الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Rheumatoid arthritis (RA) is a one from the highest common autoimmune disorders that is manifested by systemic complications, inflammatory chronic manifestations, cardiovascular risk, and increased mortality. The RA epidemiology is nearly 1% from the world’s population. Female is more affected than male by three times and its incidence increases with age development. The RA etiology depends on many factors and these factors are thought to be genetic, environmental and immunological factors.
The current study was designed to evaluate the immune-modulatory and preventive effects of milk thistle seeds hydroethanolic extract (MTHE) and silymarin in CFA-induced arthritic male Wistar rats.
The experimental animals used in this experiment are male Wistar rats, weighting 160-180 gm. Arthritis induction is attained by subcutaneous injection of double dose of 100 µl of CFA into the right hind paw footpad at the planter region of male rats at two successive days.
After arthritis induction and preparation of the hydroethanolic extract, the animals were divided in four groups as follows:
group 1 (Normal control group): It includes the normal rats, given the equivalent volume of 1% CMC as vehicle by oral administration for 9 and 18 days.
group 2 (arthritic control group): The rats in this group were injected subcutaneously with 200 µl CFA at two consecutive days and were taken the equivalent volume of 1% CMC daily for 9 and 18 days.
group 3 (arthritic treated with MTHE): The rats within this group were injected subcutaneously with CFA like group 2 and were treated with MTHE dissolved in 1% CMC by oral gavage daily for 9 & 18 days at dose level of 100 mg/kg b.w.
group 4 (Arthritic treated with silymarin): It consists of rats injected with CFA like group 2 and group 3 and treated daily with silymarin dissolved in 1% CMC at dose of 100 mg/kg b.w. by oral administration for 9 and 18 days.
After the experiment ended, right hind paw circumference (RHPC) was measured. Then, animals were anesthetized by inhalation of diethyl ether. Then, blood was collected. After dissection, tissue samples including liver, ankle joint, spleen and thymus were immediately removed for biochemical as well as for histopathological studies. EDTA solution (10%) was added to one portion of the blood for determination of some hematological parameters (TLC & DLC) and the other blood portion was left in room temperature without anticoagulant to coagulate. The coagulated blood was centrifuged at 3000 r.p.m for a period of 15 minutes and the supernatant was separated and then used for determination of serum RF, TNF-α, IL-1β, IL-17, PGE2 and IL-4 levels. Liver of each animal was removed after dissection and put in 0.9% NaCl. Half gram from liver of each rat was homogenized in 5 ml of NaCl (0.9%). The obtained homogenate was used in determination of antioxidative and oxidative stress parameters including LPO and (SOD, GPX & GST) activities, GSH content. Ankle joint, spleen and thymus were removed immediately after dissection and fixed in 10% NBF for 2 days. Subsequently, decalcification of the hind ankle joint was performed for a period of (2-3) weeks in 10% EDTA. Then, decalcified ankle, as well as fixed spleen and thymus were processed for histopathological examination.
RHPC of CFA-induced arthritic rats exhibited a highly significant increase after 9 and 18 days. The treatment of arthritic rats with MTHE induced a highly significant decrease after 9 days and a non-significant decrease after 18 days. On the other hand, treatment with silymarin exhibited a highly significant decrease after 9 and 18 days.
TLC of arthritic rats showed a highly significant increase after 9 & 18 days of CFA injection. The treatment of arthritic rats by MTHE induced a highly significant decrease after 9 days and a non-significant decrease after 18 days. On the other hand, treatment with silymarin induced a highly significant decrease after 9 and 18 days.
Neutrophil count of CFA-induced arthritic rats exhibited a highly significant increase after 9 & 18 days of CFA injection. The treatment of arthritic rats with MTHE and silymarin showed a highly significant decrease of the neutrophil count after 9 & 18 days.
Lymphocyte count within arthritic rats exhibited a highly significant elevation after 9 & 18 days of CFA injection. The treatment of arthritic rats with MTHE induced a highly significant decrease of the lymphocyte count after 9 days and a significant decrease after 18 days. The treatment of arthritic rats with silymarin induced a highly significant decrease of lymphocyte count after 9 and 18 days.
Monocyte count of arthritic rats showed a highly significant increase after 9 & 18 days of CFA injection. The treatment of arthritic rats with MTHE and silymarin induced a highly significant decrease in the monocyte count after 9 & 18 days.
Eosinophil count of male Wístar rats displayed a highly significant increase after 9 & 18 days of CFA ịnjection. The treatment of arthritic rats with MTHE and silymarin induced insignificant decrease of the eosinophil count after 9 and 18 days.
Serum RF of CFA-induced arthritic rats exhibited a highly significant increase after 9 & 18 days. The treatment of arthritic rats with MTHE and silymarin induced a highly significant decrease after 9 & 18 days.
A highly significant increase in serum TNF-α, IL-1β, IL-17 and PGE2 levels was observed in arthritic rats after 9 & 18 days of CFA injection. The treatment of arthritic rats with MTHE and silymarin induced a highly significant decrease in serum TNF-α, IL-1β, IL-17 and PGE2 after 9 and 18 days.
The IL-4 level in arthritic rats exhibited a highly significant decrease after 9 & 18 days of CFA injection. The treatment of arthritic rats with MTHE and silymarin induced a highly significant elevation in IL-4 level after 9 & 18 days.
Liver SOD activity of arthritic rats exhibited insignificant effect after 9 and 18 days of CFA injection. The treatment of arthritic rats with MTHE and silymarin showed a non-significant change of the SOD level after 9 and 18 days.
Liver GPx activity showed a highly significant decrease after 9 and 18 days following injection by CFA. The treatment of arthritic rats with MTHE and silymarin exhibited a highly significant increase after 9 & 18 days.
Hepatic GST activity of arthritic rats displayed a significant decrease after 9 days and a non-significant decrease after 18 days. The treatment of arthritic rats with MTHE induced a highly significant increase of the GST level after 9 days and showed a non-significant increase after 18 days. The treatment of arthritic rats with silymarin induced a non-significant change of GST level after 9 & 18 days.
Liver GSH content in arthritic rats showed a highly significant decrease after 9 & 18 days of CFA injection. The treatment of arthritic rats with MTHE induced a highly significant increase of the hepatic GSH level after 9 days and significant increase after 18 days. The arthritic rats’ treatment with silymarin induced a non-significant effect of the hepatic GSH level after 9 & 18 days.
Hepatic LPO (expressed by the MDA content) within rats injected with CFA showed a highly significant increase after 9 & 18 days. The treatment of arthritic rats with MTHE and silymarin induced a highly significant decrease after 9 & 18 days.
Histopathological examination of ankle joints of arthritic rats showed many histological alterations including massive inflammatory exudate within the synovial cavity leading to pannus formation, synovial hyperplasia, fibrosis between the articular cartilage and focal necrosis of the cartilage. By comparison, the treatment of arthritic rats with MTHE showed an obvious reduction in inflammation and joint destruction. Some animals showed joint histology almost similar to the normal rats and other animals exhibited pannus formation and few inflammatory cells infiltration within the synovial cavity. On the other hand, treatment of arthritic rats with silymarin induced clear improvement of histopathological alterations. The joint histology within some animals was almost similar to normal ones and the synovial membrane became almost identical to normal synovium, While other animals exhibited focal degeneration of cartilage and chondrocytes.
Microscopical examination of spleen sections of the arthritic animals revealed lymphoblasts activation, mitotic figure appearance, lymphocytic necrosis and apoptosis. By comparison, spleen sections of arthritic rats treated with MTHE exhibited appearance almost identical with normal ones and normal lymphoid follicle for some animals, while other animals showed mitotic figure appearance. Similarly, Spleen sections of arthritic animals treated with silymarin exhibited mitotic figure for some animals and appearance like spleens of normal rats for other animals.
The thymus sections of the arthritic control rats showed mitotic figure appearance and lymphoblasts activation. The thymus of the arthritic rats treated with MTHE exhibited no histopathological alterations with normal appearance. On the other hand, the thymus of rats treated with silymarin showed normal spleen sections appearance for some animals and others displayed slight lymphocytic necrosis.
A higher degree of reduction in the arthritis severity was noticed in the rats treated with MTHE and silymarin.
Conclusion
Both of MTHE and silymarin have powerful anti-inflammatory as well as antioxidant activities in CFA-induced arthritic rats. MTHE is more potent as antioxidant than silymarin. Thus, these treatments can be used as useful agents towards treating of rheumatoid arthritis after further clinical studies on human beings.