الفهرس 
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Abstract
SUMMARY
I. Introduction
Osteoarthritis [OA] is a chronic degenerative joint disease and a leading cause of adult disability. It is characterized by damage of the articular cartilage and synovitis. The risk factors are hormonal status, gender, genetics and abnormal biomechanical loading on joints - obesity, and joint injury.
The pathological changes in OA are associated with an excessive production of proinflammatory mole¬cules such as interleukin 1β [IL-1β] and tumor necrosis factor α [TNF-α], which stimulate the expression of cyclooxygenase-2 that increases the synthesis of prostaglandin E2 [PGE2]. The cytokines up-regulate the production of nitric oxide via inducible nitric oxide synthetase [iNOS].
The cytokine cascade in turn, decreases collagen and proteoglycan synthesis and increases degenerative proteases including matrix metalloproteinases [MMPs] that promote cartilage degradation and increase susceptibility to other oxidant injuries leading eventually to cartilage degradation.
The treatment of OA consists mainly of relieving its clinical symptoms and delaying joint degeneration. Non-surgical modalities for OA management include weight loss, exercise, activity modification, analgesics, DMOADs, and intra-articular hyaluronic acid [HA] preparations. Cyclooxygenase inhibitors are the most common analgesia used for the control of OA symptoms. Unfortunately, the benefits of anti-inflammatory treatments in OA are often incomplete, not sustained, and may be associated with significant adverse events. Glucosamine is one of the DMOADs commonly used. Its effects include stimulation of physiologic proteoglycan synthesis and reduction of the activity of catabolic enzymes such as MMP-3 and aggrecanase. Statins appear to have anti-inflammatory effects independent of the cholesterol lowering activity. Statins have been shown to decrease monocyte chemotactic protein-1, NF-kB , MMP-3, IL-6 , IL-1β, and TNF-α. Green tea extract, EGCG, was found to be effective in reducing IL-1β-induced inflammatory cytokines, TNFα and IL-6. EGCG treatment resulted in inhibition of MMP-1 and MMP-3. Acetyl-L-carnitine decreases TNFα and IL-1. L-carnitine was also found to enhance cartilage matrix glycosaminoglycan component production and cell proliferation in chondrocyte culture.
II. Aim of work
The aim of the present work is to assess and compare the possible chondroprotective in osteoarthritis induced rat model effects of the following drugs alone and their combination: Epigallocatechin gallate , Glucosamine sulfate, Atorvastatin, Celecoxib, and L- carnitine
III. Materials and Methods
1. Animals
The current work was carried out on 80 albino male rats weighing 190-210 g at the beginning of the experiment, they were purchased from Moassat Animal House, Faculty of Medicine, Alexandria University. The study protocol was approved by the Ethics Committee, Faculty of Medicine; Alexandria University, Egypt.
2. MIA-Induced OA
Anaesthetized rats in group II, III and IV received intra-articular injection, in left knee, of 1 mg of monosodium iodoacetate dissolved in normal saline, in a total volume of 25 μl. While group I rats received equivalent intra-articular injection of 25 μl normal saline in the left knee.
3. Animal groups
Animals were divided randomly into the following groups.
group I [control group]: Eight rats that received 1ml of 2% gum acacia, orally daily.
group II [OA untreated group]: Eight rats with MIA-induced OA, which received 1ml of 2% gum acacia, orally daily.
group III [treated groups]: Rats with MIA-induced OA randomly assigned into 5 subgroups, 8 rats in each subgroup. Each rat in each group had the corresponding drug daily orally for 4 weeks.
• group III [A]: Epigallocatechin gallate treatetd group: 250 mg/kg EGCG.
• group III [B]: Glucosamine sulfate treated group: 250 mg/kg glucosamine sulfate.
• group III [C]: Atorvastatin treated group; 25 mg/kg atorvastatin.
• group III [D]: Celecoxib treated group; 30 mg/kg celecoxib.
• group III [E]: L- carnitine treated group; 100 mg/kg L- carnitine .
group IV combination treated groups: Rats with MIA-induced OA randomly assigned into 3 subgroups, 8 rats in each subgroup. Each rat in each group had the corresponding drug daily orally for 4 weeks.
• group IV[A] EGCG+ Glucosamine + L- carnitine treated group. Epigallocatechin gallate 250 mg/kg plus Glucosamine sulfate 250 mg/kg plus L-carnitine 100 mg/kg
• group IV[B] EGCG + Glucosamine + L- carnitine + Atorvastatin treated group. epigallocatechin gallate 250 mg/kg plus Glucosamine sulfate 250 mg/kg plus L-carnitine 100 mg/kg plus 50 mg/kg Atorvastatin
• group IV[C] EGCG+ Glucosamine + L- carnitine + celecoxib treated group. epigallocatechin gallate 250 mg/kg plus Glucosamine sulfate 250 mg/kg plus L- carnitine 100 mg/kg plus 30 mg/kg celecoxib
4. Animal Assessment
The weight, Knee diameter and knee bending score [5 flexion and extensions for both knee joint, where, 0 = no response, 0.5 = struggle to maximal manipulation, 1= struggle to moderate manipulation, 2 = vocalization], were recorded on day 0, 3, 7, 14 and 28.
5. Histopathologic analysis
The left knee joints of the rats were excised after 4 weeks, and the soft tissue around it was removed. The knee joint was fixed and stained by H & E and toluidine blue for histopathological assessment. Both Mankin and OARSI scores were calculated.
6. Biochemical analysis
On day 28, rats were anaesthetized. Synovial fluid was collected from left knee for biochemical analysis of IL-1, MMP-13 and COMP.
7. Statistical analysis
All experimental results are given as mean ± standard deviation [S.D] and median. The IL-1β, COMP, MMP-13 levels were tabulated and analyzed statistically by kruskal-wallis . While the weight, knee diameter and knee bending score were tabulated and analyzed statistically using repeated measures ANOVA test. P values of less than 0.05 were considered significant.
IV. Results
A. Animal assessment
1. Body weight
Rats injected with MIA, showed a significant decrease in the body weight gain on day 7, as compared to normal control group [p < 0.05]. However, the weight gain returned to normal on day 28. There was non-significant changes in body weight for all studied groups as compared to normal control group by the end of experimentation period.
2. Left knee diameter
Groups treated with EGCG, glucosamine, and atorvastatin, had significant reduction in left knee diameter on day 7, and celecoxib group on day 14, compared to untreated OA induced group, p <0.05.
3. Knee bend score
The knee bend score was significantly decreased on day 7 onward, p < 0.05, in all treated groups, compared to OA-untreated group.
B. Biochemical Markers
1. IL-1β level
The IL-1β level in the synovial fluid of untreated OA model group was significantly increased as compared to normal control group [149.27 ± 3 pg/ml and 56.18 ± 19.67 pg/ml respectively, p < 0.05]. Groups treated with EGCG, glucosamine sulfate, atorvastatin, celecoxib and L-carnitine had a significant decrease in the IL-1β level [90.29 ± 54.42, 65.09 ± 17.65, 78.2 ± 30.56, 80.69 ± 58.8, and 76.27 ± 16.9 pg/ml respectively] compared to untreated OA induced group, p < 0.05. IL-1β level was significantly decreased in the group treated by a combination of EGCG, glucosamine and L- carnitine as compared to compared to untreated OA-induced group the mean level was 84.53 ±24.07, 72.8± 15.85 and 68.68± 38.51 versus 149.27 ± 3 pg/ml, respectively.
2. COMP level
The COMP level in the synovial fluid of untreated OA model group was significantly increased as compared to normal control group [188.23 ± 21.67 and 53.08 ± 14.29 ng/ml respectively, p < 0.05]. EGCG, glucosamine sulfate, and L-carnitine resulted in a significant decrease in COMP level in these groups as compared to untreated OA-induced group [114.77 ± 39.29, 136.51 ± 9.64, and 108.93 ± 15.17 versus 188.23 ± 21.67 ng/ml respectively, p <0.05]. A significantly decreased in the COMP level was also observed in groups treated by a combination of EGCG, glucosamine and L- carnitine [group IVA] and when celecoxib was added [ group IVC], p< 0.05, compared to untreated OA group.
3. MMP-13 level
The MMP-13 level was significantly increased in non-treated OA-induced group as compared to normal control group [2.36 ± 0.98 and 9 ± 3.6 ng/ml respectively, p < 0.05]. Treatment with EGCG, glucosamine sulfate, celecoxib and L-carnitine resulted in a significant decrease in MMP-13 level in these groups as compared to untreated OA-induced group [3.57 ± 2.48, 3.1 ± 1.05, 3.711 ± 1.69 and 3.37 ± 1.22 ng/ml respectively, p< 0.05.]. Treatment with combination of EGCG, glucosamine and L- carnitine, or when adding either atorvastatin or celecoxib, caused a significant decrease in MMP-13 level, 7.55 ± 3.16, 7.067 ± 3.45 and 5.1 ± 4.7 ng/ml respectively, p< 0.05, compared to untreated OA induced model group.
C. Histological Results
1. Hematoxylin and Eosin and Toluidine blue stain
The untreated OA-induced group revealing significant difference in articular cartilage thickness with irregular surface, abnormal matrix intensity and chondrocytes, compared to normal control group. The toluidine blue stain was moderately reduced in OA-untrated group. Subchondral bone showed irregularity in matrix and shape, with pannus formation and hyperplastic synovial villus which extended over the surface of the articular cartilage. The EGCG-treated group showed some preservation of the cartilage smooth surface. The toluidine blue stain was slightly reduced. In Glucosamine-treated group the cartilage revealed smooth surface, while the toluidine blue stain was slightly reduced. The Atorvastatin group showed localized area of complete loss of cartilage, while the toluidine blue stain was moderately reduced. The celecoxib treated group shows partial recovery of knee joint with preservation of cartilage smooth surface, while the toluidine blue stain was slightly reduced. The L-carnitine-treated group showed increased cartilage thickness, decreased number of chondrocytes, the toluidine blue stain was slightly reduced. The EGCG, Glucosamine and L- carnitine-treated groups showed cartilage with smooth surface, increased thickness and hypocellularity, while the toluidine blue stain was moderately reduced untreated OA, atorvastatin and drug combination group, while it was slightly reduced in the other groups.
2. OARSI grading and Mankin score
Both OARSI grading or Mankin score were significantly increased in untreated OA model group [p< 0.05] compared to other groups [control, treated with EGCG, glucosamine sulfate, atorvastatin, celecoxib or L-carnitine]. In groups treated with combinations, OARSI grading showed non significantly decrease in groups treated by EGCG, glucosamine and L- carnitine group when compared to untreated OA-induced group. However the OARSI grading showed significantly decrease in groups treated by EGCG, glucosamine and L- carnitine group when either atorvastatin or celcoxib was added to the combination, as compared to untreated OA-induced group.. While Mankin score was significantly reduced in all combination treated groups when compared to OA-untreated group