**3. Results**

#### *3.1. FP-MD Reduces the OA-Induced Joint Pain in Rats*

We initially tested the e ffect of oral administration of FP-MD on joint pain in an MIA-induced rat model of OA. Since OA is accompanied by pain, the severity of pain is mainly determined by the asymmetric weight-bearing distribution in the hind limbs. Figure 1 demonstrates the latency di fferences between right (ipsilateral) and left (contralateral) hind-paws in the sham control (Sham) and experimental groups, which were analyzed to evaluate OA-induced pain using a capacitance tester for 3 weeks. The MIA-induced OA control (MIA) group animals showed a quick reduction of weight-bearing distribution in the OA-induced limbs compared to non-OA-induced knees due to pain induced by MIA injection, while the weight distribution did not change in the sham control group. In contrast, FP-MD- and Celecoxib-treated group animals showed decreased ipsilateral latency throughout the experimental period as compared with MIA group. Moreover, rats treated with doses of 50 and 100 mg/kg of FP-MD and the Celecoxib-treated rats were able to balance the right and left hind-paws and practically returned to the normal control condition. These data indicate the significant restoration of hind-limb weight bearing in the FP-MD-treated rats. In addition, we found that FP-MD treatment does not a ffect regular body weight gain in rats (Supplementary Figure S2).

**Figure 1.** Effects of oral administration of FlexPro MD® (FP-MD) on changes in hind-paws weight-bearing distribution in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) rats. The weight-bearing distribution ratio was measured for 21 days after injection of MIA using an incapacitance tester, compared to that of the vehicle-treated MIA-induced group. Data are expressed as the mean ± S.E.M (*n* = 8). # *p* < 0.05 versus vehicle-treated Sham group and \* *p* < 0.05 versus MIA.

#### *3.2. FP-MD Suppressed Articular Cartilage Damage in MIA-Induced OA Rats*

As articular cartilage degeneration is the major histopathological feature of OA joints, the effect of FP-MD administration on the histopathological changes and severity of damage in the articular cartilage were evaluated using H&E and Safranin O-fast green staining in the MIA-induced OA rats. As shown in Figure 2A, the sham control group animals exhibited normal articular cartilage structures with smooth articular surfaces, normal chondrocytes with columnar orientation, and intact tide marks and subchondral bone. In contrast, the MIA group showed the severity of surface irregularity and cleft, matrix loss of articular cartilage, degeneration of columnar orientation, degeneration of the tide mark, and the penetration of subchondral bones. We found that oral administration of FP-MD attenuated the structural morphological changes in the articular cartilage, reduced the penetration of the subchondral bones, and reduced the degeneration of the tide marks in comparison to the MIA group. Proteoglycans as one of the major components of the extra cellular matrix (ECM) have various functions in the cartilage. Safranin O-fast green staining revealed that the joints of the MIA-induced OA rats showed joint space narrowing with marked proteoglycan depletion, whereas sham control rats showed the presence of intense proteoglycan in the ECM. Administration with FP-MD and Celecoxib effectively decreased the loss of proteoglycan in the knee joints compared to MIA group. Furthermore, the severity of OA lesions, graded using the modified Mankin's scoring system, and the overall modified Mankin's scores were significantly lower in FP-MD and Celecoxib-treated groups compared with MIA group (Figure 2B).

**Figure 2.** Histological evaluation of joints activity after administration with FlexPro MD® (FP-MD) in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) rats. (**A**) Knee joints of the OA rats were stained with hematoxylin and eosin (H&E) and Safranin O-Fast green. (**B**) The joint lesions were graded on a scale of 0–13 using the modified Mankin's scoring system, giving a combined score for cartilage structure. Data are expressed as the mean ± S.E.M (*n* = 8). # *p* < 0.05 versus vehicle-treated Sham group and \* *p* < 0.05 versus MIA. CLX; celecoxib 3 mg/kg-treated.

#### *3.3. FP-MD Suppressed the Pro-Inflammatory Cytokine Levels in MIA-Induced OA Rats*

The inflammatory response is an important factor associated with OA pathogenesis, and pro-inflammatory cytokines play a prominent role in the maintenance of tissue injury and chronic inflammation during the progression of OA [38]. We, therefore, examined the effect of FP-MD on the production of inflammatory cytokines associated with OA, such as TNF-<sup>α</sup>, IL-1β, and IL-6, in MIA-induced OA rats. As shown in Figure 3, the MIA group showed increased serum levels of TNF-<sup>α</sup>, IL-1β, IL-6, and IFN-γ compared with those in the sham control group. In the FP-MD- and Celecoxib-treated groups, the serum levels of pro-inflammatory cytokine were significantly decreased in a dose-dependent manner when compared to the MIA group. These results sugges<sup>t</sup> that FP-MD protects cartilage in the MIA-induced OA model by modifying these pro-inflammatory cytokines.

**Figure 3.** Effects of oral administration of FlexPro MD® (FP-MD) on the level of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) rats. The serum concentrations of (**A**) TNF-<sup>α</sup>, (**B**) IL-1β, and (**C**) IL-6 in FP-MD (25–100 mg/kg) + MIA- or Celecoxib (CLX) (3 mg/kg)+ MIA-induced OA rats were compared to those of the vehicle-treated MIA group. Data are expressed as the mean ± S.E.M (*n* = 8). # *p* < 0.05 versus vehicle-treated Sham group and \* *p* < 0.05 versus MIA.

#### *3.4. FP-MD Reduced Biomarkers of Chondrocyte Death in MIA-Induced OA Rats*

The effect of FP-MD on the levels of COMP and CTX-II, which are well-established biomarkers for OA diagnosis and progression, which are degradation products of joint tissues, especially the cartilage ECM, during progressive destruction of articular cartilage in OA [39,40], was also investigated. The serum levels of COMP and CTX-II were increased in OAC group by MIA-injection compared with those in the sham control group. In contrast, the serum levels of COMP (Figure 4A) and CTX-II (Figure 4B) were significantly lower in the OA rats treated with FP-MD at doses of 50 and 100 mg/kg, but not 25 mg/kg, than in the OAC group. The MIA-induced overproduction of COMP and CTX-II was also decreased by Celecoxib treatment.

**Figure 4.** Effects of oral administration of FlexPro MD® (FP-MD) on the production of cartilage oligomeric matrix protein (COMP) and C-telopeptide of type II collagen (CTX-II) in monosodium iodoacetate (MIA)- induced osteoarthritis (OA) rats. The serum concentrations of ( **A**) COMP and (**B**) CTX-II in FP-MD (25–100 mg/kg) + MIA- or Celecoxib (CLX) (3 mg/kg) + MIA-induced OA rats were compared to those of the vehicle-treated MIA group. Data are expressed as the mean ± S.E.M (*n* = 8). # *p* < 0.05 versus vehicle-treated Sham group and \* *p* < 0.05 versus MIA.

#### *3.5. FP-MD Suppressed mRNA Expression of Inflammatory Mediators and Matrix-Degrading Ezzymes in MIA-Induced OA Rats*

We next investigated the e ffect of FP-MD on the mRNA expression levels of inflammatory mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and matrix-degrading enzymes, matrix metalloproteinase (MMP)-2 and MMP-9, in the knee joint tissues of MIA-induced OA rats. The mRNA expression of iNOS, COX-2, MMP-2, and MMP-9 increased in OAC group by MIA-injection compared with those in the sham control group (Figure 5). In the FP-MD- and Celecoxib-treated groups, the expression levels of those genes were significantly suppressed. These results suggested that FP-MD inhibited inflammatory responses and articular cartilage damage by inhibiting inflammatory cytokines and MMPs in MIA-induced OA rats.

**Figure 5.** Effects of oral administration of FlexPro MD ® (FP-MD) on the expression of inflammatory mediators and metalloproteinases (MMPs) in the knee joint in monosodium iodoacetate (MIA)-induced osteoarthritis (OA) rats. The mRNA expression of ( **A**) inducible nitric oxide synthase (iNOS), (**B**) cyclooxygenase-2 (COX-2), ( **C**) MMP-2, and ( **D**) MMP-9 in FP-MD (25–100 mg/kg) + MIA- or Celecoxib (CLX) (3 mg/kg) + MIA-induced OA rats were compared to those of the vehicle-treated MIA group. Data are expressed as the mean ± S.E.M (*n* = 8). # *p* < 0.05 versus vehicle-treated Sham group and \* *p* < 0.05 vs. MIA.
