## 3 Results
### AyuFlex(r) Decreased the Production of MMP-2, -3, and -13 in Arthrodial cartilage  
To identify the effects of AyuFlex(r) on MMPs in the cartilage tissues of OA incurred rats, the protein levels of MMP-2, -3, and -13 were evaluated. As shown in Figure 9, the injection of MIA considerably upregulated MMP-2, -3, and -13 expression than the normal control group. The protein expression of MMP-2, -3, and -13 was significantly reduced with AyuFlex(r) and ibuprofen treatment compared with the MIA treatment (control group). When compared with the MIA-injected group, the 25 mg/kg AyuFlex(r)-treated group reduced the expression levels of MMP-2, -3, and -13, by 0.1%, 14%, and 36%, respectively. Treatment with 50 mg/kg AyuFlex(r) substantially decreased (\(p<0.01\), for all conditions) the expression of MMP-2, -3, and -13, by 32%, 32%, and 48%, respectively, with respect to the expression levels in the MIA-treated group. The 100 mg/kg AyuFlex(r) evidently downregulated (\(p<0.01\), for all conditions) the MMP-2, -3, and -13 expression levels to 24%, 33%, and 52%, respectively, of those observed in the MIA-treated group (Figure 9). Figure 8: Effects of AyuFlex® on joint pathology in MIA-incurred OA in rats. (**A**) Knee joints were stained with H&E and (**B**) Safranin-O, (**C**) and graded on a scale 0-13 scale using the Mankin scoring system. (a) Normal Control group, (b) MIA Control group, (c) MIA + AyuFlex® 25 mg/kg, (d) MIA + AyuFlex® 50 mg/kg, (e) MIA + AyuFlex® 100 mg/kg, and (f) MIA + Ibuprofen 20 mg/kg. The results are expressed as the mean \(\pm\) SEM (\(n=5\)/group). A, AyuFlex®; I, Ibuprofen. Scale bar = 300 \(\mu\)m. \({}^{*}\)\(p<0.05\) and \({}^{**}\)\(p<0.01\) in comparison with the MIA-stimulated control group; \(\#\)\(p<0.01\), in comparison with the non-MIA induced control group. AyuFlex(r) Treatment Attenuated the Degradation of Collagen Synthesis-Involved Proteins in Arthroidal Cartilage  
To investigate the impacts of AyuFlex(r) on the expression of collagen synthesis-involved proteins in cartilage tissues of OA induced rats, the protein levels of SOX9, aggrecan, COL1A1, and COL2A1 were evaluated. As shown in Figure 10, injection of MIA considerably diminished SOX9, aggrecan, COL1A1, and COL2A1 expression compared with that in the normal control group. When it was compared with the MIA-injected group, the 25 mg/kg AyuFlex(r)-treated group upregulated the expression levels of SOX9, aggrecan, COL1A1, and COL2A1 by 1.3-fold, 1.3-fold, 3.0-fold, and 1.1-fold,  
Figure 10: AyuFlex® treatment attenuated the degradation of collagen synthesis-involved proteins in arthroidal cartilage. The expression of SOX9 (65 kDa), aggrecan (110 kDa), COL1A1 (220 kDa), and COL2A1 (190 kDa) was determined by western blotting. Protein bands were quantified utilizing ImageJ. As the control for normalization, \(\beta\)-actin was utilized. The results are expressed as the mean \(\pm\) SEM of independent experiments (\(n=3\)/group). A, AyuFlex®; I, Ibuprofen. * \(p<0.05\) and ** \(p<0.01\), in comparison with the MIA-induced control group; # \(p<0.01\), in comparison with the non-MIA induced control group. Figure 9: AyuFlex® decreased the production of MMP-2, -3, and -13 in arthroidal cartilage. The expression of MMP-2 (62, 72 kDa), MMP-3 (54 kDa), and MMP-13 (54 kDa) was determined by western blotting. Protein bands were quantified using ImageJ. As the control for normalization, \(\beta\)-actin was utilized. The results are expressed as the mean \(\pm\) SEM of independent experiments (\(n=3\)/group). A, AyuFlex®; I, Ibuprofen. * \(p<0.01\), in comparison with the MIA-induced control group; # \(p<0.01\), in comparison with the non-MIA induced control group. respectively. Treatment with 50 mg/kg AyuFlex(r) considerably upregulated (\(p<0.01\), for all conditions) the expression of SOX9, aggrecan, COL1A1, and COL2A1 by 1.8-fold, 8.0-fold, 3.6-fold, and 1.9-fold, respectively, with respect to the expression levels in the MIA-treated group.