**3. Results**

### *3.1. E*ff*ects of AyuFlex* ® *on Cell Viability in Primary Human Chondrocytes (HCHs)*

The influence of AyuFlex ® on the cell viability of HCHs was confirmed by using the MTT analysis. AyuFlex ® did not show cytotoxicity at any of the concentrations (5, 10, and 20 μg/mL) investigated in this study (Figure 1).

**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 μg/mL) for 24 h. Cell viability was evaluated utilizing the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay.

### *3.2. AyuFlex*® *Repressed the Expression of iNOS, 5-LOX, LTB4, and IL-6 in IL-1*β*-Treated HCHs*

To confirm the anti-inflammatory effects of AyuFlex®, HCHs were simultaneously treated with each concentration of AyuFlex® (5, 10, and 20 μg/mL) and IL-1β (10 ng/mL) for 24 h. The protein presenting of iNOS, 5-LOX, LTB4, and IL-6 was investigated by western blotting. IL-1β was revealed to significantly induce the protein expression of iNOS, 5-LOX, LTB4, and IL-6 against the vehicle control. In contrast, the expression of iNOS, 5-LOX, LTB4, and IL-6 in HCHs treated with a combination of AyuFlex® and IL-1β decreased in the range of 14–46% (*p* < 0.01), 20–26% (*p* < 0.01), 44–58% (*p* < 0.01), and 58–74% (*p* < 0.01), respectively (Figure 2).

**Figure 2.** AyuFlex® repressed the expression of iNOS, 5-LOX, LTB4, and IL-6 in IL-1β-treated HCHs. HCHs were treated with IL-1β (10 ng/mL) single or in combination with AyuFlex® (5, 10, and 20 μg/mL) for 24 h. Effects of AyuFlex® on the expression of iNOS (135 kDa), 5-LOX (78 kDa), LTB4 (36 kDa), and IL-6 (25 kDa) in IL-1β-treated HCHs were analyzed. Protein bands were quantified utilizing ImageJ. As a control for normalization, β-actin was utilized. ## *p* < 0.01, in comparison with the vehicle control group. \*\* *p* < 0.01, in comparison with the IL-1β-treated control group.

### *3.3. AyuFlex*® *Diminished the Production of MMP-2, -3, and -13 in IL-1*β*-Treated HCHs*

As MMPs play major roles in cartilage destruction, we appraised the effects of AyuFlex® on MMP-2, -3, and -13 expression in IL-1β-treated HCHs by western blot analysis. Our results revealed

that IL-1β 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® and IL-1β 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 3.** AyuFlex® diminished the production of MMP-2, -3, and -13 in IL-1β-treated HCHs. HCHs were treated with IL-1β (10 ng/mL) single or in combination with AyuFlex® (5, 10, and 20 μg/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, β-actin was utilized. ## *p* < 0.01, in comparison with the vehicle control group. \*\* *p* < 0.01, in comparison with the IL-1β-treated control group.

**Figure 4.** AyuFlex® treatment attenuated the degradation of collagen synthesis-involved proteins in IL-1β-treated HCHs. HCHs were exposed with IL-1β (10 ng/mL) single or in combination with AyuFlex® (5, 10, and 20 μg/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, β-actin was utilized. ## *p* < 0.01, in comparison with the vehicle control group. \*\* *p* < 0.01, in comparison with the IL-1β-treated control group.

### *3.4. AyuFlex*® *Treatment Attenuated the Degradation of Collagen Synthesis-Involved Proteins in IL-1*β*-Treated HCHs*

The effects of AyuFlex® on the expression of collagen synthesis-involved proteins in IL-1β-treated HCHs was investigated using western blotting. The protein levels of SOX9, aggrecan, COL1A1, and COL2A1 were assessed. As shown in Figure 4, IL-1β significantly reduced SOX9, aggrecan, COL1A1, and COL2A1 expression in contrast with that in the vehicle control. As shown in Figure 4, AyuFlex® treatment considerably upregulated the expression of SOX9, aggrecan, COL1A1, and COL2A1 compared to that in IL-1β-stimulated HCHs. Treatment with AyuFlex® (5, 10, and 20 μg/mL) increased SOX9, aggrecan, COL1A1, and COL2A1 expression compared with that in the IL-1β-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).

### *3.5. E*ff*ects of AyuFlex*® *on the NF-*κ*B and MAPK Mechanisms in IL-1*β*-Treated HCHs*

The impacts of AyuFlex® on IL-1β-triggered NF-κB and MAPK activation was confirmed with western blotting. In Figure 5A,B, it was confirmed that IL-1β conspicuously triggered phosphorylation of NF-κB p65 and ERK. In contrast, treatment with AyuFlex® alleviated this IL-1β-induced phosphorylation of NF-κB p65 and ERK in HCHs (42–49% and 53–63%, respectively), without affecting the total form of NF-κB p65 and ERK expression.

**Figure 5.** Effects of AyuFlex® on the NF-κB and MAPK mechanisms in IL-1β-treated HCHs. HCHs were pretreated with AyuFlex® (5, 10, and 20 μg/mL) for 24 h and then incubated with IL-1β (10 ng/mL) for 30 min. (**A**) The expression of NF-κB p65 (65 kDa) and phospho-NF-κ<sup>B</sup> p65 (65 kDa) was examined with western blotting. (**B**) The expression of ERK (42, 44 kDa) and phospho-ERK (42, 44 kDa) was determined by western blotting. Western blot bands were quantified utilizing ImageJ. As the control for normalization, β-actin was utilized. ## *p* < 0.01, in comparison with the vehicle control group. \*\* *p* < 0.01, in comparison with the IL-1β-treated control group.

### *3.6. E*ff*ects of AyuFlex*® *on Changes in the Body Weight of Rats with Monosodium Iodoacetate (MIA)-Incurred Osteoarthritis (OA)*

The effects of AyuFlex® on body weight in rats with MIA-incurred OA was investigated. The body weight of all animals was measured once a week and the changes were recorded for three weeks. No significant differences were observed between the six groups (Figure 6) for three weeks, and these results demonstrated that the body weight was not influenced by AyuFlex®.

**Figure 6.** Effects of AyuFlex® on changes in body weight of rats with monosodium iodoacetate (MIA)-incurred osteoarthritis (OA). Body weight was evaluated once a week for three weeks and the data are presented as the mean ± SEM (*n* = 8/group). A, AyuFlex®; I, Ibuprofen. No significant difference was discovered between any of the groups.

### *3.7. E*ff*ects of AyuFlex*® *on Weight-Bearing Distribution in the Hind Paw for 21 Days in MIA-Incurred OA in Rats*

To confirm progression of OA, hind paw weight-bearing capabilities and the ratio of weight distribution between the right (healthy) and left (MIA-incurred osteoarthritis) limbs was confirmed utilizing an incapacitance meter tester at days 0, 7, 14, and 21. The ratio in the MIA control group on day 7 was obviously lower than that in the normal control group, and this difference was endured until day 21. However, the ratio of the weight distribution between the left and right limbs increased in the AyuFlex® (26%, 36%, 40%) and ibuprofen (32%) groups in comparison with the MIA control group on day 7. In particular, the weight distribution of the animals treated with AyuFlex® at 25 mg/kg (42.60 ± 0.72), 50 mg/kg (45.50 ± 0.29), and 100 mg/kg (46.21 ± 0.77) returned to the normal level and showed similar results to the ibuprofen (45.34 ± 0.74) group at day 21 (Table 1). Our results revealed that AyuFlex® might alleviate OA-associated pain symptoms.

**Table 1.** Effects of AyuFlex® on weight-bearing distribution in the hind paw for 21 days in MIA-incurred OA in rats. The results are expressed as the mean ± SEM (*n* = 8/group). A, AyuFlex®; I, Ibuprofen. \* *p* < 0.05, \*\* *p* < 0.01, in comparison with MIA-induced control group; ## *p* < 0.01, in comparison with the non-MIA induced control group.


### *3.8. E*ff*ects of AyuFlex*® *on Arthritis Index (AI) for 21 Days in MIA-Incurred OA in Rats*

To assess OA-associated symptoms such as limping and swelling, we observed every animal on a weekly basis. The MIA control group showed a high AI index including swelling and limping score than all other groups, which gradually decreased over time (Table 2). Rats treated with AyuFlex® at 50 mg/kg and 100 mg/kg had noticeably downregulated the AI index after day 14 compared with those treated with MIA (control group) (respectively, *p* < 0.01). Furthermore, AyuFlex® administered at 50 mg/kg and 100 mg/kg downregulated the AI index to a similar level as ibuprofen—the positive

control—after day 14. We confirmed that the increased AI index due to OA was significantly reduced by AyuFlex®.

**Table 2.** Effects of AyuFlex® on arthritis index (AI) for 21 days in MIA-incurred OA in rats. The results are expressed as the mean ± SEM (*n* = 8/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.


### *3.9. E*ff*ects of AyuFlex*® *on the Expression of iNOS, 5-LOX, LTB4, and IL-6 in Arthrodial Cartilage*

To indicate the impacts of AyuFlex® on OA-upregulated inflammation, the protein expression of iNOS, 5-LOX, LTB4, and IL-6 was detected in the cartilage tissue. MIA-incurred OA enormously increased the protein levels of iNOS, 5-LOX, LTB4, and IL-6 in comparison with that in the normal control. The expression of iNOS, 5-LOX, LTB4, and IL-6 was significantly reduced in response to AyuFlex® and ibuprofen than in response to the MIA-injected group. The 25 mg/kg AyuFlex®-treated group mitigated the expression levels of iNOS, 5-LOX, LTB4, and IL-6 increased by MIA injection (19%, 7%, 3%, and 35%, respectively). Treatment with 50 mg/kg AyuFlex® obviously decreased (*p* < 0.01, for all conditions) the expression of iNOS, 5-LOX, LTB4, and IL-6 by 55%, 38%, 43%, and 39%, respectively, with respect to the expression levels in the MIA-treated group. The 100 mg/kg AyuFlex® significantly alleviated (*p* < 0.01, for all conditions) the iNOS, 5-LOX, LTB4, and IL-6 expression levels to 67%, 32%, 34%, and 44%, respectively, of those observed in the MIA-treated group (Figure 7).

**Figure 7.** Effects of AyuFlex® on the expression of iNOS, 5-LOX, LTB4, and IL-6 in arthrodial cartilage. The expression of iNOS (135 kDa), 5-LOX (78 kDa), LTB4 (36 kDa), and IL-6 (25 kDa) was measured by western blotting. Protein bands were quantified utilizing ImageJ. As the control for normalization, β-actin was utilized. The results are expressed as the mean ± 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.

### *3.10. E*ff*ects of AyuFlex*® *on Joint Pathology in MIA-Incurred OA in Rats*

To observe the morphological alterations and seriousness of the articular destruction in the joint tissue, H&E and Safranin-O staining were conducted in rats with MIA-incurred OA. H&E staining revealed that the MIA control group revealed serious alterations in the cartilage, synovial membrane, and fibrous tissue. However, it was confirmed that administration with AyuFlex® and ibuprofen effectually relieved the structural morphological alterations in arthrodial cartilage when it was compared with the MIA (control group; Figure 8A). Additionally, we stained the proteoglycan layer with safranin-O to confirm the cartilage breakdown (Figure 8B). In the MIA control group, normal cartilage (red) was destructed and the proteoglycan layer was reduced. Conversely, the proteoglycan layer was clearly visible in animals treated with AyuFlex® at 50 mg/kg, 100 mg/kg, and ibuprofen. In addition, using the Mankin scoring system, the seriousness of OA lesions was scored as shown in Figure 8C. The Mankin score was reduced in the AyuFlex® 100 mg/kg treated group by 60% and was noticeably reduced in the AyuFlex® 50 mg/kg and ibuprofen 20 mg/kg treated groups by 64% and 71%, respectively, compared with that in response to MIA (control group).

**Figure 8.** Effects of AyuFlex® on joint pathology inMIA-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 ± SEM (*n* = 5/group). A, AyuFlex®; I, Ibuprofen. Scale bar = 300 μm. \* *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.

### *3.11. AyuFlex*® *Decreased the Production of MMP-2, -3, and -13 in Arthrodial Cartilage*

To identify the effects of AyuFlex® 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® and ibuprofen treatment compared with the MIA treatment (control group). When compared with the MIA-injected group, the 25 mg/kg AyuFlex®-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® 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® 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 9.** AyuFlex® decreased the production of MMP-2, -3, and -13 in arthrodial 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, β-actin was utilized. The results are expressed as the mean ± 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.

**Figure 10.** AyuFlex® treatment attenuated the degradation of collagen synthesis-involved proteins in arthrodial 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, β-actin was utilized. The results are expressed as the mean ± 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.

### *3.12. AyuFlex*® *Treatment Attenuated the Degradation of Collagen Synthesis-Involved Proteins in Arthrodial Cartilage*

To investigate the impacts of AyuFlex® 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®-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, respectively. Treatment with 50 mg/kg AyuFlex ® 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. The 100 mg/kg AyuFlex ® significantly increased (*p* < 0.01, for all conditions) the SOX9, aggrecan, COL1A1, and COL2A1 expression levels to 1.7-fold, 8.0-fold, 4.2-fold, and 1.9-fold, respectively, of those observed in the MIA-treated group (Figure 10).
