**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 arthrodial 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 e ffects when administered for a long time. Therefore, there is a need for e fficacious 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 di fferent diseases. It exhibits significant bioactive e ffects such as antioxidant and anti-inflammatory activities [18–29]. In many studies, *T. chebula* has been revealed to exhibit antioxidant effects by inhibiting ROS and NO production [21–24]. Therefore, we investigated the anti-osteoarthritic effects of a standardized aqueous extract of the *T. chebula* fruit (AyuFlex ®) in IL-1β-treated chondrocytes and in a rat model of MIA-incurred OA.

IL-1β, one of the most crucial inflammatory cytokines, is mainly utilized for OA research. Chondrocytes stimulated by IL-1β provoke 5-LOX and iNOS, producing large amounts of LTB4 and NO [39]. iNOS is a major nitric oxide synthase (NOS) enzyme and is responsible for synthesizing NO [40]. In addition, 5-LOX induces LTB4, which upregulates the induction and secretion of pro-inflammatory cytokines such as IL-6 from the synovial membranes. The accumulated NO and LTB4 can stimulate cells to synthesize and release MMPs that inhibit the production of type I and II collagen, eventually leading to cartilage degradation [41–45]. Therefore, this inflammatory factor a ffects both bone absorption and joint pain. In this study, we showed that AyuFlex ® downregulated iNOS and 5-LOX expression in chondrocytes stimulated by IL-1β. Furthermore, it was confirmed that AyuFlex ® also inhibited LTB4 and IL-6 expression. These findings demonstrate that AyuFlex ® inhibited LTB4 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-1β 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 [46–49]. Therefore, drugs that can suppress MMP expression can be used for the treatment of arthritis. Interestingly, our study showed that AyuFlex ® prevents IL-1β-induced MMP-2, -3, and -13 expression at the protein level in chondrocytes. Collagens and proteoglycans are the essential constituent of the ECM. Most of the collagen in arthrodial cartilage is type I and II collagen, which offers the tensile strength in tissue [50,51]. The main proteoglycan in arthrodial cartilage is aggrecan, which o ffers 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. These conditions promote the progression of OA [53]. Meanwhile, proteins related to matrix synthesis including aggrecan, SOX9, and collagens I and II were markedly inhibited by IL-1β. The results of the current study show that AyuFlex® can inhibit the reduction of aggrecan, SOX9, and the collagen I and II levels in IL-1β-stimulated chondrocytes. These results indicate that AyuFlex® suppresses MMPs due to downregulation of oxidative stress and inflammation development by IL-1β.

MAPK and NF-κB 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-κB triggers a variety inflammatory reaction in OA. Previous studies have also shown that NF-κB is an crucial regulatory factor in the production of iNOS expression in chondrocytes [13–16]. 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 [54–57]. Therefore, the mechanisms of NF-κB and MAPK are very important in inhibiting OA progress and we evaluated the efficacy of AyuFlex® in NF-κB and MAPK mechanisms based on these studies. In our study, IL-1β conspicuously demoted the phosphorylation of ERK and NF-κB p65. In contrast, this process could be reversed by AyuFlex® without affecting the total protein expression.

Taken together, AyuFlex® 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® on OA. Therefore, we confirmed the effects of AyuFlex® 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 [58–61]. MIA-incurred OA is an established model to affirm the anti-osteoarthritic 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® 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® and ibuprofen treatment protected against OA symptoms in rats with MIA-incurred OA. These results indicate that the administration of AyuFlex® 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®. In our study, we confirmed that AyuFlex® treatment reduced the MIA-triggered increase in cytokine levels, and these results indicate that AyuFlex® 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® can provide anti-inflammatory effects in OA treatment.

Additionally, cartilage and proteoglycan damage were confirmed in rats with MIA-incurred OA through H&E and Safranin-O staining of the arthrodial cartilage. AyuFlex® treatment markedly inhibited MIA-induced synovial membrane damage and cartilage destruction. Moreover, it was confirmed that the expression of MMP proteins such as MMP-2, -3, and -13 was increased by MIA in cartilage tissue, whereas AyuFlex® treatment reduced MMP expression. These results indicate that AyuFlex® suppresses MMPs due to downregulation of oxidative stress and inflammation development by MIA. Therefore, based on in vitro and in vivo studies, it is suggested that the use of AyuFlex® could inhibit the decomposition of cartilage by inhibiting MMPs in OA treatment. Arthrodial cartilage

damage occurs because the main component of the extracellular matrix, proteoglycan, is degraded by the MMPs. In the present study, we observed collagen synthesis-related proteins in the arthrodial cartilage. We also confirmed that AyuFlex® reduced cartilage destruction through an increase in SOX9, aggrecan, and collagen I and II levels in arthrodial cartilage. Furthermore, based on in vitro and in vivo studies, AyuFlex® revealed collagen preserving properties shown by the significantly suppressed MMPs and upregulated collagen synthesis-related proteins.

Therefore, in our research, AyuFlex® showed anti-osteoarthritic effects through the suppression of NF-κB and MAPK mechanisms, which contribute to a further understanding of the effectiveness of AyuFlex® on OA treatment. In addition, if there are additional studies such as in vivo studies of MIA-induced rat models for ellagic acid, an indicator component of AyuFlex®, we believe that the efficacy of AyuFlex® in the treatment of OA could be further demonstrated.
