| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 93 === Steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs) block the action of cyclo-oxygenase (COX) to relieve pain and inflammation for osteoarthritis (OA) patients. However, investigators have indicated that NSAIDs might damage the cartilage. Previous in vivo studies demonstrated that several glucocorticoids reduce proliferation of chondrocytes and the synthetic rate of glycosaminoglycan in articular cartilage. Previous in vitro studies also demonstrated that several NSAIDs accelerate the damage of osteoarthritic cartilage by ways of inhibiting the synthesis of proteoglycan and/or facilitating the effects of cytokines. Recently, COX-2 inhibitors have been prescribed for long-term treatment of OA for their lesser side effects on gastrointestines. However, the influences of COX-2 inhibitors on normal articular chondrocytes were rarely investigated. On the other hand, it has been found that one of the main pathogenesis of OA is that articular chondrocytes undergo terminate differentiation, so that the chondrocytes become hypertrophy, synthesize collagen type X, form mineral formation and eventually undergo apoptosis. However, the influences of anti-inflammatory drugs on this terminal differentiation of articular chondrocytes remain unclear. Accordingly, in this study, we investigated the effects of anti-inflammatory drugs on the expressions of normal functional genes and the marker genes of terminal differentiation in cultured human articular chondrocytes. Cytotoxicity and apoptosis induced by anti-inflammatory drugs on chondrocytes were also tested.
In this study, we isolated human articular cartilage from a 23-year-old male cavader and encapsulated chondrocytes in alginate beads as a 3 dimensional culture. The effects of dexamethasone (10-9~10-7M); diclofenac, indomethacin, ketorolac and piroxicam（10-5~10-4M）; and DFU (an analogue of refecoxib) and celecoxib (10-6~10-5M) were tested. The expressions of normal functional genes, collagen type II, SOX9, aggrecan, and the marker genes of terminal differentiation, collagen type X, Indian hedgehog (Ihh) and PTHrP, were examined in human articular chondrocytes by RT-PCR. Total sulfated glucosaminoglycan was quantified by DMMB assay. Cytotoxicity was tested by lactate dehydrogenase (LDH) leakage. Apoptosis was examined by TUNEL stain.
Our results showed that 1-, 5- and 7-day treatments of dexamethasone (10-9~10-7M), DFU and celecoxib (10-6~10-5M) significantly inhibited the expressions of collagen type II, SOX9 and aggrecan. However, non-selective NSAIDs, diclofenac, indomethacin, ketorolac and piroxicam （10-5~10-4M）, showed no significant effect on the expression of these functional genes of articular chondrocytes. We also found that the three classes of anti-inflammatory drugs induced the mRNA expressions of collagen type X, but inhibited that of PTHrP. The result of DMMB assay showed that dexamethasone (10-9~10-7M), DFU and celecoxib (10-6~10-5M) significantly inhibited the total sulfated glucosaminoglycan content. However, the treatment of either diclofenac, indomethacin, ketorolac or piroxicam （10-5~10-4M）for 7 days showed no significant effect in comparison to the control cultures. The results of glycosaminoglycan were consistent with the gene expression of aggrecan. Our results showed that anti-inflammatory drugs showed no significantly effects on cytotoxicity and apoptosis of chondrocytes.
These results showed that dexamethasone and COX-2 inhibitors inhibited functional genes expressions, suggesting that dexamethasone and COX-2 inhibitors may inhibit the synthesis of extracellular matrix in normal human articular chondrocytes. We also suggest that these drugs may inhibited the gene expressions of type II collagen and aggrecan through inhibiting SOX9 expression. In addition, these three classes of anti-inflammatory drugs induced marker genes of terminal differentiation, suggesting that they may help to promote cell undergoing terminal differentiation.
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