| Osteoarthritis is the main reason for disability in the elderly, and theprevalence of osteoarthritis increase as aging population grows all over the world.Currently, there are no satisfactory therapy or prevention regimens forosteoarthritis, due to lack of the understanding of underlying pathomechanism.Multiple joints can be affected in osteoarthritis, but mostly in hip and kneejoints. Osteoarthritis is characterised by destruction of cartilage, subchondral boneeburnation, cyst formation, fibrilation of capsule, and eventually, jointdeformation and function loss.In osteoarthritis, cartilage matrix metabolism imbalance is directly related tocartilage destruction. Enhancement of anabolism and inhibition of catabolism mayretard or even reverse the process of cartilage degradation, and is highlighted inmany studies. One key fator in cartilage catabolism, the matrix metalloproteinase,is of much higher activity in osteoarthritis, and are targeted in the exploration ofdrugs for the therapy of osteoarthritis. Statins can significantly reduce the mortality of patients with artherosclerosis,and the mechanism was that statins can lower the risk of artherosclerosis plaquerupture, by inhibiting the activity of matrix metalloproteinase and thus stabilizingthe collagen matrix in plaques. This matrix metalloproteinase-inhibiting effectwas also observed in many other tissues, including in vivo and in vitro studies.Statins also have anti-inflammation properties. We thus hypothesized that statinsmay inhibit the cartilage destruction in osteoarthritis by this mechanism.This research work was designed to explore the possible therapeutic effectsof simvastatin in osteoarthritis at both in vitro and in vivo levels.Thechondroprotectivity of simvastatin was our main consideraton in these studies.Our work began with the question that whether simvastatin can influence theactivity of MMP produced by chondrocytes cultured in vitro by using gelatinzymography. And then an in vitro cartilage explant culture model was used toobserve the effect of simvastatin on degradation of cartilage matrix. In the last, arabbit knee joint osteoarthritis model was established by anterior cruciate ligamenttransection, and efficacy of simvastatin in treating osteoarthritis was evaluated.The contents and results of this work were as follows.1. Effects of simvastatin on the viability of rabbit articular chondrocytescultured in vitroBoth sides of knee articular cartilage were harvested from a 4-week old NewZealand rabbit. Chondrocytes were isolated by digestion with type-II collagenaseand cultured routinely. Cells of the third passage were used in this experiment.They were treated with simvastatin, diclofenac, or dexmethasone, with a finalconcentration of 0.01μM, 0.1μM, 1μM, and 10μM. After 48 h, the viability of cells was measured using MTT method. It was revealed that all agents at givenconcentration have no detectable influence on the viability of chondrocytes.2. Effect of simvastatin on MMP-2 acitivity of chondrocytes induced by IL-1αChondrocytes were stimulated by IL-1αwith a final concentration of 5ng/ml,and treated with simvastatin, sodium diclofenac and dexmethasone. Theconditioned mediums were collected after 48 hours in culture. Gelatinzymography was used to evaluate the quantity of gelatinase released into themedium. The results indicated that IL-1αstimulation can cause upregulation ofMMP-2 in chondrocytes; and this can be inhibited by simvastatindose-dependantly; while sodium diclofenac and dexmethasone showed no effects.3. Effects of simvastatin on proteoglycan degradation of rabbit articularcartilage induced by IL-1αRabbit knee joint articular cartilage were used to establish an in vitrocartilage explant culture model, and they were pretreated with simvastatin, sodiumdiclofenac or dexmethasone, then stimulated by IL-1αwith a final concentrationof 5 ng/ml, the conditioned medium were collected after 48 hours in culture.Glucosaminoglycan that had been released into the medium were measured toevaluate the extent of proteoglycan degradation. the results showed thatsimvastatin can inhibit the proteoglycan degradation within a concentration scopeof 1μM to 10μM, and this effect is concentration dependant; whereas sodiumdiclofenac and dexmethasone showed no effects.4. Effects of simvastatin on collagen degradation of rabbit articular cartilageinduced by IL-1αCartilage explants were pretreated with simvastatin, sodium diclofenac or dexmethasone, then stimulated by IL-1αwith a final concentration of 5 ng/ml, theconditioned medium were collected after 15 days. Hydroxyproline that had beenreleased into the medium were measured to evaluate the extent of collagendegradation. The results showed that simvastatin can inhibit the collagendegradation within a concentration scope of 1μM to 10μM, and this effect isconcentration dependant; whereas sodium diclofenac showed no effects, and onlyat a high concentration did dexmethasone show inhibitory effect.5. Establishment of rabbit osteoarthritis model and its evaluation20 adult New Zealand rabbits weighting about 2.5-3Kg were included in thisstudy. They were randomly divided into two groups: the sham-operation groupserved as normal control, and the anterior cruciate ligament transection (ACLT)group as model. ACLT was performed in the right knee joint of the model groupto induce OA, while the control group were operated on without ACLT. Allanimals were bred in boxes and allowed acitivity.8 weeks after surgery the rabbitswere necropsied. The articular cartilage was evaluated by ink-dye gradesmacroscopically and also histologically scored. The results showed that rabbits ofthe ACLT group had more severe degeneration of articular cartilage compared tothat of control group.Macroscopic analysis showed well relevance to thehistological scores. In all the four compartments of articular cartilage, the cartilagelesions of the lateral femoral condyle (LFC) were most significant. So LFC shouldbe the choice of site when evaluating osteoarthritis6. Evaluations of simvastatin therapy on rabbit osteoarthritis40 adult New Zealand rabbits weighting about 2.5 ~ 3.0 kg were included inthis study. They were randomly divided into 4 groups: the sham-operation group served as normal control; and the Anterior cruciate ligament transection (ACLT)groups, which was subdivided into simvastain, diclofenac and placebo group. 1week after surgery the rabbits were treated with simvastatin (n=10) or sodiumdiclofenac (n=10) or placebo (n=10) daily for 8 weeks and then necropsied. Thearticular cartilage was evaluated macroscopically and histologically. The synovialmembrane was collected to evaluate the level of inflammation. The level ofMMP-2 in OA cartilage chondrocytes was evaluated by immunohistochemistry.Macroscopic analysis showed that cartilage lesions were significantly reduced inthe simvastatin treated OA joints compared with that of placebo treated OA joints.It was also shown that in cartilage from placebo and sodium diclofenac treatedOA rabbits, a significantly higher percentage of chondrocytes stained positive forMMP-2 compared with unoperated control. Rabbits treated with simvastatinrevealed a significant reduction in the level of MMP-2. It was also showed thatsimvastatin and sodium diclofenac group had a relatively low severity of synovialinflammation, compared to that of placebo group.
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