Osteoclasts were measured in the subchondral bone area. P< 0.05, and P< 0.01 compared with the vehicle-treated MIA-induced OA group.We next investigated the effects of eupatilin on the expression of catabolic and anticatabolic molecules in IL-1-stimulated human OA chondrocytes. Expression of MMP-3, and -13 in IL1-stimulated OA Berbamine (dihydrochloride) structure chondrocytes were significantly reduced in response to eupatilin treatment in a dose-dependent manner similar reduction in ADAMTS5 expression in OA chondrocytes were also observed following eupatilin treatment. The inhibitory properties of eupatilin on these catabolic molecules in chondrocytes were comparable with those of celecoxib, a potent inhibitor of MMPs and NO production [30] (Fig 6A). In contrast, expression of anticatabolic factor TIMP-1 in IL-1-induced OA chondrocytes increased significantly after eupatilin treatment in a dose-dependent manner (Fig 6B). Cell viability, as determined by MTT assay, was not altered at any of the doses used in this study (Fig 6C). Taken together, these data show that eupatilin exert chondroprotective properties in vivo by modulating specific catabolic and anticatabolic molecules in OA chondrocytes.To understand the molecular mechanisms by which effect of eupatilin, we investigated whether the eupatilin modulate JNK signaling pathway in IL-1-stimulated OA chondrocytes. Phosphorylation of JNK was reduced with eupatilin treatment in a dose dependent manner (data in S1 Fig).Fig 4. Delayed efficacy of eupatilin in MIA-induced OA. Rats were injected with 3 mg of monosodium iodoacetate (MIA) in the right knee. Eupatilin was administered orally daily for 14 days after MIA injection. (A) The knee joints from the OA rats treated with either eupatilin or vehicle control were stained with HE, Safranin O, and toluidine blue. The joint lesions were graded on a scale of 03 using the modified Mankin scoring system, giving a combined score for cartilage structure, cellular abnormalities, and matrix staining. The data are expressed as means SEM for six animals per group. (B) Osteoclasts were measured in the subchondral bone region. P< 0.05, and P< 0.01 compared with the vehicle-treated OA animals.Fig 5. Effects of eupatilin on the expression of MMP-13, IL-1, IL-6, iNOS, nitrotyrosine in OA joints. Rats were injected with 3 mg of monosodium iodoacetate (MIA) in the right knee. Eupatilin was administered orally daily for 7 days after MIA injection. Immunohistochemical staining was used to identify the expression of MMP13, IL-1, IL-6 (A), iNOS, and nitrotyrosine (B) in the articular cartilage. The data are expressed as means SEM for six animals per group. P < 0.01 P < 0.001 compared with the MIA-induced OA group.Fig 6. Recovery of anabolic and catabolic activities in human OA chondrocytes following the19467704 treatment 
with eupatilin. Human articular chondrocytes from OA patients were cultured with IL-1 in the presence or absence of eupatilin for 48 h following 24 h in serum-free medium. The mRNA expression of catabolic (MMP-3, and MMP-13, and ADAMTS5) (A) and anabolic (TIMP-1) factor (B) were measured by quantitative real-time PCR -actin was used as the internal control.