*2.5. Animals*

Sixty male Sprague-Dawley (SD) rats (6-week-old; 130-190 g) were o ffered from Samtako Bio, Inc. (Osan, Korea). Whole animals were acclimated for seven days and normal animals were sorted out for experiments. The experiment was progressed under optimal conditions (22 ± 2 ◦C and 12 h light/dark cycles). Animals were free to consume sterile water and food. The study was conducted in compliance following the national guidelines for the managemen<sup>t</sup> and utilization of experimental animals permitted by the Animal Ethics Committee (permission number: IV-RB-02-1910-21 of INVIVO Co. Ltd. (Chungnam, Korea)). Once a week, alterations in body weight and alterations in water and food consumption were observed.

### *2.6. Monosodium iodoacetate (MIA)-Incurred Osteoarthritis (OA) and Drug Administration*

The left knee was shaved and then 50 μL of 0.9% sodium chloride including 3 mg monosodium iodoacetate (MIA) was injected once into the synovial cavity utilizing an insulin syringe to induce OA. After three days, the rats were randomly arranged to six groups, comprising eight rats each. A non-MIA-stimulated control group was also used: (1) Non-MIA-stimulated control + Vehicle; (2) MIA-stimulated control + Vehicle; (3) MIA + AyuFlex ® 25 mg/kg; (4) MIA + AyuFlex ® 50 mg/kg; (5) MIA + AyuFlex ® 100 mg/kg; and (6) MIA + Ibuprofen 20 mg/kg. The test substances were homogenized in a carboxymethyl cellulose sodium salt (CMC-Na) of 0.5%. Then, the test substances were administrated orally once a day for three weeks.

### *2.7. Progression of OA and Hind Paw Weight-Bearing Distribution*

After 0, 7, 14, and 21 days after treatment of the test substances, the whole rats were left free to roam the cage, and the walking and knee joint swelling aspect, for instance, gait disorder was precisely assessed in rats. Limping and swelling were categorized as: No change (0), Mild swelling (1), Moderate swelling (2), and Severe swelling (3). All evaluations were performed by an identical proficient evaluator, who blinded the type of test substance administered to the rats during the study period.

The normal balance of weight bearing capacity in the hind paws was impaired after OA occurrence. Rats were cautiously located in the measurement chamber of the incapacitance meter tester (IITC Life Science, Woodland Hills, CA, USA) to assess alterations in weight-bearing tolerance and the force applied by each hind limb was averaged for 10 s. The following formula: % weight distribution of left hind paw = weight on left hind limb/(weight on right hind limb + weight on left hind limb) × 100 was used to analyze the percentage distribution of the left hind paw.

### *2.8. Histological Examination of Joints*

To determine the e ffectiveness of AyuFlex ® on knee joint cartilage atrophy, we evaluated the histological alteration in a rat model with MIA-incurred OA. After euthanizing the animals at the end of the experiments, the knee joint was incised, fixed with 10% formalin for 24 h at 4 ◦C, and decalcified using 5% hydrochloric acid (Sigma-Aldrich, St. Louis, MO, USA) for four days. After removal of calcification, acetone was utilized to dehydrate the specimens, which were then embedded in para ffin. Para ffin-embedded knee joints were sliced 5 μm thick along the sagittal axis. Hematoxylin and Eosin (H&E) and Safranin-O fast green staining (Sigma-Aldrich, St. Louis, MO, USA) were utilized to stain the sliced sections. Whole stained sections were scanned utilizing the Motic EasyScan (Meyer Instrument, Houston, TX, USA) and were assessed and graded on a 0-13 scale depending on the Mankin scoring system by a double-blinded observer.

### *2.9. Cartilage Protein Expression*

Cartilage tissue was excised and cleaned three times in cold PBS. Cartilage was frozen by liquid nitrogen briefly and then pulverized. Protein was extracted with RIPA reagen<sup>t</sup> (Tris-HCl of 50 mM, pH 7.5; sodium chloride (NaCl) of 150 mM; ethylenediaminetetraacetic acid (EDTA) of 2 mM; Triton X-100 of 1%; sodium deoxycholate of 0.5%; SDS of 0.1%; protease inhibitor of 0.1% (Roche, Mannheim, Germany)) and then centrifuged at 10,000× *g* during 15 min at 4 ◦C. Protein concentration was calculated by the same method as described above. The protein expression profiles of β-actin, 5-LOX, LTB4, IL-6, MMP-2, -3, and -13, iNOS, SOX9, aggrecan, COL1A1, and COL2A1 were confirmed by western blotting as described above.

### *2.10. Statistical Analysis*

The results are presented as the means ± standard error of the mean (SEM) and were assessed with the SPSS program (version 22.0, SPSS Inc., Chicago, IL, USA). Student's *t*-test and one-way analysis of variance (ANOVA) were utilized to compare di fferent treatment groups, followed by multiple comparisons correction through Dunnett's post-hoc test utilizing Origin 7.0 software (OriginLab, Northampton, MA, USA). The di fferences between mean values were regarded significant or intensely significant if *p* < 0.05 and *p* < 0.01, respectively.
