## 4 Discussion  
OA is one of the most general chronic degenerative joint illnesses in the population of old people, which is featured by the destruction of the arthroidal cartilage. Over time, the subchondral bone thickness and various inflammatory mediators are produced in the synovial membrane [36; 37]. For this reason, steroids and non-steroidal anti-inflammatory medications are mainly utilized to alleviate the progression of arthritis. However, these anti-inflammatory drugs alleviate only joint pain and swelling and have side effects when administered for a long time. Therefore, there is a need for efficacious and safe medication that not only alleviates the symptoms of OA, but also delays the progression of the disease [38]. The fruit of _T. chebula_ has been utilized widely in a variety of traditional oriental medicine for the therapy of different diseases. It exhibits significant bioactive effects such as antioxidant and anti-inflammatory activities [18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29]. In many studies, _T. chebula_ has been revealed to exhibit antioxidant effects by inhibiting ROS and NO production [21; 22; 23; 24]. Therefore, we investigated the anti-osteoarthritic effects of a standardized aqueous extract of the _T. chebula_ fruit (AyuFlex(r) in IL-1\(\beta\)-treated chondrocytes and in a rat model of MIA-incurred OA. IL-1\(\beta\), one of the most crucial inflammatory cytokines, is mainly utilized for OA research. Chondrocytes stimulated by IL-1\(\beta\) provoke 5-LOX and iNOS, producing large amounts of LTB\({}_{4}\) and NO [39]. iNOS is a major nitric oxide synthase (NOS) enzyme and is responsible for synthesizing NO [40]. In addition, 5-LOX induces LTB\({}_{4}\), which upregulates the induction and secretion of pro-inflammatory cytokines such as IL-6 from the synovial membranes. The accumulated NO and LTB\({}_{4}\) can stimulate cells to synthesize and release MMPs that inhibit the production of type I and II collagen, eventually leading to cartilage degradation [41; 42; 43; 44; 45]. Therefore, this inflammatory factor affects both bone absorption and joint pain. In this study, we showed that AyuFlex(r) downregulated iNOS and 5-LOX expression in chondrocytes stimulated by IL-1\(\beta\). Furthermore, it was confirmed that AyuFlex(r) also inhibited LTB\({}_{4}\) and IL-6 expression. These findings demonstrate that AyuFlex(r) inhibited LTB\({}_{4}\) and IL-6 production, and these results may be connected with the regulation of iNOS and 5-LOX expression, thereby reducing the progression of OA. Moreover, recent studies have explained that IL-1\(\beta\) remarkably upregulated the synthesis of cartilage matrix-degrading enzymes, for example, MMPs. MMPs have been considered one of the major enzymes that breakdown aggrecan and collagen in cartilage. Several studies have demonstrated that the appearance of MMPs is upregulated in cartilage tissues of OA patients. MMPs are enzymes that mediate the diverse role in tissue remodeling such as conversion, degradation, and destruction of the ECM [46; 47; 48; 49]. Therefore, drugs that can suppress MMP expression can be used for the treatment of arthritis. Interestingly, our study showed that AyuFlex(r) prevents IL-1\(\beta\)-induced MMP-2, -3, and -13 expression at the protein level in chondrocytes. Collaborens and proteoglycans are the essential constituent of the ECM. Most of the collagen in arthroidal cartilage is type I and II collagen, which offers the tensile strength in tissue [50; 51]. The main proteoglycan in arthroidal cartilage is aggrecan, which offers structural support by keep moisture in the matrix. SOX9 is an indispensable transcription factor for controlling the appearance of many cartilage ECM genes such as COL1A1 and COL2A1 [52]. Aggrecan and collagens I and II are synthesized and secreted by chondrocytes to prevent mechanical destruction under normal circumstances. When cartilage destruction occurs, aggrecan and collagens I and II undergo significant breakdown due to increased production of proteases.