## 4 Discussion
OA is characterized by the loss of AC components, notably PGs, leading to tissue degradation, hypocellularity, and eventual loss of joint function [12]. The inflammation-induced degradation of ECM components such as collagen, aggrecan, and the transcription factor SOX9--essential for chondrogenic differentiation and cartilage integrity--contributes to homeostasis disruption and functional limitations in OA [54]. SOX9, a transcription factor that plays a crucial role in chondrogenic differentiation, along with aggrecan and collagen, which are essential components of a healthy cartilage ECM [53], are crucial for maintaining cartilage integrity. Preventing ECM degradation represents a critical target for anti-OA treatments aimed at preserving the structural and functional integrity of AC [55; 56]. In this context, our study demonstrates that NEM treatment notably improved the expression of Col-2, SOX9, and aggrecan, which were diminished in the OA model. Interestingly, while Col-2 expression was upregulated in the synovial membrane of control OA models, it was downregulated in the DS, NEM100, and NEM50 groups, potentially contributing to the mitigation of joint fibrosis. These results provide clear and direct evidence that NEM has beneficial effects on ECM formation, facilitated through its anti-inflammatory and chondrocyte-protective activities in OA. Furthermore, the observed increase in mRNA expression of Col-2, SOX9, and aggrecan in the femoral and tibial AC in the DS group suggests secondary changes attributable to potent anti-inflammatory and cell-protective activities, rather than direct proliferative effects on cells. The absence of significant increases in BrdU-immunlabeled cells in both femoral and tibial AC aligns with our in vivo observations, providing compelling evidence of NEM's beneficial effects on OA through mechanisms beyond mere cell proliferation. The Mankin scoring system, a widely recognized histopathological tool, evaluates AC injuries based on criteria including surface damage, chondrocyte density, clone formation, and safranin O staining intensity. Higher scores signify a more severe progression of OA [57]. Our findings indicate that OA notably increased Mankin scores in femoral and tibial AC, alongside a reduction in cartilage thickness. Remarkably, treatment with DS or NEM, particularly at the NEM100 dosage, effectively reversed these pathological changes. This reversal suggests the potential of DS and NEM, especially NEM100, as therapeutic agents in mitigating the histopathological manifestations of OA, underscoring their role in preserving cartilage integrity and offering a promising avenue for OA treatment strategies. PARP is a marker of apoptosis, and excessively increased apoptosis induces damage to various organs, including the bone (OA) [58]. Meanwhile, among the methods used to evaluate cell proliferation in histological sections, BrdU staining is simple and enables specific observation of cell proliferation [59; 60; 61] This study demonstrated that NEM100 improves OA by protecting the cartilage and SM cells through the inhibition of apoptosis and promotion of cartilage proliferation. In future studies, given its proven anti-inflammatory, cytoprotective, and chondrogenic properties, NEM could be evaluated for its efficacy in addressing other musculoskeletal disorders characterized by inflammation and tissue degeneration. Additionally, if NEM is utilized for biocompatible materials, it can provide new research materials for regenerative medicine and orthopedic applications. To exploit the potential of NEM as a versatile therapeutic agent, further studies focusing on elucidating the underlying mechanism of action and assessing long-term safety are essential. To assess the extent of the in vivo absorption of the active components of NEM, we conducted observations using various dosages, and the effectiveness was evident at specific dosages, helping to address doubts regarding absorption.