**4. Discussion**

In the present study, we evaluated whether or not FP-MD exerts an anti-osteoarthritic e ffect in the MIA-induced OA rat model, which is one of the well-established animal models for human OA and joint pain. In rats, injection of MIA into joints successfully induces acute inflammation, articular cartilage degradation, and joint pain by the direct interruption of chondrocyte metabolism and the subsequent induction of chondrocyte death, representative of the changes observed in patients with OA [41]. Our data show that oral administration of FP-MD led to a significant reduction in MIA-induced joint pain and a decrease in structural changes, including joint space narrowing and cartilage destruction, which were associated with the reduction of pro-inflammatory cytokines and articular cartilage degeneration biomarkers.

Several clinical studies have demonstrated that daily consumption of krill oil (2 g/day for 30 days) improves the subjective symptoms of knee pain in adults with mild knee pain [42], mitigates the subjective symptoms of OA as assessed by Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and reduces C-reactive protein (CRP) levels in patients with rheumatoid arthritis or OA with CRP levels greater than 1.0 mg/dL [43]. In addition, the anti-arthritis, anti-pain, and anti-inflammatory effects of krill oil were also observed in a carrageenan-induced mouse model of inflammatory pain [44,45]. Furthermore, current studies have demonstrated that astaxanthin is a promising anti-inflammatory and anti-pain agen<sup>t</sup> against carrageenan-induced paw edema and pain behavior, as well as neuropathic pain [46–48]. Hyaluronic acid is also known as a useful treatment option for OA that can modify symptoms and relieve joint pain in OA [49–51]. Interestingly, krill oil, astaxanthin, and hyaluronic acid have also been shown to play a role in gu<sup>t</sup> flora modulation, an emerging area of investigation that may also influence the development and progression of osteoarthritis, and symptom managemen<sup>t</sup> [52–54]. Considering the beneficial therapeutic properties of krill oil, astaxanthin, and hyaluronic acid on arthritis, we have developed a novel multi-ingredient dietary supplement formulation, FP-MD, consisting of krill oil, natural astaxanthin, and proprietary lower molecular weight hyaluronic acid, to address the key factors involved in maintaining joint health. In the present study, we performed in vivo animal studies to further investigate the therapeutic potential of FP-MD for pain relief and chondroprotection in an MIA-induced OA rat model by measuring weight-bearing distribution, histopathological changes, serum levels of pro-inflammatory cytokines, and biomarkers for degradation products of articular cartilage.

Here, we demonstrated that oral administration of FP-MD has pain-relieving effects inMIA-induced OA in rats, consistent with our previous findings in a clinical trial that showed remarkable pain relief in subjects suffering from chronic mild-to-moderate knee joint pain [26]. Moreover, histopathological analysis revealed that the FP-MD-treated group exhibited marked suppression of structural changes, bone resorption, and proteoglycan degradation in the MIA-treated rat knee joint. Taken together, these observations sugges<sup>t</sup> that FP-MD possesses potential pain-relieving activity in OA by reducing articular cartilage damage.

The degradation of articular cartilage, including the degradation of cartilage cells and matrix, is the main pathological characteristic of OA, and matrix degeneration mainly results in the losses of proteoglycans and type II collagen [55]. Several previous studies have demonstrated that the serum levels of some structural molecules and fragments derived from articular cartilage, bone, and the synovium, all of which are affected by OA, are elevated in OA patients and OA animal models, thereby they can be used as diagnostic biomarkers which can potentially predict the increased risk and progression of OA [56,57]. COMP and CTX-II are two biochemical markers that are degradation products of joint tissues, especially the cartilage extracellular matrix, and can potentially predict the destruction of articular cartilage in OA. COMP, a tissue-specific pentametric glycoprotein, is one of the essential components of the extracellular matrix of the cartilage which binds to type II collagen fibers and stabilizes the collagen fiber network of articular cartilage in cooperation with other matrix proteins. However, when the articular cartilage is destroyed during the development of OA and under inflammatory conditions, the levels of COMP is noticeably increased in synovial fluid and serum and is positively correlated with joint damage in knee OA [58]. In addition, levels of CTX-II, a degradation product of type II collagen produced by proteases activated by cartilage injury or degeneration, are also elevated in OA patients as compared with normal individuals and are associated with both the prevalence and progression of OA [59].

Accordingly, the serum levels of these two factors, COMP and CTX-II, have the potential to be prognostic biomarkers for monitoring cartilage degradation in patients with OA. Thus, the serum levels of COMP and CTX-II were analyzed to assess the effects of FP-MD on the degradation of articular cartilage. Consistent with previous studies [60,61], serum levels of both COMP and CTX-II were significantly higher in the OAC group than in the sham control group; however, treatment with FP-MD significantly reduced this increase. These findings sugges<sup>t</sup> that the reduction in COMP and CTX-II serum levels by FP-MD most likely represents suppressed MIA-induced degradation of cartilage, as damaged cartilage is a major contributor to circulating COMP and CTX-II levels.

Increasing evidence has demonstrated that the significantly elevated levels of pro-inflammatory cytokines observed in OA patients play a critical role in the promotion of the catabolic processes in OA, causing cartilage degradation [62,63]. High levels of pro-inflammatory cytokines, such as TNF-<sup>α</sup>, IL-1β, IL-6, and IL-10, have been found in synovial fluid from OA patients and several experimental animal models of cartilage degradation [64]. Among these cytokines, TNF-α and IL-1β are highly overexpressed in the cartilage as well as in the synovial tissue and are considered the major mediators in the OA pathogenesis [65,66]. These cytokines are known to drive the inflammatory cascade, and their increased production induces catabolic events as they downregulate the synthesis of ECM structural components, including proteoglycan, by inhibiting the anabolic activities of chondrocytes and enhancing MMPs, resulting in the loss of cartilage and increased bone resorption during the development and progression of OA [65,67]. IL-6 has also been reported to play a major role in OA. Although the production of IL-6 by chondrocytes is considerably low in physiological conditions, its production can be stimulated by the number of other cytokines and inflammatory mediators, including TNF-<sup>α</sup>, IL-1β, IFN-γ, and prostaglandin E2, resulting in decreased production of type II collagen [68].

Many previous studies have demonstrated that anti-inflammatory agents capable of inhibiting the production of these cytokines might have the potential to control or treat OA [65–67]. Hence, we investigated the anti-inflammatory effects of FP-MD by measuring the serum levels of the pro-inflammatory cytokines TNF-<sup>α</sup>, IL-1β, and IL-6 in MIA-induced OA rats, and found that oral administration of FP-MD effectively decreased the serum levels of these cytokines. In addition, FP-MD significantly reduced the mRNA expression levels of inflammatory mediators, iNOS and COX-2, and matrix-degrading enzymes, MMP-2 and MMP-9, in the knee joint tissue. Taken together, the present results indicate that FP-MD has the potential to blunt inflammatory responses, and which may subsequently reduce articular cartilage damage.
