into rats induced renal toxicity as evidenced by a significant sharp increase in serum creatinine level in comparison with control values. On the other hand, it has been shown from the present work that treatment of arthritic rats with either low dose γ-irradiation or MnBNPs revealed a significant reduction in the the elevated serum creatinine concentration by 26.32% and 34.21 %, respectively, in comparison with arthritic untreated group. Data also revealed that low dose γ-irradiation of arthritic rats along with MnBNPs administration produced the most pronounced significant decrease in serum creatinine concentration (35.53%) compared to arthritic untreated group. It was found that both MnBNPs and low dose radiotherapy didn‟t cause any kidney impairment as evident by our results for healthy rats either exposed to whole body γ-radiation or orally administered MnBNPs for 30 successive days.