## 4 Discussion
MMPs are pivotal proteolytic enzymes in the degradation of extracellular matrix components, including proteoglycans, within chondrocytes and bone tissues [40]. Since MMPs act as enzymes that degrade the extracellular matrix, particularly proteoglycans (PGs) [41], they represent a significant target for OA treatment [9; 31; 42]. In this study, control rats with OA demonstrated significant increases in knee thickness, reductions in the thickness of femoral and tibial articular cartilage, and a marked rise in COX-2- and TNF-\(\alpha\)-immunolabeled cells upon histopathological examination. Furthermore, these OA rats exhibited significant increases in COX-2- and TNF-\(\alpha\)-immunopositive cells within the synovial membrane, alongside notable elevations in PGE\({}_{2}\), 5-LPO, MMP-2, and MMP-9 levels in both femoral and tibial articular cartilage and the synovial membrane. Our findings reveal that the administration of DS and NEM effectively inhibited the activity of proinflammatory enzymes (PGE\({}_{2}\), 5-LPO, TNF-\(\alpha\), and COX-2) and enzymes responsible for cartilage extracellular matrix degradation (MMP-2 and MMP-9), which were upregulated due to OA in the femoral and tibial cartilage and synovial membrane. Overall, NEM can prevent OA-induced cartilage damage by reducing MMPs that degrade proteoglycans, which are essential components of articular cartilage, and by reducing inflammation. Notably, the results indicate that NEM100 demonstrates more potent anti-inflammatory effects compared to DS, offering substantial evidence of its therapeutic potential in mitigating OA-induced inflammatory and degradative processes. In OA, fibrosis arising from chronic inflammatory processes imposes limitations on joint motion, with joint stiffness being a prominent symptom. In assessing joint stiffness, we evaluated the maximum extension angle of the joint, considering 0\({}^{\circ}\) as the maximum extension. A lower value indicates better knee function [29; 30; 31; 43]. In our study, the maximum extension angles significantly increased in OA-induced knees, strongly indicating the successful induction of OA through appropriate surgical procedures. Moreover, animals with OA demonstrated substantial hypertrophy and hyperplasia in the SM; both processes are associated with SM fibrosis and joint stiffness [31; 44; 45]. These findings were also evident in our OA rats. Overall, such findings provide strong evidence that, similar to the effects of DS, NEM has favorable antifibrotic and spasticity-relieving activities in OA. As a part of the OA process, physical damage results in significant loss in the joint region, cartilage erosion, and the formation of osteophytes, which are easily detected through gross examination and X-ray imaging [46]. Hence, the reduction or inhibition of cartilage damage observed through gross and radiographic assessments provides a straightforward and valuable metric for evaluating the effectiveness of anti-OA medications [47]. BMD provides good predictable information regarding anti-OA drug efficacy in bone or bone-related diseases [48]. In addition, the focal BMDs of joint surfaces are useful in diagnosing OA progression [49; 50]. Bone strength also provides good predictable information on the efficacy of drugs targeting bone or bone-related diseases [51; 52]. Similarly, we considered that the compressive strength of the surface cartilage provides information on ECM composition and chondrocyte status on the joints. In our study, subcutaneous administration of DS or oral administration of NEM significantly mitigated surgically induced OA-related edematous changes in the knee joint, femur, and tibia, as well as erosive signs in the AC. Additionally, we noted a considerable suppression of related decreases in BMD and joint surface compression strength with these treatments. The ECM of AC is primarily composed of collagen and glycosaminoglycans, including proteoglycans (PGs), aggrecan, and glycosaminoglycan (GAG) [8; 53].