Experimental Study On Duyiwei Against Articular Cartilage Influence Of Rabbit Knee OA Models

Objective To explore the protection and treatment mechanism of Duyiwei capsule on rabbit KOA model of cartilage degeneration and to provide theoretical support for the clinical application of the Duyiwei capsule on osteoarthritis.Methods40New Zealand rabbits were numbered and divided into five groups at random:the control group (A), model control group (B), the low dose group(C), the middle dose group(D) and the high dose group (E),8for each group. Group A was simulated operation as sham operation group. Group B, C, D, and E were duplicated into the rabbit model of knee osteoarthritis by Hulth’s technique. All the rabbits moved with goad for30min evryday after1week. A week after the Modeling, the drug was given to the rabbits through stomach by Meeh-Rubner. The dosage of administration was converted through the surface area of human and the rabbit. Duyiwei capsule was dissolved in the2ml distilled water. The low dose group, the middle dose group and the high dose group were respectively given the drug (60mg/kg、120mg/kg、240mg/kg),one time a day.At each time point, the control group and model control group were respectively given the equivalent normal saline. After6weeks of treatment the animals were given the last dose of the drugs before fasting for24h, Then the rabbits were sacrificed. The articular cartilage, synovial fluid of rabbit KOA model were measured and evaluated by histological techniques, immunohistochemistry, RT-PCR and other techniques.After opening the rabbit knee joint in experimental side, observed and scorded the articular cartilage of the rabbit’ s knee joint according to previous literature and compared the differences between each two groups. The articular cartilage was prepared for optical microscope sections. Observed and scorded the optical microscope sections by Mankin scoring system and compared the differences between each two groups. The the articular cartilage was determined with transmit electron microscope to observe the ultrastructural pathologic changes.Synovial fluid was taken before opening the rabbit knee joint. The IL-1β and NO levels in synovial fluid were measured and compared the differences between each two groups.After therapy, the MMP-3, MMP-13and TIMP-1mRNA expression levels were measured of articular cartilage and compared the differences between each two groups.The GAG levels, changes in the positive staining area of type Ⅱ collagen in articular cartilage were measured by immunohistochemical method and compared the differences between each two groups.ResultsThe rabbit body weight changed before and after admini-stration:Compared with that before administration, all the body weight of treatment groups increased at different degrees, but there is no significant differences compared with normal group (P>0.05). The range of motion:The results of the knee joint mobility determination showed that:All the knee joint mobility of treatment groups recovered in varying degrees, especial ly the middle dose group and the high dose group (P<0.01)General observation, compared with model control group, the cartilage abrasion, degeneration of the middle dose group and the high dose group were less obvious (P<0.01).Light microscopy observation, compared with model control group, the scores of the low dose group by Mankin scoring system were less obvious (P<0.05), so did the middle dose group and the high dose group (P<0.01),Electron microscopy observation, Cells morphology, structure, arrangement of the middle dose group and the high dose groupwere better than the model control group.After Duyiwei capsule therapy, compared with the model control group, the IL-1β and NO levels in synovial fluid of each group was decreased. Compared in each group, the IL-1β and NO levels in synovial fluid of the middle dose group and the high dose group were less obvious, and there was significant difference in the comparison between the groups (P<0.01). Compared with the model control group, the IL-1β and NO levels of the low dose group were less (P<0.05). These results suggested that the IL-1(3and NO levels in synovial fluid decreased with the elevation of the dose.After therapy, compared with the model control group, the MMP-3and MMP-13mRNA expression levels of the middle dose group and the high dose group were less obvious, whi le the TIMP-1mRNA expression levels were obvious. The MMP-3and MMP-13mRNA expression levels in synovial fluid of the model control group were significantly increased, while the TIMP-1mRNA expression levels were decreased significantly. There was significant difference in the comparison between the groups (P<0.01).After positive staining of type Ⅱ collagen, positive staining aere was increased significantly with uneven distribution and a breach appeared in the superficial zone. After Duyiwei capsule therapy, compared with the model control group, the positive staining area of type Ⅱ collagen of the middle dose group and the high dose group were increased significantly, and there was significant difference in the comparison between the groups (P<0.05). Compared with the model control group, the GAG levels of type Ⅱ collagen of the low dose group was increased significantly (P<0.05), the GAG levels of type Ⅱ collagen of the middle dose group and the high dose group were obvious increased (P<0.01).Conclusions1. Build the model of rabbit knee osteoarthritis by Hulth’s technique with favorable stability, high success rate, shorter inducement duration and good reproducibility.2. Duyiwei capsule inhibits the expression of MMPs, promotes the TIMPs expression, advance the GAG content in ECM, promote synthesis of type Ⅱ collagen, delays and prevents the development of rabbit knee osteoarthritis to some extent, improves osteoarthritis of rabbit knee by decreasing the IL-1β and NO levels in synovial fluid of rabbit KOA model.3. There Maybe some factors Duyiwei capsule correlates to the therapeutic mechanism on OA as follow: After different doses of Duyiwei capsule therapy, there are same changes on KOA model. experimental models, especially the middle dose group and the high dose group. The knee joint mobility was improved. Cartilage degeneration wear degree were reduced or delayed.The formation of the cartilaginous osteophyte was decreased and the scores by Mankin was reduced.After the Intervention Effect of Duyiwei capsule on KOA model, the IL-1β and NO levels in synovial fluid of each group were decreased. It showed that The synovial fluid and inflammatory response of synovial were reduced by Duyiwei capsule reducing the IL-1β and NO levels inside the lesion joints, and it showed a relationship with dose-dependent manner. Duyiwei capsule reduced destruction of articular cartilage by reducing the NO levels in synovial fluid, reducing or delaying the destructive action of NO on articular cartilage. decreasing the chondrocyte destruction, promoting the synthesis of extracellular matrix, inhibiting apoptosis of rabbit chondrocytes. Duyiwei capsule delayed the process of OA by the IL-1β levels in synovial fluid, controlling the anabolism and catabolism of chondrocyte and the powerful resistance to inflammation.The experimental results show that the MMP-3and MMP-13mRNA expression levels of the control group lower than the model control group, while the TIMP-1mRNA expression levels of treatment groups were higher than the model control group. This conclusion was further verified that MMPs and TIMPs were related to OA. After Duyiwei capsule therapy, the MMP-3and MMP-13mRNA expression levels of each group lower than the model control group obviously. It showed that Duyiwei capsule could inhibit the MMP-3and MMP-13mRNA expression with dose-dependent manner. It proved that Duyiwei capsule could delay the process of OA and the destruction of cartilage matrix and protect articular cartilage.Duyiwei capsule may delay the process of OA and the destruction of articular cartilage to some extent and has played a certain action of protection and repair by decreasing the effect of IL-1β and other inflammatory cytokines on chondrocytes, reducing the secretion of MMPs, promoting the synthesis of type Ⅱ collagen and proteoglycan and the ex-pression of procollagen type Ⅱ mRNA, preventing degradation of matrix collagen.