| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 92 === Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) block the action of cyclo-oxygenase (COX) to relieve pain and inflammation. Steroids have been reported to inhibit phospholipase A2 and reduce prostaglandin synthesis. However, it has been reported that both steroids and NSAIDs suppress bone repair and remodeling in vivo. The recent developed selective COX-2 inhibitors, such as celecoxib and refecoxib were also reported to decrease bone repair in animal studies. Our previous studies showed that NSAIDs inhibited osteoblast proliferation and induced cell death in fetal rat osteoblast cultures. However, the effects of steroid, NSAIDs and COX-2 inhibitors on the proliferation and cell death of human osteoblast are rarely investigated.
In this study, the effects of steroid, NSAIDs and COX-2 selective inhibitors on the functions of human osteoblasts are examined. Normal human osteoblasts were purchased from Clonetics®. Indomethacin, ketorolac, piroxicam and diclofenac (10-5 and 10-4M); dexamethasone (10-7 and 10-6M); Celecoxib and DFU, an analogue of refecoxib, (10-7-10-4M) were used in this study. Drugs were treated for 24 or 48 hr in human osteoblast cultures. DNA synthesis was examined by thymidine incorporation. Cell cycle kinetics was measured by flow cytometry. Cytotoxicity was tested by lactate dehydrogenase (LDH) leakage. RNA expressions of cell cycle regulators and pro-apoptotic factors were analyzed by RT-PCR.
Our results showed that the tested 4 NSAIDs, dexamethasone and both the COX-2 selective inhibitors significantly inhibited proliferation of human osteoblast. The results of cell cycle kinetics showed that the tested 4 NSAIDs, dexamethasone and COX-2 selective inhibitors arrested cell cycle of human osteoblasts at G0/G1 phase. The inhibition effect on proliferation of celecoxib was completely reversed at 24 hr after removal of the agents, while DFU was partially reversed.
Our results showed that celecoxib and DFU significantly increased LDH leakage in cultures upon 24 hr treatments, while NSAIDs and dexamethasone showed non-significant effects. After 48 hr treatments, 10-4M of diclofenac and 10-7-10-6 M of dexamethasone showed cytotoxic in human osteoblast cultures. Furthermore, our results showed that dexamethasone 10-7M increased the mRNA expression of p27Kip1 (Cdk inhibitors). Dexamethasone 10-6M increased the mRNA expressions of p53, p21Waf1 and p27Kip1. Indomethacin and diclofenac significantly increased the mRNA expressions of p21Waf1 and p27Kip1. Celecoxib 10-5M increased the mRNA expression of p27Kip1, while 5x10-5M of celecoxib increased the mRNA expressions of p53, p27Kip1, p21Waf1 and Bax. However, DFU only significantly increased the mRNA expressions of p27Kip1 and Bax.
In this study, we found that NSAIDs (indomethacin, ketorolac, diclofenac and piroxicam), steroid (dexamethasone) and COX-2 inhibitors (celecoxib and DFU) all significantly inhibited proliferation and arrested cell cycle at G0/G1 phase. Steroid and COX-2 inhibitors had cytotoxicity in human osteoblasts. However, NSAIDs had no cytixic effects in human osteoblasts. The effects of NSAIDs, steroid and COX-2 inhibitors on proliferation and cell cycle progression in human osteoblasts may undergo through went influencing the expressions of different cell cycle regulators and pro-apoptosis factors. The results suggest that the effects of anti-inflammatory drugs on the proliferation inhibition and cytotoxicity of human osteoblasts may cause the suppression of bone formation. These effects of steroid, NSAIDs and COX-2 inhibitors on osteoblastic functions may through different mechanisms.
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