## 3 Results
### AyuFlex(r) Diminished the Production of MMP-2, -3, and -13 in IL-1\(\beta\)-Treated HCHs  
As MMPs play major roles in cartilage destruction, we appraised the effects of AyuFlex(r) on MMP-2, -3, and -13 expression in IL-1\(\beta\)-treated HCHs by western blot analysis. Our results revealed  
Figure 1: Effects of AyuFlex® on cell viability in primary human chondrocytes (HCHs). HCHs were treated with each concentration of AyuFlex® (5, 10, and 20 \(\upmu\)g/mL) for 24 h. Cell viability was evaluated utilizing the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay.  
Figure 2: AyuFlex® repressed the expression of iNOS, 5-LOX, LTB\({}_{4}\), and IL-6 in IL-1\(\beta\)-treated HCHs. HCHs were treated with IL-1\(\beta\) (10 ng/mL) single or in combination with AyuFlex® (5, 10, and 20 \(\upmu\)g/mL) for 24 h. Effects of AyuFlex® on the expression of iNOS (135 kDa), 5-LOX (78 kDa), LTB\({}_{4}\) (36 kDa), and IL-6 (25 kDa) in IL-1\(\beta\)-treated HCHs were analyzed. Protein bands were quantified utilizing ImageJ. As a control for normalization, \(\beta\)-actin was utilized. \(\#p<0.01\), in comparison with the vehicle control group. \(\text{{}^{**}}\)\(p<0.01\), in comparison with the IL-1\(\beta\)-treated control group.  
that IL-1\(\beta\) considerably upregulated the expression of MMP-2, -3, and -13 contrasted with that in the untreated- and vehicle control treated-HCHs. Treatment of HCHs with a combination of AyuFlex(r) and IL-1\(\beta\) considerably reduced the protein expression of MMP-2, -3, and -13 in the range of 20-43% (\(p\) < 0.01), 39-72% (\(p\) < 0.01), and 38-77% (\(p\) < 0.01), respectively (Figure 3).  
Figure 4: AyuFlex® treatment attenuated the degradation of collagen synthesis-involved proteins in IL-1\(\beta\)-treated HCHs. HCHs were exposed with IL-1\(\beta\) (10 ng/mL) single or in combination with AyuFlex® (5, 10, and 20 \(\upmu\)g/mL) for 24 h. 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. #\(p\) < 0.01, in comparison with the vehicle control group. **\(p\) < 0.01, in comparison with the IL-1\(\beta\)-treated control group.  
Figure 3: AyuFlex® diminished the production of MMP-2, -3, and -13 in IL-1\(\beta\)-treated HCHs. HCHs were treated with IL-1\(\beta\) (10 ng/mL) single or in combination with AyuFlex® (5, 10, and 20 \(\upmu\)g/mL) for 24 h. The expression of MMP-2 (62, 72 kDa), MMP-3 (54 kDa), and MMP-13 (54 kDa) was detected by western blot analysis. Protein bands were quantified utilizing ImageJ. As the control for normalization, \(\beta\)-actin was utilized. #\(p\) < 0.01, in comparison with the vehicle control group. **\(p\) < 0.01, in comparison with the IL-1\(\beta\)-treated control group.  
AyuFlex(r) Treatment Attenuated the Degradation of Collagen Synthesis-Involved Proteins in IL-1\(\beta\)-Treated HCHs  
The effects of AyuFlex(r) on the expression of collagen synthesis-involved proteins in IL-1\(\beta\)-treated HCHs was investigated using western blotting. The protein levels of SOX9, aggrecan, COL1A1, and COL2A1 were assessed. As shown in Figure 4, IL-1\(\beta\) significantly reduced SOX9, aggrecan, COL1A1, and COL2A1 expression in contrast with that in the vehicle control. As shown in Figure 4, AyuFlex(r) treatment considerably upregulated the expression of SOX9, aggrecan, COL1A1, and COL2A1 compared to that in IL-1\(\beta\)-stimulated HCHs. Treatment with AyuFlex(r) (5, 10, and 20 ug/mL) increased SOX9, aggrecan, COL1A1, and COL2A1 expression compared with that in the IL-1\(\beta\)-stimulated HCHs (1.9-2.1-fold, \(p<0.01\); 1.9-2.3-fold, \(p<0.01\); 1.6-1.8-fold, \(p<0.01\); 4.7-11.1-fold, \(p<0.01\), respectively).