Non-steroidal Anti-inflammatory Drugs Inhibit Osteoblasts Proliferation And Differentiation

Objective To investigate the effects of non-steroidal anti-inflammatory drugs(NSAIDs) in the induced differentiation of osteoblasts, and to discuss the mechanism of canonical Wnt signaling pathways in this process.Methods(1) The rat bone marrow mesenchymal stem cells(BMSCs) were isolated by Ficoll density gradient centrifugation and expanded in vitro.(2) Continuous 3-5 generations of rat bone marrow mesenchymal stem cells were collected and cultured in L – DMEM conditioned medium which contained dexamethasone(10-8 mol/L), beta glycerin sodium phosphate(10-2 mol/L) and vitamin C(0.05 g/L) for induced differentiation of osteoblasts. During this period, different concentration of diclofenac sodium or celecoxib(each group n = 5) was added respectively.(3) The effects of diclofenac sodium or celecoxib on the proliferation of BMSCs were evaluated by CCK8 assay.(4) Alkaline phosphatase(ALP) staining was used to analyze the effect of diclofenac sodium on the ALP expression of BMSCs when induced to differentiate into osteoblasts. Calcium salt dyeing was used to analyze the effect of celecoxib on the calcium salt deposition in BMSCs during induced osteoblasts differentiation. Image Pro Plus 6.0 software was used to analyse and calculated the IOD sum/Area sum value.(5) PT-PCR was used to analyze the effect of celecoxib on the expression of RUNX2 m RNA, a critical transcription factor of osteogenesis, and the expression of beta-catenin m RNA, a critical factor in canonical Wnt signaling pathways. Image J software was used to analyze RUNX2 luminance value/GAPDH luminance value and beta-catenin luminance value/GAPDH luminance value.Results(1) BMSCs were observed under inverted phase contrast microscope. They were large mononuclear round cells with morphological diversity. After 24 hours culturing, adherence began in parts of cells, the shape of adherent cells included elliptical, tears, spindle and fibrous, uneven distribution. The medium was changed on day 5 for the first time. On day 10, fusiform or fibrous cells were found. These cells were big and had big, circular or elliptic nucleus. Cytoplasm is full of particles. When the aggregated cells accounted for 80%- 90%, passage was needed.(2) The results of CCK8 assay showed that 1.25 umol/L, 2.5 umol/L, 5.0 umol/L and 10.0 umol/L diclofenac sodium inhibited the proliferation of BMSCs with the inhibition rates of 7.08%, 12.42%, 16.28% and 12.42%, respectively(comparing two adjoining dose groups, P < 0.001). The inhibition rate of 1.25 umol/L, 2.5 umol/L, 5.0 umol/L and 10.0 umol/L celecoxib were 7.27%, 11.87%, 12.78% and 11.87%(comparing two adjoining dose groups, P < 0.001), respectively which indicated that diclofenac sodium or celecoxib inhibited proliferation of BMSCs and the inhibition depended on the agent concentration. The inhibition rate of 1.25 umol/L diclofenac sodium and celecoxib were 7.08%, respectively 7.27%.The inhibition rate of 2.5 umol/L diclofenac sodium and celecoxib on BMSCs proliferation were 12.42% and 11.87%, respectively,showing these two agents were similar in inhibition of proliferation of BMSCs(P > 0.05).(3) On day 7 of culture in conditioned medium, ALP expression in 10 umol/L diclofenac sodium group was obviously lower than the control group. IOD sum/Area sum value were 0.270±0.256 and 0.412±0.113(P < 0.05), respectively. The results of alizarin red S staining of BMSCs on day 14 of culture showed that calcium salt deposition of 10 umol/L celecoxib group was obviously less than control group. IOD sum/Area sum value were 0.319±0.018 and 0.412±0.113, respectively(P < 0.05). These results indicated that the diclofenac sodium inhibited the expression of alkaline phosphatase and celecoxib inhibited calcium salt deposition in the process of the osteoblasts differentiation.(4) RT-PCR showed that RUNX2 luminance value/GAPDH luminance value in 0 umol/L, 1.25 umol/L, 2.5 umol/L, 5.0 umol/L and 10.0 umol/L celecoxib group were 0.7985 ± 0.0356, 0.6131 ± 0.0244, 0.3926 ± 0.0189, 0.2553 ± 0.0294 and 0.1348±0.0071, respectively(comparing two adjoining concentrations, P < 0.001). Betacatenin luminance value/GAPDH luminance value were 0.7959 ± 0.03136, 0.4002 ± 0.0177, 0.3388 ± 0.0336, 0.2209 ± 0.0102 and 0.2162 ± 0.0140, respectively(comparing two adjoining concentrations, P < 0.001), which indicated that celecoxib inhibited the expression of RUNX2 m RNA and beta- catenin m RNA, and the inhibition depended on agent concentration.Conclusion Diclofenac sodium and celecoxib might inhibit the proliferation of BMSCs and suppress their differentiation into osteoblasts. The Inhibition may be associated with the change of canonical Wnt signaling pathways.