## 3 Results and Discussion
The dose of 10 mg/kg was based both on a previous study on the anti-inflammatory and antinociceptive activities of  
Figure 3: (**A**) Immunohistochemical expression pattern of cell positivity for IL-1\(\beta\) in the experimental groups treated with oral administration of vehicle only (Vehicle), formononetin at 10 mg/kg (FNT), and ibuprofen at 10 mg/kg (LSAB, 400\(\times\)). (**B**) Assessment of the mean number of IL-1\(\beta\)-positive cells for histological field (0.025 mm\({}^{2}\)). Differences in comparison with Vehicle are expressed as \({}^{**}p\) < 0.01, whereas differences in comparison with Sham are expressed as \({}^{**}p\) < 0.011 (ANOVA and [PERSON]’s multiplex comparison test). formonentin administered by the oral route [6] and on a pilot test in experimental OA (data not shown) to provide a rational use of experimental animals. Safranin-O dye was chosen for the histological analysis. This dye is based on the combination of an anion (e.g., keratan sulfate or chondro sulfate) and a cationic chromophyll, which stains red the proteoglycans distributed in the cartilaginous tissue; hence, the intensity of staining is proportional to the concentration of PG in the tissue [29]. The reduced and irregular pattern of stain observed in Group Vehicle in opposition to the homogeneous pattern seen in Group Sham has also been reported in previous studies [23; 30], suggesting that the experimental model was successful. On the other hand, the groups treated with the oral administration of commonetin showed a more intense pattern of dye impregnation, especially in the deepest third of the tissue, which is suggestive of a possible chondrocyte effect. To confirm the protective effect of the oral administration of commonetin on joint cartilage, the grading system recommended by the Osteoarthritis Research Society International (OARSI) for histological assessments of osteoarthritis in rats was used in the current study. The simple semi-quantitative score method used in this system is accurate, reproducible, and practical for most studies, as it permits a more complete assessment of the cartilage histopathology [23; 31]. Based on the OARSI histopathological grading system, commonetin significantly attenuated the cartilage-matrix-loss width, degeneration scores, and wear depth, supporting the hypothesis that the isoflavoid can prevent, at least partially, the joint damage induced by medial meniscectomy. The content of proteoglycan in the joints is closely related to the type II collagen content [32]. Type II collagen is the main molecular component of the extracellular framework of adult articular cartilage [33], and previous studies have demonstrated that the pathological degenerative changes observed in the articular cartilage in OA are associated with the reduction of the tissue contents of type II collagen [32], which can be observed both in experimental models [23; 30] and in clinical trials [34]. The similarities in the immunohistochemical expression of type II collagen and the content of proteoglycan assessed by Safranin-O impregnation observed in the current study fully supported this theory and seemed to suggest that the chondrocyte effects played by formonetin might be related to a possible modulation of type II collagen catabolism. Previous studies have demonstrated increased levels of metalloproteinase (MMP)- 3 and MMP-13, enzymes that degrade the extracellular substrates of the cartilage matrix and collagen type II, respectively, in OA [35; 36]. As commonetin has been previously demonstrated to effectively reduce the expression of MMP-3 and MMP-13 and increase proteoglycan content by antagonizing the IL-1\(\beta\)-induced catabolic effects in chondrocytes in vitro [10; 37], we investigated the immunohistochemical expression of IL-1\(\beta\) in the chondrocytes of the articular joint in vivo. We found that the immunohistochemical expression of IL-1\(\beta\) was significantly reduced by the oral administration of commonetin.