## 4 Discussion
These conditions promote the progression of OA [53]. Meanwhile, proteins related to matrix synthesis includingaggrecan, SOX9, and collagens I and II were markedly inhibited by IL-1\(\beta\). The results of the current study show that AyuFlex(r) can inhibit the reduction of aggrecan, SOX9, and the collagen I and II levels in IL-1\(\beta\)-stimulated chondrocytes. These results indicate that AyuFlex(r) suppresses MMPs due to downregulation of oxidative stress and inflammation development by IL-1\(\beta\). MAPK and NF-\(\kappa\)B are two major mechanisms in the onset of OA. Activated MAPK elevated MMPs, and this mechanism is associated with cartilage destruction. The transcription factor NF-\(\kappa\)B triggers a variety inflammatory reaction in OA. Previous studies have also shown that NF-\(\kappa\)B is an crucial regulatory factor in the production of iNOS expression in chondrocytes [13; 14; 15; 16]. As this signaling pathway is closely related to OA, it is recognized as a promising target in OA. Many recent studies have revealed that substances such as resveratrol and curcumin are activated as intracellular signaling molecules during anti-inflammatory action through these various signaling pathways [54; 55; 56; 57]. Therefore, the mechanisms of NF-\(\kappa\)B and MAPK are very important in inhibiting OA progress and we evaluated the efficacy of AyuFlex(r) in NF-\(\kappa\)B and MAPK mechanisms based on these studies. In our study, IL-1\(\beta\) conspicuously demoted the phosphorylation of ERK and NF-\(\kappa\)B p65. In contrast, this process could be reversed by AyuFlex(r) without affecting the total protein expression. Taken together, AyuFlex(r) showed antioxidant, anti-inflammatory, and anti-degenerative effects in OA in an in vitro model. However, in vitro studies are not sufficient to prove the therapeutic effect of AyuFlex(r) on OA. Therefore, we confirmed the effects of AyuFlex(r) in protection against cartilage destruction in a rat model with MIA-incurred OA. MIA is known to inhibit glycolytic signaling in cells by obstructing with glyceraldehyde-3-phosphatase activity and causes inflammation along with cartilage degeneration. Inflammatory stimulation triggers the release of cytokine and its complex biochemical interaction with other mediators, leading to OA and promoting illness symptoms, for example, pain, swelling, and stiffness [58; 59; 60; 61]. MIA-incurred OA is an established model to affirm the anti-osteoarthritis effectiveness of candidate therapeutic agents on OA pathology. Therefore, a rat model with MIA-induced OA has been used in many studies associated with OA. Weight bearing distribution is utilized for pain measurement and as an index of joint discomfort. MIA injections reduce it in the affected limb, indicating joint pain [62; 63]. In this study, it was found that treatment with AyuFlex(r) and ibuprofen considerably upregulated the weight bearing distribution, indicating symptoms of pain relief. In addition, after inducing MIA, general OA symptoms such as swelling and limping in the knee joint were confirmed. In our present study, we discovered that AyuFlex(r) and ibuprofen treatment protected against OA symptoms in rats with MIA-incurred OA. These results indicate that the administration of AyuFlex(r) and ibuprofen showed a marked improvement in OA symptoms and pain-related behavior. Exacerbation of OA symptoms is accompanied by pain and joint destruction, which is related to the expression of inflammatory cytokines [64]. Therefore, we confirmed the expression of iNOS, 5-LOX, LTB4, and IL-6 proteins in cartilage tissues in rats with MIA-incurred OA to affirm the antioxidant and anti-inflammatory effects of AyuFlex(r). In our study, we confirmed that AyuFlex(r) treatment reduced the MIA-triggered increase in cytokine levels, and these results indicate that AyuFlex(r) may reduce the inflammatory response, and then the mediators can decrease cartilage damage. Therefore, based on the in vitro and in vivo studies, the use of AyuFlex(r) can provide anti-inflammatory effects in OA treatment.